Plant tissue imaging with bipyramidal upconversion nanocrystals by introducing Tm3+ ions as energy trapping centers.

Plant cell imaging is critical for agricultural production and plant pathology study. Advanced upconversion nanoparticles (UCNPs) are being developed as fluorescent probes for imaging cells and tissues in vivo and in vitro. Unfortunately, the thick cellulosic walls as barriers together with hemicelluloses and pectin hinder the entrance of macromolecules into the epidermal plant cell. Hence, realizing satisfactory temporal and spatial resolution with UCNPs remains an arduous task. Here, bipyramidal LiErF4:1%Tm3+@LiYF4 core-shell UCNPs with a super-bright red emission upon 980 nm laser excitation are explored, where the introduction of Tm3+ ions permits alleviation of the energy loss at defective sites and a significant improvement of the upconversion output. The as-obtained bipyramidal UCNPs could readily puncture plant cell walls and further penetrate into cell membranes, facilitating improved tissue imaging of cellular internalization, as demonstrated with the luminescence images obtained by multiphoton laser-scanning microscopy. Hence our work opens up a new avenue for exploring effective upconversion nanoparticles for achieving high resolution imaging of plant tissues.

Construction of NF-κB-targeting RNAi adenovirus vector and the effect of NF-κB pathway on proliferation and apoptosis of vascular endothelial cells.

To construct a recombinant adenovirus vector expressing a RNAi for the Nuclear Factor kappa B (NF-κB)/p65 gene and use it to explore the role of the NF-κB pathway on the regulation of proliferation and apoptosis of vascular endothelial cells. A recombinant adenovirus containing a RNAi cassette targeting the p65 gene was constructed, and its silencing effect on p65 was detected by Western blot analysis in ECV304 cells. Expression of the p65 protein in ECV304 cells was efficiently down-regulated by the RNAi adenovirus for more than 6 days. ECV304 cells proliferation and apoptosis were measured using the MTT assay and flow cytometry, respectively. Blocking the NF-κB pathway with the RNAi adenovirus substantially decreased the proliferation of ECV304 cells, but only slightly affected cell apoptosis. We used a NF-κB/p65-targeting RNAi adenovirus to demonstrate the role of the NF-κB pathway in the regulation of ECV304 cell proliferation. This adenovirus may serve as an important tool to study the NF-κB pathway.

High FFA-induced proliferation and apoptosis in human umbilical vein endothelial cell partly through Wnt/beta-catenin signal pathway.

Free fatty acids (FFA)-induced proliferation and apoptosis was studied in human umbilical vein endothelial cells (HUVECs). A recombinant adenovirus containing a RNAi cassette targeting the GSK-3beta gene was produced and its silencing effect on GSK-3beta gene was detected by Western blot analysis and immunohistochemistry assay in HUVECs. The effect of the RNAi on the protein level of beta-catenin was explored by transfecting the RNAi adenovirus to inhibit the expression of GSK-3beta protein. The subsequent effect on the Wnt/GSK-3beta/beta-catenin signal pathway and on proliferation and apoptosis of HUVECs cultured with FFAs, was analyzed by BrdU assay, Annexin V-FITC/PI Apoptosis Detection Kit, and 4',6-diamidino-2- phenylindole(DAPI) to explore the possible connection between the signaling pathway and FFA-induced proliferation and apoptosis. The Western blot results showed that the expression of GSK-3beta protein in HUVECs could be inhibited efficiently by the RNAi adenovirus, and that the protein level of beta-catenin was increased by RNAi adenovirus transfection. The results of the BrdU assay suggested that knockdown of GSK-3beta with the RNAi adenovirus may stimulate the proliferation of HUVECs. Apoptosis was observed in HUVECs exposed to FFAs (0.75 mmol/L) for 72 h, and this effect could be partly reversed when interfering with the RNAi adenovirus. It may be concluded that the RNAi adenovirus specific to GSK-3beta may partly protect HUVECs from apoptosis induced by FFAs, probably through the up-regulation of the Wnt/beta-catenin signal pathway.

Ablation of NF-kappaB expression by small interference RNA prevents the dysfunction of human umbilical vein endothelial cells induced by high glucose.

Diabetes is a major independent risk factor for cardiovascular disease and stroke. High glucose (HG) reduces endothelial cell (EC) proliferation with a concomitant increase in apoptosis. HG also induces the translocation of nuclear factor (NF)-kappaB in human umbilical vein endothelial cells (HUVECs). However, data regarding the relationship between NF-kappaB signaling and HG-induced endothelial dysfunction are limited. In the present study, we constructed an NF-kappaB-targeting RNA interference (RNAi) adenovirus vector and cultured HUVECs in 5.5, 20.5, or 30.5 mM D: -glucose or in daily alternating 5.5 or 30.5 mM D: -glucose. We assessed the effects of the NF-kappaB pathway on proliferation under HG conditions by measuring bromodeoxyuridine incorporation and conducting methyl thiazolyltetrazolium assays. We also tested apoptosis by performing flow cytometry and terminal deoxynucleotidyl transferase nick-end labeling assay. The RNAi adenovirus effectively downregulated expression of the p65 protein in HUVECs for more than 6 days. Blockage of the NF-kappaB pathway with the RNAi adenovirus substantially protected HUVECs from decreased proliferation and reduced cellular apoptosis in HG conditions. These findings may explain how hyperglycemia promotes dysfunction of ECs and could elucidate a potential new target for therapeutic interventions.

[Construction of GSK-3beta-targeting RNAi adenovirus vector and the influence of Wnt/beta-catenin pathway in proliferation of human thyrocytes].

OBJECTIVE: To construct RNAi recombinant adenoviral expressive vectors specific to glycogen synthase kinase-3beta (GSK-3beta) and to observe its gene knockdown effect on the expression of GSK-3beta, and to explore the effect of Wnt/beta-catenin pathway on the proliferation of human thyrocytes using the RNAi adenovirus vector. METHODS: An adenovirus plasmid that contained the RNAi cassette targeting the GSK-3beta gene was constructed by homologous recombination and cloning techniques, transfected into human embryo kidney (HEK) 293A cells to product adenovirus, and then was used to infect the HEK293A cells to amplify the adenoviral stock. Plaque forming assay was used to titer the adenoviral stock. Normal human thyrocytes fart from thyroid adenoma were obtained during resection of adenoma, cultured, and infected by the GSK-3beta specific RNAi adenovirus. The GSK-3beta gene silencing effect induced by the RNAi adenovirus was detected by Western blotting 0, 24, 48, 72, 120, and 144 hours later. BrdU method was used to detect the cell proliferation. Another HEK293A cells were divided into 3 groups: infected with recombinant adenovirus plasmid Ad-1457, infected with un-recombinant framework plasmid pAd-DEST, and un-infected. 72 hours later Western blotting was used to examine the level of beta-catenin. RESULTS: The GSK-3beta expression of the thyrocytes infected with the recombinant adenovirus plasmid Ad-1457 were significantly lower than those of the thyrocytes infected with Ad-DEST (all P<0.05). The expression of beta-catenin of the thyrocytes infected with Ad-DEST was significantly higher than those of the Ad-DEST group and un-infected group (both P<0.05). BrdU assay suggested that the proliferation rates 1, 3, 5, and 7 days after infection of the thyrocytes infected with Ad1457 plasmid were significantly higher than those of the thyrocytes infected with the plasmid pAd-DEST (all P<0.05). CONCLUSION: RNAi adenovirus is an important tool inhibiting the expression of target gene efficiently. The Wnt/beta-catenin pathway plays an important role in the regulation of proliferation of human thyrocytes.