Modulation of Microglia Polarization through Silencing of NF-κB p65 by Functionalized Curdlan Nanoparticle-Mediated RNAi.

Microglia polarization plays an important role in poststroke recovery. Inhibition of proinflammatory (M1) polarization and promotion of anti-inflammatory (M2) polarization of microglia are potential therapeutic strategies for inflammation reduction and neuronal recovery after stroke. Here, we evaluated the central nervous system (CNS)-targeted short interfering RNA (siRNA) delivery ability of functionalized curdlan nanoparticles (CMI) and investigated the nuclear factor-κB (NF-κB) p65 silencing efficiency of CMI-mediated siRNA in microglia, as well as the resulting neuroprotective effect of microglia polarization and neuroprotection in vitro and in vivo. The systemic delivery of NF-κB p65 siRNA (sip65) complexed to CMI nanoparticles in the mouse model of transient middle cerebral artery occlusion (tMCAO) resulted in the distribution of siRNA in microglia and significant silencing in NF-κB p65 in the peri-infarct region. Knockdown of NF-κB p65 resulted in M1 to M2 phenotypic transition of microglia, evidenced by the change in the expression pattern of signature cytokines as well as inducible nitric oxide synthase and CD206. Moreover, the CMI-mediated silencing of p65 increased the density of neurons and decreased pyknosis and edema in the peri-infarct region. Assessment of the neurological deficit score on the Bederson scale revealed a significantly reduced score in the mouse model of tMCAO treated with the sip65/CMI complex. Collectively, our data suggest that CMI nanoparticles are a promising CNS-targeting siRNA delivery system, and NF-κB p65 may be a potential therapeutic target for inflammation reduction and poststroke recovery.

Receptor-mediated delivery of therapeutic RNA by peptide functionalized curdlan nanoparticles.

Natural carbohydrate polymer-based nanoparticles have great biocompatibility that is required for the safe delivery of various drugs including nucleic acid therapeutics. Herein, we designed curdlan-based nanoparticles for cancer cell targeted delivery of short interfering RNA (siRNA). iRGD peptide conjugated 6-amino-6-deoxy curdlan specifically delivered siRNA to integrin expressing cancer cells. Incubation of cancer cells with free iRGD peptide competitively blocked cellular uptake of the iRGD functionalized curdlan nanoparticles. Chloroquine but not nystatin inhibited cellular uptake of iRGD functionalized curdlan nanoparticles, indicating that the iRGD peptide conjugated curdlan nanoparticles were internalized through the receptor (clathrin)-mediated endocytosis. Moreover, a disease related gene Plk1 was substantially knocked down by siRNA carried by 6AC-iRGD nanoparticles in HepG2 cells. Our data suggested that iRGD functionalized curdlan may provide a biocompatible carrier for siRNA delivery.

Structural and Functional Analysis of a Bidirectional Promoter from Gossypium hirsutum in Arabidopsis.

Stacked traits have become an important trend in the current development of genomically modified crops. The bidirectional promoter can not only prevent the co-suppression of multigene expression, but also increase the efficiency of the cultivation of transgenic plants with multigenes. In Gossypium hirsutum, Ghrack1 and Ghuhrf1 are head-to-head gene pairs located on chromosome D09. We cloned the 1429-bp intergenic region between the Ghrack1 and Ghuhrf1 genes from Gossypium hirsutum. The cloned DNA fragment GhZU had the characteristics of a bidirectional promoter, with 38.7% G+C content, three CpG islands and no TATA-box. Using gfp and gus as reporter genes, a series of expression vectors were constructed into young leaves of tobacco. The histochemical GUS (Beta-glucuronidase) assay and GFP (green fluorescence protein) detection results indicated that GhZU could drive the expression of the reporter genes gus and gfp simultaneously in both orientations. Furthermore, we transformed the expression vectors into Arabidopsis and found that GUS was concentrated at vigorous growth sites, such as the leaf tip, the base of the leaves and pod, and the stigma. GFP was also mainly expressed in the epidermis of young leaves. In summary, we determined that the intergenic region GhZU was an orientation-dependent bidirectional promoter, and this is the first report on the bidirectional promoter from Gossypium hirsutum. Our findings in this study are likely to enhance understanding on the regulatory mechanisms of plant bidirectional promoters.

Cloning and characterization of a Ca(2+)/H(+) exchanger from the halophyte Salicornia europaea L.

The calcium ion (Ca(2+)), which functions as a second messenger, plays an important role in plants' responses to various abiotic stresses, and Ca(2+)/H(+) exchangers (CAXs) are an important part of this process. In this study, we isolated and characterized a putative Ca(2+)/H(+) exchanger gene (SeCAX3) from Salicornia europaea L., a succulent, leafless euhalophyte. The SeCAX3 open reading frame was 1368 bp long and encoded a 455-amino-acid polypeptide that showed 67.9% similarity to AtCAX3. SeCAX3 was expressed in the shoots and roots of S. europaea. Expression of SeCAX3 was up-regulated by Ca(2+), Na(+), sorbitol, Li(+), abscisic acid, and cold treatments in shoots, but down-regulated by Ca(2+), sorbitol, abscisic acid, and cold treatments in roots. When SeCAX3 was transformed into a Ca-sensitive yeast strain, the transformed cells were able to grow in the presence of 200 mM Ca(2+). Furthermore, SeCAX3 conferred drought, salt, and cold tolerance in yeast. Compared with the control strains, the yeast transformants expressing SeCAX3 were able to grow well in the presence of 30 mM Li(+), 150 mM Mg(2+), or 6 mM Ba(2+). These results showed that the expression of SeCAX3 in yeast suppressed its Ca(2+) hypersensitivity and conferred tolerance to Mg(2+) and Ba(2+). Together, these findings suggest that SeCAX3 might be a Ca(2+) transporter that plays a role in regulating cation tolerance and the responses of S. europaea to various abiotic stresses.

Glutamate receptor-like channel3.3 is involved in mediating glutathione-triggered cytosolic calcium transients, transcriptional changes, and innate immunity responses in Arabidopsis.

The tripeptide reduced glutathione (GSH; γ-glutamate [Glu]-cysteine [Cys]-glycine) is a major endogenous antioxidant in both animal and plant cells. It also functions as a neurotransmitter mediating communication among neurons in the central nervous system of animals through modulating specific ionotropic Glu receptors (GLRs) in the membrane. Little is known about such signaling roles in plant cells. Here, we report that transient rises in cytosolic calcium triggered by exogenous GSH in Arabidopsis (Arabidopsis thaliana) leaves were sensitive to GLR antagonists and abolished in loss-of-function atglr3.3 mutants. Like the GSH biosynthesis-defective mutant PHYTOALEXIN DEFICIENT2, atglr3.3 showed enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Pathogen-induced defense marker gene expression was also decreased in atglr3.3 mutants. Twenty-seven percent of genes that were rapidly responsive to GSH treatment of seedlings were defense genes, most of which were dependent on functional AtGLR3.3, while GSH suppressed pathogen propagation through the AtGLR3.3-dependent pathway. Eight previously identified putative AtGLR3.3 ligands, GSH, oxidized glutathione, alanine, asparagine, Cys, Glu, glycine, and serine, all elicited the AtGLR3.3-dependent cytosolic calcium transients, but only GSH and Cys induced the defense response, with the Glu-induced AtGLR3.3-dependent transcription response being much less apparent than that triggered by GSH. Together, these observations suggest that AtGLR3.3 is required for several signaling effects mediated by extracellular GSH, even though these effects may not be causally related.

[Structure characteristics of ORF2a gene of potato leafroll virus Chinese isolate].

According to the genomic sequence of foreign four PLRV isolates, three pairs of specific primer were designed and synthesized. The cDNA of the ORF2a gene of PLRV-Ch was synthesized by reverse transcription and followed by Polymerase Chain Reaction amplication. The synthesized 3' and 5' cDNA fragment of the PLRV-Ch ORF2a gene were inserted into pUC19 and cloned in E. coli JM109 and were sequenced respectively. The middle cDNA fragment were directly sequenced. The homology of nucleotide sequence of PLRV-Ch compared with PLRV-S (Scotland, UK), PLRV-N(Netherlands), PLRV-A(Australia) and PLRV-C(Canada) were 98.96%, 98.70%, 94.79%, 97.5%, the homology of putative amino acid sequence are 97.97%, 97.97%, 89.69%, 95.94%. In 3' region of ORF2a gene a slippery sequence for-1 frameshift and its downstream "stem-loop" or "pseudoknot" and upstream nucleotide sequence repeats were found. Authors suggested that the nucleotide repeat sequences characteristic for PLRV could form a tight successively folded complementary double stranded regions and hairpins. This structure possibly has something to do with-1 frameshift. The amino acid sequence of C terminus region of 70 kD protein translated by motif IV has a protease characteristic motif and a helicase motif IV. The amino acid sequence of polypeptide translated by ORF2a gene undergoing frameshift has a single-stranded nucleic acid binding protein-like characteristic motif.