Transcriptome analysis revealed differentially expressed genes in rice functionally associated with brown planthopper defense in near isogenic lines pyramiding BPH14 and BPH15.

Although rice has many pests, brown planthopper (BPH) in particular is known to cause substantial damage. The pyramiding application of BPH-resistance genes BPH14 and BPH15 has proven effective in enhancing rice defense against BPH. However, the molecular mechanisms underlying BPH14/BPH15-conferred resistance remain unexplained. In this investigation, we analyzed the transcriptomes of near isogenic lines (NILs) containing either BPH14 (B14), BPH15 (B15), or BPH14/BPH15 (B1415), as well as their recurrent parent (RP) 'Wushansimiao'. In total, we detected 14,492 differentially expressed genes (DEGs) across 12 mRNA profiles of resistant NILs and RP at different feeding stages. In the transcriptomic analysis, 531 DEGs appeared to be common among the resistant NILs compared to RP before and after BPH feeding. These common DEGs were enriched in defense response, phosphorylation, and salt stress response. In addition, 258 DEGs shared only in resistant NILs were obtained among the different feeding stages, which were enriched in oxidative stress response, karrikin response, and chloroplast organization. Considering the expression patterns and relevant research reports associated with these DEGs, 21 were chosen as BPH resistance candidates. In rice protoplasts, the candidate DEG OsPOX8.1 was confirmed to increase reactive oxygen species (ROS) accumulation by chemiluminescence measurement. Our results provide valuable information to further explore the defense mechanism of insect-resistant gene pyramiding lines and develop robust strategies for insect control.

Genetically Dissecting the Novel Powdery Mildew Resistance Gene in Wheat Breeding Line PBDH1607.

Powdery mildew is one of the most destructive diseases in wheat production. Identifying novel resistance genes and deploying them in new cultivars is the most effective approach to minimize wheat losses caused by powdery mildew. In this study, wheat breeding line PBDH1607 showed high resistance to powdery mildew at both the seedling and adult plant stages. Genetic analysis of the seedling data demonstrated that the resistance was controlled by a single dominant gene, tentatively designated PmPBDH. The ΔSNP index based on bulked segregant RNA sequencing indicated that PmPBDH was associated with an interval of about 30.8 Mb (713.5 to 744.3 Mb) on chromosome arm 4AL. Using newly developed markers, we mapped PmPBDH to a 3.2-cM interval covering 7.1 Mb (719,055,516 to 726,215,121 bp). This interval differed from those of Pm61 (717,963,176 to 719,260,469 bp), MlIW30 (732,769,506 to 732,790,522 bp), and MlNSF10 (729,275,816 to 731,365,462 bp) reported on the same chromosome arm. PmPBDH also differed from Pm61, MlIW30, and MlNSF10 by its response spectrum, origin, or inheritance mode, suggesting that PmPBDH should be a new Pm gene. In the candidate interval, five genes were found to be associated with PmPBDH via time course gene expression analysis, and thus they are candidate genes of PmPBDH. Six closely linked markers, including two kompetitive allele-specific PCR markers, were confirmed to be applicable for tracking PmPBDH in marker-assisted breeding.

Rice SUT and SWEET Transporters.

Sugar transporters play important or even indispensable roles in sugar translocation among adjacent cells in the plant. They are mainly composed of sucrose-proton symporter SUT family members and SWEET family members. In rice, 5 and 21 members are identified in these transporter families, and some of their physiological functions have been characterized on the basis of gene knockout or knockdown strategies. Existing evidence shows that most SUT members play indispensable roles, while many SWEET members are seemingly not so critical in plant growth and development regarding whether their mutants display an aberrant phenotype or not. Generally, the expressions of SUT and SWEET genes focus on the leaf, stem, and grain that represent the source, transport, and sink organs where carbohydrate production, allocation, and storage take place. Rice SUT and SWEET also play roles in both biotic and abiotic stress responses in addition to plant growth and development. At present, these sugar transporter gene regulation mechanisms are largely unclear. In this review, we compare the expressional profiles of these sugar transporter genes on the basis of chip data and elaborate their research advances. Some suggestions concerning future investigation are also proposed.

Bulked Segregant RNA-Seq Reveals Distinct Expression Profiling in Chinese Wheat Cultivar Jimai 23 Responding to Powdery Mildew.

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive fungal diseases threatening global wheat production. Host resistance is well known to be the most efficient method to control this disease. However, the molecular mechanism of wheat powdery mildew resistance (Pm) is still unclear. To analyze the molecular mechanism of Pm, we used the resistant wheat cultivar Jimai 23 to investigate its potential resistance components and profiled its expression in response to powdery mildew infection using bulked segregant RNA-Seq (BSR-Seq). We showed that the Pm of Jimai 23 was provided by a single dominant gene, tentatively designated PmJM23, and assigned it to the documented Pm2 region of chromosome 5DS. 3,816 consistently different SNPs were called between resistant and susceptible parents and the bulked pools derived from the combinations between the resistant parent Jimai23 and the susceptible parent Tainong18. 58 of the SNPs were assigned to the candidate region of PmJM23. Subsequently, 3,803 differentially expressed genes (DEGs) between parents and bulks were analyzed by GO, COG and KEGG pathway enrichment. The temporal expression patterns of associated genes following Bgt inoculation were further determined by RT-qPCR. Expression of six disease-related genes was investigated during Bgt infection and might serve as valuable genetic resources for the improvement of durable resistance to Bgt.

Discovery of DC_H31 as potential mutant IDH1 inhibitor through NADPH-based high throughput screening.

IDH1 mutations are early events in the development of IDH-mutant gliomas and leukemias and are associated with various regulation of molecular process. Mutations of active site in IDH1 could lead to high levels of 2-HG and the suppression of cellular differentiation, while these changes can be reversed by molecule inhibitors target mutant IDH1. Here, through in-house developed enzymatic assay-based high throughput screening platform, we discovered DC_H31 as a novel IDH1-R132H/C inhibitor, with the IC50 value of 0.41 µmol/L and 2.7 µmol/L respectively. In addition, saturable SPR binding assay indicated that DC_H31 bound to IDH1-R132H/C due to specific interaction. Further computational docking studies and structure-activity relationship (SAR) suggest that DC_H31 could occupy the allosteric pocket between the two monomers of IDH1-R132H homodimer, which accounts for its inhibitory ability. And it is possible to conclude that DC_H31 acts via an allosteric mechanism of inhibition. At the cellular level, DC_H31 could inhibit cell proliferation, promote cell differentiation and reduce the production of 2-HG with a dose-dependent manner in HT1080 cells. Taken together, DC_H31 is a potent selective inhibitor of IDH1-R132H/C both in vitro and in vivo, which can promote the development of more potent pan-inhibitors against IDH1-R132H/C through further structural decoration and provide a new insight for the pharmacological treatment of gliomas.

HIV-1 Tat enhances purinergic P2Y4 receptor signaling to mediate inflammatory cytokine production and neuronal damage via PI3K/Akt and ERK MAPK pathways.

BACKGROUND: HIV-associated neurocognitive disorders (HANDs) afflict more than half of HIV-1-positive individuals. The transactivator of transcription (Tat) produced by HIV virus elicits inflammatory process and is a major neurotoxic mediator that induce neuron damage during HAND pathogenesis. Activated astrocytes are important cells involved in neuroinflammation and neuronal damage. Purinergic receptors expressed in astrocytes participate in a positive feedback loop in virus-induced neurotoxicity. Here, we investigated that whether P2Y4R, a P2Y receptor subtype, that expressed in astrocyte participates in Tat-induced neuronal death in vitro and in vivo. METHODS: Soluble Tat protein was performed to determine the expression of P2Y4R and proinflammatory cytokines in astrocytes using siRNA technique via real-time PCR, Western blot, and immunofluorescence assays. Cytometric bead array was used to measure proinflammatory cytokine release. The TUNEL staining and MTT cell viability assay were analyzed for HT22 cell apoptosis and viability, and the ApopTag® peroxidase in situ apoptosis detection kit and cresyl violet staining for apoptosis and death of hippocampal neuron in vivo. RESULTS: We found that Tat challenge increased the expression of P2Y4R in astrocytes. P2Y4R signaling in astrocytes was involved in Tat-induced inflammatory cytokine production via PI3K/Akt- and ERK1/2-dependent pathways. Knockdown of P2Y4R expression significantly reduced inflammatory cytokine production and relieved Tat-mediated neuronal apoptosis in vitro. Furthermore, in vivo challenged with Tat, P2Y4R knockdown mice showed decreased inflammation and neuronal damage, especially in hippocampal CA1 region. CONCLUSIONS: Our data provide novel insights into astrocyte-mediated neuron damage during HIV-1 infection and suggest a potential therapeutic target for HANDs.

MiR-409-3p and MiR-1896 co-operatively participate in IL-17-induced inflammatory cytokine production in astrocytes and pathogenesis of EAE mice via targeting SOCS3/STAT3 signaling.

Th17 cells and interleukin-17 (IL-17) have been found to play an important role in the pathology of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Response to IL-17, reactive astrocytes accompany with immune cells infiltration and axonal damage in MS/EAE. However, the role and the regulatory mechanism of IL-17-activated astrocytes in inflammation and in the EAE process still remain largely unknown. Here, we elucidated that miR-409-3p and miR-1896, as co-upregulated microRNAs in activated astrocytes and in EAE mice, targeted suppressor of cytokine signaling proteins 3 (SOCS3). Overexpression of miR-409-3p or miR-1896 significantly reduced SOCS3 expression and increased phosphorylation of STAT3 as well as induced the inflammatory cytokines production (IL-1ß, IL-6, IP-10, MCP-1, and KC), CD4+ T cells migration and demyelination, in turn aggravating EAE development. Importantly, the effects of co-overexpression of miR-409-3p and miR-1896 in vitro or in vivo are strongly co-operative. In contrast, simultaneously silenced miR-409-3p and miR-1896 co-operatively ameliorates inflammation and demyelination in the central nervous system of EAE mice. Collectively, our findings highlight that miR-409-3p and miR-1896 co-ordinately promote the production of inflammatory cytokines in reactive astrocytes through the SOCS3/STAT3 pathway and enhance reactive astrocyte-directed chemotaxis of CD4+ T cells, leading to aggravate pathogenesis in EAE mice. Co-inhibition of miR-409-3p and miR-1896 may be a therapeutic target for treating MS and neuroinflammation.

HIV-1 Nef-induced lncRNA AK006025 regulates CXCL9/10/11 cluster gene expression in astrocytes through interaction with CBP/P300.

BACKGROUND: HIV-associated neurocognitive disorder (HAND) is a neurodegenerative disease associated with persistent neuroinflammation and subsequent neuron damage. Pro-inflammatory factors and neurotoxins from activated astrocytes by HIV-1 itself and its encoded proteins, including the negative factor (Nef), are involved in the pathogenesis of HAND. This study was designed to find potential lncRNAs that regulate astrocyte functions and inflammation process. METHODS: We performed microarray analysis of lncRNAs from primary mouse astrocytes treated with Nef protein. Top ten lncRNAs were validated through real-time PCR analysis. Gene ontology (GO) and KEGG pathway analysis were applied to explore the potential functions of lncRNAs. RIP and ChIP assays were performed to demonstrate the mechanism of lncRNA regulating gene expression. RESULTS: There were 638 co-upregulated lncRNAs and 372 co-downregulated lncRNAs in primary astrocytes treated with Nef protein for both 6 h and 12 h. GO and KEGG pathway analysis showed that the biological functions of top differential-expressed mRNAs were associated with inflammatory cytokines and chemokine. Knockdown of lncRNA AK006025, not AK138360, inhibited significantly CXCL9, CXCL10 (IP-10), and CXCL11 expression in astrocytes treated with Nef protein. Mechanism study showed that AK006025 associated with CBP/P300 was enriched in the promoter of CXCL9, CXCL10, and CXCL11 genes. CONCLUSIONS: Our findings uncovered the expression profiles of lncRNAs and mRNAs in vitro, which might help to understand the pathways that regulate astrocyte activation during the process of HAND.

Analysis of Long Noncoding RNA and mRNA Expression Profiles in IL-9-Activated Astrocytes and EAE Mice.

BACKGROUND/AIMS: Multiple sclerosis (MS) is an autoimmune disease in the central nervous system associated with demyelination and axonal injury. Astrocyte activation is involved in the pathogenesis of MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. This study was designed to find potential lncRNAs in EAE mice and activated astrocytes. METHODS: we performed microarray analysis of lncRNAs from the brain tissues of EAE mice and primary mouse astrocytes treated with IL-9(50 ng/ml). 12 lncRNAs were validated through real-time PCR. Gene ontology and KEGG pathway analysis were applied to explore the potential functions of lncRNAs. RESULTS: Differentially expressed 3300 lncRNAs and 3250 mRNAs were in the brain tissues of EAE mice, and 3748 lncRNAs and 3332 mRNAs were in activated astrocytes. Notably, there were 2 co-up-regulated lncRNAs and 3 co-down-regulated lncRNAs both in the brain tissues of EAE mice and in activated astrocytes, including Gm14005, Gm12478, mouselincRNA1117, AK080435, and mouselincRNA0681, which regulate the ER calcium flux kinetics, zinc finger protein and cell apoptosis. Similarly, there were 7 mRNAs co-up-regulated and 2 mRNAs co-down-regulated both in vivo and in vitro. Gene ontology and KEGG pathway analysis showed that the biological functions of differentially expressed mRNAs were associated with metabolism, development and inflammation. The results of realtime PCR validation were consistent with the data from the microarrays. CONCLUSIONS: Our data uncovered the expression profiles of lncRNAs and mRNAs in vivo and in vitro, which may help delineate the mechanisms of astrocyte activation during MS/EAE process.

An Integrative Survival Analysis with Identification of Prognostic Factors in the Patients with Coexisting Glioma and Intracranial Aneurysm.

OBJECTIVE: This study aimed to perform an integrative clinical and survival analysis for patients with coexisting glioma and intracranial aneurysm and to assess the influence of prognostic factors on overall survival (OS). METHODS: A systematic literature search on PubMed and Web of Science was performed for literature in English published from 1951 to August 2017. Detailed information including clinical characteristics, treatments, critical events, and time to events for survival analysis was extracted from the included articles. Clinical characteristics of included patients were reviewed, and survival analysis was performed to identify prognostic factors of OS. RESULTS: A total of 56 cases from 38 articles published in English-language journals were included in this study, and relative details were selected for integrative analysis. Univariate analysis demonstrated that age (<47/≥47 years), ruptured aneurysm (yes/no), glioma type (glioblastoma multiforme [GBM]/no GBM), World Health Organization (WHO) grade (low/high), and radiotherapy (yes/no) had a statistically significant correlation with OS (log-rank P = 0.004, P = 0.037, P = 0.004, P < 0.001, and P < 0.001, respectively). Further, multivariate analysis revealed that WHO grade (hazard ratio [HR], 22.383; 95% confidence interval [CI], 1.795-279.151; P = 0.016) and receiving radiotherapy (HR, 0.054; 95% CI, 0.009-0.333; P = 0.002) were the independent prognostic factors for OS. CONCLUSIONS: This integrative survival analysis revealed that WHO grade and receiving radiotherapy were independent prognostic factors for OS, and patients with low-grade glioma and receiving radiotherapy had longer survival than counter groups. Nevertheless, similar clinical studies which should be larger samples, multicenter, and collaborative are needed further.

Synthesis of pentacyclic iminosugars with constrained butterfly-like conformation and their HIV-RT inhibitory activity.

Novel pentacyclic iminosugars 1 and 2 with the constrained butterfly-like conformation were first synthesized by the key intramolecular click reaction from the tricyclic iminosugars fused benzo[e][1,3]thiazin-4-one 3 and 4. The pentacyclic iminosugar was constructed by fusing both benzo[e][1,3]thiazin-4-one and triazolo[5,1-c][1,4]oxazepine scaffolds. Their structures were determined by their 1H, 13C NMR, and HRMS (ESI) spectra and X-ray. The pentacyclic iminosugars 1(a-c), 2(a-b) and their corresponding protected precursors 13(a-c) and 14(a-b) were examined for their HIV reverse transcriptase (RT) inhibitory activities. The result showed that all compounds could effectively inhibit RT activity. Among them, compound 13c was the best one with the IC50 value of RT inhibitory activity of 0.69 µM. Structure-activity relationship analysis suggested that the improvement of the hydrophilicity of the pentacycles was of benefit to their anti-HIV RT activity.

Expression of microRNA-30c via lentivirus vector inhibits the proliferation and enhances the sensitivity of highly aggressive ccRCC Caki-1 cells to anticancer agents.

The clear cell renal cell carcinoma (ccRCC) is one of the most fatal urologic tumors, and the prognosis remains very poor for advanced or metastatic ccRCC. This study reveals the roles of microRNA (miR)-30c in regulating a highly aggressive ccRCC cell line proliferation by targeting MTA-1, which is a key mediator for human cancer metastasis. Results from quantitative real-time polymerase chain reaction showed that the expression of MTA-1, the target of miR-30c, was significantly higher in metastatic ccRCC specimens than in nonmetastatic ccRCC or nontumor specimens. Accordingly, endogenous miR-30c is at a much lower level in highly aggressive ccRCC Caki-1 cells than nontumor or ccRCC cell lines. Expression of miR-30c via lentivirus vector inhibits the proliferation, anchorage-independent growth, in vitro invasion or migration, or in vivo growth of Caki-1 cells by repressing MTA-1 protein expression. miR-30c also enhances the sensitivity of Caki-1 cells to anticancer agents, including sorafenib and paclitaxel. These data reveal the potential application of miR-30c and that its targeting gene, MTA-1, would be a potential target in metastatic ccRCC treatment.

Crystal structure of the mismatch-specific thymine glycosylase domain of human methyl-CpG-binding protein MBD4.

Methyl-CpG (mCpG) binding domain protein 4 (MBD4) is a member of mammalian DNA glycosylase superfamily. It contains an amino-proximal methyl-CpG binding domain (MBD) and a C-terminal mismatch-specific glycosylase domain, which is an important molecule believed to be involved in maintaining of genome stability. Herein, we determined the crystal structure of C-terminal glycosylase domain of human MBD4. And the structural alignments of other helix-hairpin-helix (HhH) DNA glycosylases show that the human MBD4 glycosylase domain has the similar active site and the catalytic mechanisms as others. But the different residues in the N-terminal of domain result in the change of charge distribution on the surface of the protein, which suggest the different roles that may relate some diseases.

[Determination of bismuth pectin by flow-injection hydride-generation atomic absorption spectrometry].

A new method was developed by flow-injection hydride-generation atomic absorption spectrometry (FI-HG-AAS) for direct determination of bismuth in bismuth pectin, which is mostly composed of pectin and bismuth and serves as one of pharmaceuticals for protecting stomach mucous membrane. The influences of some experimental parameters of this method, such as the acidity of sample solution, the reductant of KBH4 and NaOH that served as the stabilizer of KBH4, were studied and these parameters were optimized. Bismuth was detected directly with the linear range of 0.00-44.00 ng.mL-1. The equation of working curve was A = 0.012 + 0.017c (c:ng.mL-1), r = 0.9995, the detection limit was 0.095 ng.mL-1 (3 sigma), and the recovery was 97.3%-103.3%. The method proposed has been used in the analysis of practical sample for bismuth with 96.6%-100.1% founded and 2.1% of RSD. Experiments showed that this method was simple, rapid, accurate and suitable for the assay of bismuth in bismuth pectin.

[Direct resolution of clenbuterol enantiomers with amide derivative as chiral stationary phase].

A simple and rapid high performance liquid chromatographic method has been developed for the direct resolution of clenbuterol enantiomers. The method involved the use of an amide type chiral stationary phase(CSP) made of (R)-1-naphthylglycine and 3,5-dinitrobenzoic acid known as the Chirex 3005 column. The effects of different contents of n-hexane, 1,2-dichloroethane and methanol in mobile phase on the chiral separation are discussed. The effects of column temperature and flow rate of mobile phase were also studied. The separation factor obtained was 1.32 and the resolution factor was 1.81 when using the optimized mobile phase composed of n-hexane-1,2-dichloroethane-methanol (54:38:8, volume ratio) at 17 degrees C and 1.0 mL/min. The mechanism of separation is also discussed.

[The studies on spectrum properties of LB film of Schiff base].

Two Schiff bases with different alkyl chains length, N-dodecyl (2-hydroxy-5-nitro-benzylideneaminato) (TA12) and N-hexadecyl (2-hydroxy-5-nitro-benzylideneaminato) (TA16), were synthesized. Their monolayers behavior and LB films characteristics were investigated and compared by pi-A isotherm, UV-Vis absorption spectra, fluorescence spectra and Micro-IR spectra. The results indicate that TA12 and TA16 have good formation properties. J-aggregate was found in LB films of Schiff bases that have photoluminesence properties. The Schiff base with longer alkyl chains is more orderly in LB films. But the whole films are not so perfect in microscopic state.