In search of active nucleoside 5'-triphosphate mimics, we have synthesized a series of AZT triphosphate mimics (AZT P3Ms) and evaluated their inhibitory effects on HIV-1 reverse transcriptase as well as their stability in fetal calf serum and in CEM cell extracts. Reaction of AZT with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, followed by treatment of the phosphite intermediate 2 with pyrophosphate analogues, yielded the cyclic triphosphate intermediates 4b-4f, which were subjected to boronation and subsequent hydrolysis to give AZT 5'-alpha-borano-beta,gamma-bridge-modified triphosphates 6b-6f in moderate to good yields. Reaction of the cyclic intermediate 4d with iodine, followed by treatment with a series of nucleophiles, afforded the AZT 5'-beta,gamma-difluoromethylene-gamma-substituted triphosphates (7b-7i). Several different types of AZT P3Ms containing alpha-P-thio (or dithio) and beta,gamma-difluoromethylene (13,14), alpha,beta-difluoromethylene and gamma-P-methyl(or phenyl) (15,16), and alpha-borano-beta,gamma-difluoromethylene and gamma-O-methyl/phenyl (11,12) were also synthesized. The effectiveness of the compounds as inhibitors of HIV-1 reverse transcriptase was determined using a fluorometric assay and a poly(A) homopolymer as a template. A number of AZT P3Ms exhibited very potent inhibition of HIV-1 reverse transcriptase. Modifications at the beta,gamma-bridge of triphosphate rendered the AZT P3Ms 6b-6f with varied activities (K(i) from 9.5 to >>500 nM) while modification at the alpha,beta-bridge of triphosphate led to weak AZT P3M inhibitors. The results imply that the AZT P3Ms were substrate inhibitors, as is AZT triphosphate. The most active compound, AZT 5'-alpha-R(p)()-borano-beta,gamma-(difluoromethylene)triphosphate (AZT 5'-alphaB-betagammaCF(2)TP) (6d-I), is as potent as AZT triphosphate with a K(i)() value of 9.5 nM and at least 20-fold more stable than AZT triphosphate in the serum and cell extracts. Therefore, for the first time, a highly active and stable nucleoside triphosphate mimic has been identified, which is potentially useful as a new type of antiviral drug. The promising triphosphate mimic, 5'-alpha-borano-beta,gamma-(difluoromethylene)triphosphate, is expected to be valuable to the discovery of nucleotide mimic antiviral drugs.

Whether hispidulin, a flavone from traditional Chinese medicine, can modulate the anticancer effects of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), the cytokine currently in clinical trials was investigated. In the present study, we found that hispidulin potentiated the TRAIL-induced apoptosis in human ovarian cancer cells and converted TRAIL-resistant cells to TRAIL-sensitive cells. When examined for its mechanism, we found that hispidulin was highly effective in activation of caspases 8 and caspase 3 and consequent poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, we found that hispidulin downregulated the expression of Mcl-1, Bcl-2, and Bcl-xL. Whereas the downregulation of Bcl-2 and Bcl-xL was less pronounced, the downregulation of Mcl-1 was quite dramatic and was time-dependent. This sensitization is controlled through the adenosine monophosphate (AMP)-activated protein kinase (AMPK), which is the central energy-sensing system of the cell. Interestingly, we determined that AMPK is activated upon hispidulin treatment, resulting in mammalian target of rapamycin (mTOR) inhibition leading to Mcl-1 decrease. Therefore, our results show a novel mechanism for the sensitization to TRAIL-induced apoptosis linking hispidulin treatment to Mcl-1 downexpression. In addition, this study provides a rationale for the combined use of death receptor (DR) ligands with AMPK activators or mTOR inhibitors in the treatment of human cancers.

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A surface-enhanced Raman scattering (SERS) measurement of 3,3',4,4'-tetrachlorobiphenyl (PCB77) with aptamer capturing in a microfluidic device was demonstrated. To construct the microfluidic chip, an ordered Ag nanocrown array was fabricated over a patterned polydimethylsiloxane (PDMS) that was achieved by replicating an anodic aluminum oxide (AAO) template. The patterned PDMS sheet was covered with another PDMS sheet having two input channel grooves to form a close chip. The Ag nanocrown array was used for the SERS enhancement area and the detection zone. PCB 77 aptamers were injected into one channel and the other allows for analytes (PCBs). The mercapto aptamers captured the targets in the mixed zone and were immobilized to the SERS detection zone via S-Ag bonds so as to further improve both the SERS sensitivity and selectivity of PCB77. Such an aptamer-based microfluidic chip realized a rapid SERS detection. The lowest detectable concentration of 1.0 × 10(-8) M was achieved for PCB77. This work demonstrates that the aptamer-modified SERS microfluidic sensor can be utilized for selective detections of organic pollutants in the environment.

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Aim:To investigate whether sequestosome 1/p62 (p62), a key cargo adaptor protein involved in both the ubiquitin-proteasome system and the autophagy-lysosome system, could directly regulate autophagy in vitro.Methods:HEK 293 cells or HeLa cells were transfected with p62-expressing plasmids or siRNA targeting p62. The cells or the cell lysates were subsequently subjected to immunofluorescence assay, immunoprecipitation assay, or immunoblot analysis. In vitro pulldown assay was used to study the interaction of p62 with Bcl-2.Results:Overexpression of p62 significantly increased the basal level of autophagy in both HEK 293 cells and HeLa cells, whereas knockdown of p62 significantly decreased the basal level of autophagy. In vitro pulldown assay showed that p62 directly interacted with Bcl-2. It was observed in HeLa cells that p62 co-localized with Bcl-2. Furthermore, knockdown of p62 in HEK 293 cells significantly increased the amount of Beclin 1 that co-immunoprecipitated with Bcl-2.Conclusion:p62 induces autophagy by disrupting the association between Bcl-2 and Beclin 1.

We propose a highly sensitive and selective surface-enhanced Raman scattering (SERS) method for determining lead ions based on a DNAzyme-linked plasmonic nanomachine. A metallic nanoparticle-on-a-film structure was built through a rigid double-stranded bridge linker composed of a DNAzyme and its substrate. This DNAzyme could be activated by lead ions and catalyze a fracture action of the substrate. Thus, the double chain structure of DNA would turn into a flexible single strand, making the metal nanoparticles that connected to the terminal of DNAzyme fall to the surface of the metal film. Hereby, a narrow gap close to 2 nm generated between metal nanoparticles and the metal film, exhibiting a similar effect of a "hot spot" and remarkably enhancing the signal of randomly dispersed Raman-active molecules on the surface of metal film. By measuring the improvement of SERS intensity of the Raman-active molecules, we realized the lowest detection concentration of Pb(2+) ions to 1.0 nM. This SERS analytical method is highly selective and can be extended universally to other targets via the accurate programming of corresponding DNA sequences.

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