Development of an agroinfiltration-based transient hairpin RNA expression system in pak choi leaves (Brassica rapa ssp. chinensis) for RNA interference against Liriomyza sativae.

The vegetable leafminer (Liriomyza sativae) is a devastating invasive pest of many vegetable crops and horticultural plants worldwide, causing serious economic loss. Conventional control strategy against this pest mainly relies on the synthetic chemical pesticides, but widespread use of insecticides easily causes insecticide resistance development and is harmful to beneficial organisms and environment. In this context, a more environmentally friendly pest management strategy based on RNA interference (RNAi) has emerged as a powerful tool to control of insect pests. Here we report a successful oral RNAi in L. sativae after feeding on pak choi (Brassica rapa ssp. chinensis) that transiently express hairpin RNAs targeting vital genes in this pest. First, potentially lethal genes are identified by searching an L. sativae transcriptome for orthologs of the widely used V-ATPase A and actin genes, then expression levels are assessed during different life stages and in different adult tissues. Interestingly, the highest expression levels for V-ATPase A are observed in the adult heads (males and females) and for actin in the abdomens of adult females. We also assessed expression patterns of the target hairpin RNAs in pak choi leaves and found that they reach peak levels 72 h post agroinfiltration. RNAi-mediated knockdown of each target was then assessed by letting adult L. sativae feed on agroinfiltrated pak choi leaves. Relative transcript levels of each target gene exhibit significant reductions over the feeding time, and adversely affect survival of adult L. sativae at 24 h post infestation in genetically unmodified pak choi plants. These results demonstrate that the agroinfiltration-mediated RNAi system has potential for advancing innovative environmentally safe pest management strategies for the control of leaf-mining species.

[Remediation of Three Oxidants on Polycyclic Aromatic Hydrocarbons in Coking Contaminated Soil and Its Response to Indigenous Microorganisms].

To explore the influences of chemical oxidation on the physiological and ecological functions of indigenous microorganisms during contaminated soil remediation, three oxidants, including KMnO4, Na2S2O8, and O3, were selected to investigate their remediation effects on PAHs and the responses to indigenous microorganisms under different liquid-solid ratios, in this study. The results showed that:when the ΣPAHs concentration was 679.1 mg·kg-1 and the dosage of KMnO4 and Na2S2O8 was 1%, the removal efficiency of ΣPAHs reached up to 96.9% and 95.7% under the liquid-solid ratio of 6:1; for the O3 treatment, the removal efficiency of ΣPAHs was the highest(82.3%) at the O3 dosage and the liquid-solid ratio of 72 mg·min-1 and 8:1, respectively. The removal efficiency of low ring(3-4 rings) PAHs was higher than that of high ring(5-6 rings) PAHs under different liquid-solid ratios. The highest removal efficiencies were observed for phenanthrene and acenaphthene, whereas for benzo[a]pyrene, only the KMnO4treatment provided an effective performance, showing the highest removal efficiency of 97.4%. The microbial quantity analysis indicated that the quantity of soil microorganisms in the soil dropped sharply after being treated with KMnO4, decreasing from 108 copies·g-1 to 105 copies·g-1, whereas it changed only slightly after being treated with Na2S2O8 and O3. The community structure analysis showed that Proteobacteria were predominant in the contaminated soil, with the relative abundance of 99.5%. The addition of KMnO4 and Na2S2O8 significantly increased the microbial diversity; in particular, the relative abundance of a variety of microorganisms(such as Ralstonia and Acinetobacter) that can degrade PAHs was remarkably increased. The analysis of microbial metabolic function pathways revealed that chemical oxidation could simultaneously increase the relative abundance of PAHs-degrading bacteria and improve the ability of organic metabolism. Overall, the KMnO4 treatment greatly altered the quantity of microorganisms and the structure of the microbial community and the relative abundance of PAHs-degrading microorganisms at the liquid-solid ratio of 6:1.

Functional differentiation of two general odorant-binding proteins to sex pheromones in Spodoptera frugiperda.

A precise chemosensory system can help insects complete various important behavioral responses by accurately identifying different external odorants. Therefore, deeply understanding the mechanism of insect recognition of important odorants will help us develop efficient and environmentally-friendly behavioral inhibitors. Spodoptera frugiperda is a polyphagous pest that feeds on >350 different host plants worldwide and also harms maize production in China. However, the molecular mechanism of the first step for males to use odorant-binding proteins (OBPs) to recognize sex pheromones remains unclear. Here, we obtained 50 OBPs from the S. frugiperda genome, and the expression level of SfruGOBP1 in females was significantly higher than that in males, whereas SfruGOBP2 displayed male-biased expression. Fluorescence competitive binding assays showed that only SfruGOBP2 showed binding affinities for the four sex pheromones of female S. frugiperda. Subsequently, we identified some key amino acid residues that can participate in the interaction between SfruGOBP2 and sex pheromones using molecular docking and site-directed mutagenesis methods. These findings will help us explore the interaction mechanism between GOBPs and sex pheromones in moths, and provide important target genes for developing new mating inhibitors of S. frugiperda in the future.

Transcriptome profiling Revealed the potential mechanisms of Shen Lin Bai Zhu San n-butanol extract on DSS induced Colitis in Mice and LC-MS analysis.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic and recurrent inflammatory disorder in gastrointestinal tract. Shen Ling Bai Zhu San (SLBZS), which has a long history of use in Traditional Chinese Medicine (TCM), has been widely used to treat gastrointestinal diseases. The isolated fractions of TCM have also been proved to possess an important potential for treating diseases, which are due to their effective components. PURPOSE: In this study, we examined the possibility that SLBZS and its isolated active fractions may prevent DSS-induced colitis, and investigated the potential mechanisms by regulating genetic profile of colon. METHODS: Colitis mice were induced by 2.5% DSS for 7 days, and then SLBZS and different SLBZS extracts were administrated to protect the mice for 7 days. Body weight, diarrhea, bleeding in stool, colon length, spleen weight, cytokines of serum and colon and pathology of colon were assessed. The level of Ginsenoside Rg1, Re and Rb1 in different SLBZS extracts and qualitative analysis of n-butanol extract of SLBZS (S-Nb) was performed by HPLC and LC-MS, respectively. And the effects of S-Nb on the transcriptome in colitis were investigated. RESULTS: Our results showed that SLBZS and S-Nb significantly regained body weight, reduced DAI, splenomegaly and the length of colon and attenuated histological damage of the colon. Meanwhile, SLBZS and S-Nb markedly reduced the levels of TNF-α, IL-1ß and IL-6 and increased the level of IL-10 in serum and colon. These effects may be associated with the high levels of Ginsenoside Rg1, Re and Rb1 and rich variety of compounds in S-Nb including 6 ginsenosides, glycyrrhizin, L-tryptophan, and so on. Transcriptome analysis revealed that S-Nb selectively regulated 103 differentially expressed genes (DEGs), 36 of which were changed in DSS-induced mice. And the genes of Per2, Per3, Npy and Serpina3m were closely related to colitis and also restored by S-Nb with different extent. Remarkably, these DEGs modulated the biological functions of colitis mice, including extracellular region, response to external stimulus, MAPK signaling pathway and arginine and proline metabolism. CONCLUSIONS: These data indicated that SLBZS and S-Nb blunted DSS-induced colitis by modulating differentially expression gene profile and biological functions based on their ginsenosides and rich compounds.

Polysaccharides derived from Shenling Baizhu San improve colitis via modulating tryptophan metabolism in mice.

Shenling Baizhu San has beneficial effects on the metabolism of the gut microbiota, however, the mechanisms underlying microbiota metabolites mediated anti-inflammation signaling are not well understood. Previously, we have demonstrated that supplementation with Shenling Baizhu San alleviated antibiotic-associated diarrhea (AAD). The current study intends to investigate the dynamic modulation of Shenling Baizhu San polysaccharides (SP) on colitis from the gut microbiota metabolites perspective. Administration of SP effectively relieved colitis induced by DSS in mice, including alleviating body weight loss, the downregulation of colon proinflammatory mediators, and the promotion of intestinal injury repair. Whereas, the efficacy was eliminated by antibiotics, which demonstrated that the efficacy of SP was dependent on the gut microbiota. Fecal microbiota transplantation (FMT) showed that the efficacy of SP can be transferred to gut microbiota. Serum metabolomics analysis showed that supplementation with SP significantly promoted tryptophan metabolism, which was consistent with the changed structure of the gut microbiota, including Bacteroides, Bifidobacterium and Ruminococcus regulated by SP. Especially, the tryptophan metabolites-kynurenine (KYN) activated the expression of amplifying aryl-hydrocarbon receptor (AhR) and Cyp1A1 to promote IL-10 expression in colon. These data suggested that SP positively affected colitis in mice by regulating tryptophan metabolic function of their gut microbiota.

Portulaca oleracea L. extracts alleviate 2,4-dinitrochlorobenzene-induced atopic dermatitis in mice.

Atopic dermatitis (AD) is a common chronic allergic skin disease characterized clinically by severe skin lesions and pruritus. Portulaca oleracea L. (PO) is a resourceful plant with homologous properties in medicine and food. In this study, we used two different methods to extract PO, and compared the therapeutic effects of PO aqueous extract (POAE) and PO ultrasound-assisted ethanol extract (POEE) on 2,4-dinitrochlorobenzene (DNCB)-induced AD mice. The results showed that in POAE and POEE, the extraction rates of polysaccharides were 16.95% and 9.85%, while the extraction rates of total flavonoids were 3.15% and 3.25%, respectively. Compared with AD mice, clinical symptoms such as erythema, edema, dryness and ulceration in the back and left ear were alleviated, and pruritus behavior was reduced after POAE and POEE treatments. The thickness of the skin epidermis was thinned, the density of skin nerve fibers labeled with protein gene product 9.5 (PGP9.5) was decreased, and mast cell infiltration was reduced. There was a decrease in blood lymphocytes, eosinophils and basophils, a significant decrease in spleen index and a noticeable decrease in serum immunoglobulin E (Ig E). POEE significantly reduced the concentration of the skin pruritic factor interleukin (Il)-31. POAE and POEE reduced the concentration of skin histamine (His), down-regulated mRNA expression levels of interferon-γ (Ifnγ), tumor necrosis factor-α (Tnf-α), thymic stromal lymphopoietin (Tslp) and Il-4, with an increase of Filaggrin (Flg) and Loricrin (Lor) in skin lesions. These results suggested that POAE and POEE may inhibit atopic response and alleviate the clinical symptoms of AD by inhibiting the expression of immune cells, inflammatory mediators and cytokines. PO may be a potential effective drug for AD-like diseases.

STAT3 ameliorates cognitive deficits by positively regulating the expression of NMDARs in a mouse model of FTDP-17.

In tauopathies, memory impairment positively strongly correlates with the amount of abnormal tau aggregates; however, how tau accumulation induces synapse impairment is unclear. Recently, we found that human tau accumulation activated Signal Transduction and Activator of Transcription-1 (STAT1) to inhibit the transcription of synaptic N-methyl-D-aspartate receptors (NMDARs). Here, overexpressing human P301L mutant tau (P301L-hTau) increased the phosphorylated level of Signal Transduction and Activator of Transcription-3 (STAT3) at Tyr705 by JAK2, which would promote STAT3 translocate into the nucleus and activate STAT3. However, STAT3 was found mainly located in the cytoplasm. Further study found that P301L-htau acetylated STAT1 to bind with STAT3 in the cytoplasm, and thus inhibited the nuclear translocation and inactivation of STAT3. Knockdown of STAT3 in STAT3flox/flox mice mimicked P301L-hTau-induced suppression of NMDARs expression, synaptic and memory impairments. Overexpressing STAT3 rescued P301L-hTau-induced synaptic and cognitive deficits by increasing NMDARs expression. Further study proved that STAT3 positively regulated NMDARs transcription through direct binding to the specific GAS element of NMDARs promoters. These findings indicate that accumulated P301L-hTau inactivating STAT3 to suppress NMDARs expression, revealed a novel mechanism for tau-associated synapse and cognition deficits, and STAT3 will hopefully serve as a potential pharmacological target for tauopathies treatment.

Application of N-methyl-D-aspartate receptor nanopore in screening ligand molecules.

N-methyl-D-aspartate receptors (NMDARs) are crucial for excitatory synaptic transmission in the central nervous system. To study NMDARs more accurately and conveniently, we developed a stable NMDAR nanopore in a planar lipid bilayer. Pharmacological properties were validated using the allosteric modulator Ro 25-6981 and antagonist D-2-amino-5-phosphonopentanoic acid (D-APV). The cyanotoxin ß-N-methylamino-L-alanine (BMAA) found in fresh water systems is suspected to be associated with the development of neurodegenerative diseases. Therefore, BMAA and its two isomers L-2, 4-Diaminobutyric acid dihydrochloride (DAB) and N-(2-aminoethyl) glycine (AEG) and an endogenous excitotoxin, quinolinic acid (QA), were studied using the NMDAR nanopores to assess their effects on NMDAR modulation. We demonstrated that the NMDAR nanopore could reliably detect its ligand molecules at the single-channel level. The study also demonstrated the practicability of NMDAR nanopores, and results were validated using two-electrode voltage-clamp (TEVC) recording. Compared with TEVC recording, the NMDAR nanopores conducted ion channel gating at the single-channel level without being affected by other proteins on the cell membrane. The highly sensitive and accurate NMDAR nanopore technique thus has a unique advantage in screening NMDAR ligand molecules that could be associated with neurodegenerative disease.

A combination of a microemulsion and a phospholipid complex for topical delivery of oxymatrine.

Oxymatrine (OMT), a water-soluble drug, has a very low oral bioavailability because of its low membrane permeability and its biotransformation in the gastrointestinal tract. Formulated as an oxymatrine-phospholipid complex (OMT-PLC) can improve the lipid solubility and effectiveness of OMT. The purpose of this study was to explore the utility of the combination of a microemulsion and an OMT-PLC as a topical delivery vehicle for enhancing the absorption and efficacy of OMT. The solubility of OMT-PLC was determined and phase diagrams of microemulsions were constructed. Various microemulsion formulations were developed and characterized by their physicochemical properties, and their in vitro and in vivo permeability through skin. An optimal microemulsion (ME4), which presented as spherical droplets and consisted of 10.0% OMT-PLC, 8.0% isopropyl myristate, 30.0% Cremophor RH40/polyethylene glycol 400 (1:1) and 52.0% water, was selected. It possessed an average droplet size of 32.4 nm, a low viscosity of 113.7 mPa · s, and a high cloud point of 88°C. Compared to the control solution, ME4 provided better skin permeability in vitro and a higher retention ratio of OMT in skin in vivo. Moreover, ME4 significantly enhanced the antiproliferative activity of OMT on scar fibroblasts. These results indicate that the combination of a microemulsion and a phospholipid complex represents an effective vehicle for topical delivery of OMT.

Survival change of ventral mesencephalon-derived progenitor cells after grafting into unilateral intact adult rat striatum.

Neural transplantation is one of the most promising treatments for neurodegenerative disorders. Survival rates of embryonic dopamine (DA) neurons following transplantation are low, between 2% and 20% in a number of animal models. To further establish survival changes of the transplanted gestational day 13.5 ventral mesencephalic (VM) cells into left intact adult rat striata so that design strategies of increasing survival of DA neurons, the tyrosine hydroxylase (TH) expression of VM-derived progenitor cells has been examined using immunohistochemistry and Western blot analysis. TH immunostaining revealed that the grafted VM cells developed to mature TH-positive neurons strongly at 3 weeks, peaked at 4 weeks, thereafter, gradually dropped following the degenerative expression of the grafted cells at both 5 and 6 weeks after transplantation. Western blot analysis also showed that the TH proteins were maximally expressed at 4 weeks post-grafting. Our finding suggested that the peak of surviving VM-derived TH positive cells occurred approximately 4 weeks after transplantation.

Ethanol modulates GABA(B) receptor expression in cortex and hippocampus of the adult rat brain.

Using in situ hybridization, RNase protection assay and Western blot, we studied the effects of ethanol on the expression levels of GABA(B) receptor mRNA and protein in the cortex and hippocampus from adult rat brain. The results showed that ethanol significantly increased GABA(B1) and GABA(B2) receptor protein expression in the cortex, whereas only GABA(B2) was increased in the hippocampus. GABA(B) receptor agonist baclofen could partially reverse the effect of ethanol. Further studies of the mRNA levels defined that GABA(B1) mRNA levels were significantly increased in the hippocampus, with no significant changes of GABA(B2) mRNA levels. Moreover, GABA(B1) and GABA(B2) receptor mRNA levels were increased on 3-week ethanol treatment. Finally, GABA(B) agonist baclofen and antagonist phaclofen showed significant decreasing effects on GABA(B1) receptor mRNA levels in the cortex, but not in the hippocampus. These results were further confirmed by in situ hybridization. Thus, the present results showed the effects of ethanol on GABA(B) receptors in the cortex and hippocampus, implying the possible role of GABA(B) receptor in ethanol effects. The effects of GABA(B) receptor agonist and antagonist suggested that the possible mechanisms underlying that GABA(B) receptor modulated the behavioral effect induced by ethanol.

Chronic nicotine and smoke treatment modulate dopaminergic activities in ventral tegmental area and nucleus accumbens and the gamma-aminobutyric acid type B receptor expression of the rat prefrontal cortex.

Dopaminergic afferents from the mesencephalic areas, such as ventral tegmental area (VTA), synapse with the gamma-aminobutyric acid (GABA)-ergic interneurons in the prefrontal cortex (PFC). Pharmacological and electrophysiological data show that the reinforcement, the dependence-producing properties, as well as the psychopharmacologic effects of nicotine depend to a great extent on activation of nicotinic receptors within the mesolimbocortical dopaminergic projection. To explore further the relationship between the mesencephalic dopaminergic neurons and PFC GABAergic neurons, we investigated the effects of nicotine and passive exposure to cigarette smoke on the regulation of tyrosine hydroxylase (TH) in VTA and substantia nigra (SNC) and dopamine (DA) D1 receptor levels in nucleus accumbens (NAc) and caudate-putamen (CPu). Also, the simultaneous changes in GABAB receptors mRNAs in the PFC were studied. The results showed that chronic nicotine and smoking treatment differentially changed the levels of TH protein in VTA and SNC and DA D1 receptor levels in Nac and CPu. GABAB1 and GABAB2 receptor mRNA levels also showed different change patterns. Ten and thirty minutes of smoke exposure increased GABAB1 receptor mRNA to a greater extent than that of GABAB2, whereas GABAB2 was greatly enhanced after 1 hr of smoke exposure. The TH levels in VTA were closely related to DA D1 receptor levels in NAc and with GABAB receptor mRNA changes in PFC. These results suggest that the mesolimbic pathway and GABAB receptor mRNA in PFC are modulated by nicotine and cigarette smoke, implying an important role in nicotine's psychopharmacological effects.

Melatonin protects SH-SY5Y neuroblastoma cells from calyculin A-induced neurofilament impairment and neurotoxicity.

Hyperphosphorylation of cytoskeletal proteins seen in Alzheimer's disease is most probably the result of an imbalanced regulation in protein kinases and protein phosphatases (PP) in the affected neurons. Previous studies have revealed that PP-2A and PP-1 play important roles in the pathogenesis. Employing human neuroblastoma cells, we found that 10 nM calyculin A (CA), a selective inhibitor of PP-2A and PP-1, significantly increased phosphorylation and accumulation of neurofilament (NF) in the cells. Levels of NF-M (middle chain) and NF-L (light chain) mRNA decreased after CA treatment. Additionally, CA led to a decreased cell viability determined by MTT and crystal violet assay. Melatonin efficiently protects the cell from CA-induced alterations in NF hyperphosphorylation and accumulation, suppressed NF gene expression as well as decreased cell viability. It is concluded that inhibition of PP-2A/PP-1 by CA induces abnormalities in NF metabolism and cell survival, and melatonin efficiently arrests the lesions.

Differential distribution of GABA(B1) and GABA(B2) receptor mRNAs in the rat brain.

Gamma-aminobutyric acid receptors (GABA(B)Rs) are G-protein coupled receptors mediating the slow-onset and prolonged synaptic actions of GABA in the central nervous system (CNS). There are two subtypes, GABA(B1)R and GABA(B2)R, that are though to form heterodimers, with the GABA(B1)R subunit essential for ligand binding and the GABA(B2)R subunit for cell surface localization. We have analyzed the distribution of GABA(B1)R and GABA(B2)R transcripts by in situ hybridization. The two transcripts were generally expressed in parallel, and the highest levels were detected in the piriform cortex, hippocampus, medial habenula, and olfactory bulb. There was moderate expression in several thalamic nuclei and the cortex area. In contrast to the distribution of GABA(B2)R mRNA, GABA(B1)R gave weak signals throughout the thalamus, piriform cortex, and field CA2 of the hippocampus, but strong signals in the septum, superior colliculus, retrosplenial cortex area, and field CA1 of the hippocampus. Thus GABA(B2)R and GABA(B1)R mRNA expression overlapped considerably, consistent with the suggestion that the two subtypes assemble to form a heterodimer. There are nevertheless differences in their expression levels that suggest at the existence of unidentified receptor subtypes.