Identification of Functional Brassinosteroid Receptor Genes in Oaks and Functional Analysis of QmBRI1.

Brassinosteroids (BRs) play important regulatory roles in plant growth and development, with functional BR receptors being crucial for BR recognition or signaling. Although functional BR receptors have been extensively studied in herbaceous plants, they remain largely under-studied in forest tree species. In this study, nine BR receptors were identified in three representative oak species, of which BRI1s and BRL1s were functional BR receptors. Dispersed duplications were a driving force for oak BR receptor expansion, among which the Brassinosteroid-Insensitive-1 (BRI1)-type genes diverged evolutionarily from most rosids. In oak BRI1s, we identified that methionine in the conserved Asn-Gly-Ser-Met (NGSM) motif was replaced by isoleucine and that the amino acid mutation occurred after the divergence of Quercus and Fagus. Compared with QmBRL1, QmBRI1 was relatively highly expressed during BR-induced xylem differentiation and in young leaves, shoots, and the phloem and xylem of young stems of Quercus mongolica. Based on Arabidopsis complementation experiments, we proved the important role of QmBRI1 in oak growth and development, especially in vascular patterning and xylem differentiation. These findings serve as an important supplement to the findings of the structural, functional and evolutionary studies on functional BR receptors in woody plants and provide the first example of natural mutation occurring in the conserved BR-binding region (NGSM motif) of angiosperm BRI1s.

Genome-Wide Identification and Expression Analysis of the bHLH Transcription Factor Family and Its Response to Abiotic Stress in Mongolian Oak (Quercus mongolica).

The basic helix-loop-helix (bHLH) family, one of the largest families of transcription factors in plants, is extensively involved in the growth, development, and stress response of several woody plants. However, no systematic analysis of the bHLH gene family in Quercus mongolica has been reported. We characterize QmbHLH genes and identify the functions of QmbHLH proteins in Q. mongolica. We used bioinformatics approaches, qRT-PCR analysis, and RNA sequencing data to examine chromosomal distributions, gene structures, and conserved patterns, and identified 89 QmbHLH genes, which were divided into 21 subgroups based on the phylogenetic analysis of bHLH genes in Arabidopsis thaliana. Segmental replication played a more prominent role than tandem duplication in the expansion of the QmbHLH gene family. Based on patterns of tissue-specific expression, protein interactions, and cis-element analysis, QmbHLH genes may be extensively involved in the growth and development of Q. mongolica. In leaves, stems, and roots, 12 selected QmbHLH genes exhibited responsiveness to abiotic stresses (salt, cold, weak light, and drought). Our study facilitates follow-up functional investigations of the bHLH gene family in Q. mongolica and provides novel insights into bHLH superfamilies in woody plants.

Selection and validation of reference genes for quantitative real-time PCR of Quercus mongolica Fisch. ex Ledeb under abiotic stresses.

Quercus mongolica Fisch. ex Ledeb is the main species of coniferous and broadleaved mixed forests in northeast and north China, which has high ornamental, economic, and ecological value. The appropriate reference genes must be selected for quantitative real-time PCR to reveal the molecular mechanisms of stress responses and their contribution to breeding of Q. mongolica. In the present study, we chose 11 candidate reference genes (TUA, CYP18, HIS4, RPS13, ACT97, TUB1, UBQ10, UBC5, SAND, PP2A, and SAMDC) and used four programs (GeNorm, NormFinder, BestKeeper, and RefFinder) to assess the expression stability of the above genes in roots, stems, and leaves under five abiotic stress factors (cold, salt, drought, weak light, and heavy metal). The findings revealed that under various experimental environments, the most stable genes were different; CYP18, ACT97, and RPS13 ranked the highest under most experimental environments. Moreover, two genes induced by stress, CMO and P5CS2, were chosen to demonstrate the reliability of the selected reference genes in various tissues under various stress conditions. Our research provides a significant basis for subsequent gene function studies of Q. mongolica.

A chromosome-scale genome assembly of the Mongolian oak (Quercus mongolica).

Mongolian oak (Quercus mongolica Fisch.) is an ecologically and economically important white oak species native to and widespread in the temperate zone of East Asia. Here, we present a chromosome-scale reference genome assembly of Q. mongolica, a representative white oak species, by combining Illumina and PacBio data with Hi-C mapping technologies that is the first reference genome created for an Asian oak. Our results showed that the PacBio draft genome size was 809.84 Mb, with a BUSCO complete gene percentage of 92.71%. Hi-C scaffolding anchored 774.59 Mb contigs (95.65% of draft assembly) onto 12 pseudochromosomes. The contig N50 and scaffold N50 were 2.64 and 66.74 Mb, respectively. Of the 36,553 protein-coding genes predicted in the study, approximately 95% had functional annotations in public databases. A total of 435.34 Mb (53.75% of the genome) of repetitive sequences were predicted in the assembled genome. Genome evolution analysis showed that Q. mongolica is closely related to Q. robur from Europe, and they shared a common ancestor ~11.8 million years ago (Ma). Gene family evolution analysis of Q. mongolica revealed that the nucleotide-binding site (NBS)-encoding gene family related to disease resistance was significantly contracted, whereas the ECERIFERUM 1 (CER1) homologous genes related to cuticular wax biosynthesis was significantly expanded. This pioneering Asian oak genome resource represents an important supplement to the oak genomics community and will improve our understanding of Asian white oak biology and evolution.

Prognosis and immune features of pyroptosis-related RNA patterns in low-grade glioma.

Purpose: Low-grade gliomas (LGG), which are malignant primary brain tumors, are more prevalent in young adults. Pyroptosis, an inflammatory form of programmed cell death, has been shown in recent years to be directly associated with tumor growth and tumor microenvironment (TME). However, the correlation between LGG and pyroptosis remained to be explored. In this research, we explored pyroptosis-related gene expression patterns and their prognostic significance based on transcriptome profiles and clinical data in LGG. Methods: We identified 31 pyroptosis-related genes differentially expressed at the mRNA level between the data of LGG patients from TCGA and the data of normal brain tissues from GTEx. Univariate Cox regression analysis was used to screen 16 differentially expressed genes (DEGs) based on survival data. Next, the prognostic model was established using LASSO Cox regression, which divided LGG patients into high- and low- risk subgroups and showed an independent prognostic value for overall survival (OS) combined with clinical factors in the CGGA test cohort. Pyroptosis and immune cells were correlated through the CIBERSORT R package and the TIMER database. Results: Based on the analyses of 523 LGG and 1152 normal tissues, nine significant differential genes were identified. The AUC remained at about 0.74 when combined with the risk score and clinical factors. Enrichment analyses revealed that DEGs were mainly enriched in cytokine-cytokine receptor interactions, immune response and chemokine signaling pathways. Immune cell enrichment analysis demonstrated that scores for most immune cell types differed significantly between the high-and low-risk groups, and further infiltrating analysis showed obvious differences between these two risk subgroups. Conclusion: Pyroptosis-related genes play a pivotal role in LGG and are associated with tumor immunity, which may be beneficial to the prognosis and immunotherapy of LGG.

UAV path planning method for data collection of fixed-point equipment in complex forest environment.

In a complicated forest environment, it is usual to install many ground-fixed devices, and patrol personnel periodically collects data from the device to detect forest pests and valuable wild animals. Unlike human patrols, UAV (Unmanned Aerial Vehicles) may collect data from ground-based devices. The existing UAV path planning method for fixed-point devices is usually acceptable for simple UAV flight scenes. However, it is unsuitable for forest patrol. Meanwhile, when collecting data, the UAV should consider the timeliness of the collected data. The paper proposes two-point path planning and multi-point path planning methods to maximize the amount of fresh information collected from ground-fixed devices in a complicated forest environment. Firstly, we adopt chaotic initialization and co-evolutionary algorithmto solve the two-point path planning issue considering all significant UAV performance and environmental factors. Then, a UAV path planning method based on simulated annealing is proposed for the multi-point path planning issue. In the experiment, the paper uses benchmark functions to choose an appropriate parameter configuration for the proposed approach. On simulated simple and complicated maps, we evaluate the effectiveness of the proposed method compared to the existing pathplanning strategies. The results reveal that the proposed ways can effectively produce a UAV patrol path with higher information freshness in fewer iterations and at a lower computing cost, suggesting the practical value of the proposed approach.

Up-regulation of MCM3 Relates to Neuronal Apoptosis After Traumatic Brain Injury in Adult Rats.

Minichromosome maintenance complex component 3, one of the minichromosome maintenance proteins, functions as a part of pre-replication complex to initiate DNA replication in eukaryotes. Minichromosome maintenance complex component 3 (MCM3) was mainly implied in cell proliferation and tumorigenesis. In addition, MCM3 might play an important role in neuronal apoptosis. However, the functions of MCM3 in central nervous system are still with limited acquaintance. In this study, we performed a traumatic brain injury (TBI) model in adult rats. Western blot and immunohistochemistry staining showed up-regulation of MCM3 in the peritrauma brain cortex. The expression patterns of active caspase-3 and Bax, Bcl-2 were parallel with that of MCM3. Immunofluorescent staining and terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling suggested that MCM3 was involved in neuronal apoptosis. In conclusion, our data indicated that MCM3 might play an important role in neuronal apoptosis following TBI. Further understanding of these insights could serve as the basis for broadening the therapeutic scope against TBI.

Increased Expression of Ubiquitin-Specific Protease 4 Participates in Neuronal Apoptosis After Intracerebral Hemorrhage in Adult Rats.

Ubiquitinating enzymes catalyze protein ubiquitination, a reversible process countered by deubiquitinating enzyme (DUB) action. Ubiquitin-specific protease 4 (USP4) is a member of the ubiquitin-specific protease (USP) family of DUBs that has a role in spliceosome regulation. In the present study, we demonstrated that USP4 may be involved in neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). We obtained a significant up-regulation of USP4 in neurons adjacent to the hematoma following ICH by the results of Western blot, immunohistochemistry, and immunofluorescence. Increasing USP4 level was found to be accompanied by the up-regulation of active caspase-3, γH2AX, Bax, and decreased expression of Bcl-2. In addition, USP4 co-localized well with γH2AX in the nucleus in the ICH model and hemin-induced apoptosis model. Moreover, in vitro study, knocking down USP4 by USP4-specific siRNA in PC12 cells reduced active caspase-3 expression. All these results above suggested that USP4 may be involved in neuronal apoptosis after ICH.

Spatiotemporal Patterns of RING1 Expression after Rat Spinal Cord Injury.

Ring finger protein 1 (RING1) is a RING domain characterized protein belonging to the RING finger family. It is an E3 ubiquitin-protein ligase that mediated monoubiquitination of histone H2A and the core component of PRC1 complex, which is the repressive multiprotein complex of Polycomb group (PcG). Previous studies showed the important tumorigenic role of RING1 via promoting cell proliferation and the crucial function in maintaining transcriptional program stability during development. However, its mechanism for spinal cord injury (SCI) is still unknown. In our research, we established an acute SCI model in adult rats and studied the expression and function profiles of RING1. RING1 protein level detected by western blot peaked at day 3 after trauma and then decreased gradually. Immunohistochemistry showed the increase of RING1 expression displayed in the white matter more obviously than in the gray matter. Furthermore, increased co-expression of RING1 and GFAP confirmed activated astrocytes in injured spinal cord via double immunofluorescence staining. Meanwhile, we also found the co-localization of PCNA, a famous marker of proliferative cells, with RING1 and GFAP, which indicated RING1 might play a role in astrocyte proliferation after SCI. In vitro studies, RING1 protein level in C6 cells increased after LPS challenge and RING1 was required for astrocyte proliferation and activation induced by LPS. In summary, we took a new insight into the function of RING1 in the cellular and molecular mechanism underlying the pathophysiology of SCI.

nNOS Translocates into the Nucleus and Interacts with Sox2 to Protect Neurons Against Early Excitotoxicity via Promotion of Shh Transcription.

Cerebral ischemic stroke is a major public health problem leading to high mortality rates and disability in adults. The NMDA receptor (NMDAR)/neuronal nitric oxide synthase (nNOS)/NO-dependent excitotoxicity has been recognized to play an important role in cerebral ischemic stroke pathogenesis. Accumulating evidence suggests that the biological function of nNOS is associated with its ability to couple proteins and its subcellular localization. Previously, we and others determined that nNOS could translocate into the nucleus in cultured astrocytes, but the underlying mechanisms and biological significance remained unclear. In the present study, we identified a specific interaction between nNOS and Sox2 (SRY (sex determining region Y)-box 2), a member of the Sox family of transcription factors, both in vivo and in vitro. Our studies showed that nNOS is transported into the nucleus and interacted with Sox2 to form a nNOS-Sox2 complex in neurons at the early stage following glutamate stimulation. Mechanistically, via activating the transcription of Shh (Sonic hedgehog), the downstream target of Sox2, this nNOS-Sox2 complex exerted a neuroprotective function against glutamate-induced excitotoxicity. Utilizing the MCAO focal ischemia model on rats, we further verified that the 'nNOS-Sox2-Shh' axis was involved in the ischemic neuronal injury. Taken together, our studies revealed that the 'nNOS-Sox2-Shh' axis functions as a novel feedback compensatory mechanism to protect neurons against the early excitotoxicity and ischemic injury.

Involvement of dysregulated Wip1 in manganese-induced p53 signaling and neuronal apoptosis.

Overexposure to manganese (Mn) has been known to induce neuronal death and neurodegenerative symptoms. However, the precise mechanisms underlying Mn neurotoxicity remain incompletely understood. In the present study, we established a Mn-exposed rat model and found that downregulation of wild type p53-induced phosphatase 1 (Wip1) might contribute to p53 activation and resultant neuronal apoptosis following Mn exposure. Western blot and immunohistochemical analyses revealed that the expression of Wip1 was markedly decreased following Mn exposure. In addition, immunofluorescence assay demonstrated that Mn exposure led to significant reduction in the number of Wip1-positive neurons. Accordingly, the expression of Mdm2 was progressively decreased, which was accompanied with markedly increased expression of p53, as well as the ratio of Bax/Bcl-xl. Furthermore, we showed that Mn exposure decreased the viability and induced apparent apoptosis in NFG-differentiated neuron-like PC12 cells. Importantly, the expression of Wip1 decreased progressively, whereas the level of cellular p53 and the ratio of Bax/Bcl-xl were elevated, which resembled the expression of the proteins in animal model studies. Depletion of p53 significantly ameliorated Mn-mediated cytotoxic effect in PC12 cells. In addition, ectopic expression of Wip1 attenuated Mn-induced p53 signaling as well as apoptosis in PC12 cells. Finally, we observed that depletion of Wip1 augmented Mn-induced apoptosis in PC12 cells. Collectively, these findings suggest that downregulated Wip1 expression plays an important role in Mn-induced neuronal death in the brain striatum via the modulation of p53 signaling.

CAP1 was associated with actin and involved in Schwann cell differentiation and motility after sciatic nerve injury.

Adenylate cyclase-associated protein 1 (CAP1), a member of cyclase-associated proteins that regulating actin dynamics, was shown to regulate actin filaments, localize to dynamic actin structures and mediate such processes as establishment of cell polarity, motility, morphogenesis, receptor-mediated endocytosis and mRNA location. But little is known about the role of CAP1 during peripheral nervous system injury. Here, we found the spatiotemporal protein expression of CAP1 after sciatic nerve crush. After crush, CAP1 had an increased protein expression level, reached a peak at about day 5 and then returned to the normal level at 4 weeks, similar to Oct-6. Besides, in 5-day injured tissue, using double immunofluorescent staining we found CAP1 had a colocalization with S100 and Oct-6. In vitro, during the process of cAMP-induced Schwann cells differentiation, we observed enhanced expression of CAP1 and P0. Specially, CAP1-specific siRNA-tranfected SCs did not show significant actin structure which form cellure surface tension and protrusion shape after cAMP treatment. And we observed the interaction of CAP1 with actin and that CAP1-specific siRNA-transfected SCs had a decreased motility and migration. Together, all these data indicated that the change of CAP1 protein expression was associated with Schwann cells motility and differentiation after the crush of sciatic nerve.

[Effects of H1 blocker and inhaled corticosteroids on asthmatic patients with allergic rhinitis].

OBJECTIVE: To evaluate the therapeutic effects of H(1) blocker in combination with low dose inhaled corticosteroid on allergic asthma. METHODS: A multi-center, double blind, randomized, placebo control study was conducted in 67 patients with mild to moderate allergic asthma. Patients were randomized to receive either Loratadine 10 mg or placebo twice a day on the basis of inhaled beclomethasone dipropionate (400 microg/d for 14 days, then reduced to 200 microg/d) for 5.3 +/- 1.3 months. Symptom scores of asthma, frequencies of episode of rhinitis and common cold and doses of inhaled Salbutamol as rescue drug were recorded. Bronchial hyperresponsiveness (PD(20) FEV(1) in response to Histamine) and serum ICAM-1 and VCAM-1 were measured before and after the treatment. RESULTS: After treatment, there was much better improvement in symptom score (2.4 +/- 0.9 vs 3.1 +/- 0.9, P < 0.01), symptomatic days due to rhinitis (4.0 +/- 1.2 d/week vs 1.9 +/- 0.9 d/week, P < 0.001), episode of common cold symptom (0.8 +/- 0.5 time vs 1.1 +/- 0.4 time, P < 0.001), average doses of inhaled beta(2) agonist as rescue medication (2.6 +/- 0.9 puff/week vs 3.7 +/- 0.8 puff/week, P < 0.001) and bronchial responsiveness (P < 0.05) in Loratadine group as compared with the control group. However, there was no significant change in serum ICAM-1 and VCAM-1 levels after treatment in both groups (P > 0.05). CONCLUSIONS: On the basis of low dose inhaled corticosteroid, orally administered Loratadine significantly improves the therapeutic efficacy of asthma in patients with allergic asthma and rhinitis.