Applying SEM-EDX and XRD Techniques to Demonstrate the Overgrowth of Atmospheric Soot and Its Coalescence with Crystal Silicate Particles in Delhi

Scanning electron microscopy-energy dispersive X-ray analysis (SEM-EDX) and X-ray diffraction (XRD) systems were used to demonstrate the overgrowth of soot to fractal like structure and its subsequent coalescence with crystal shaped silicate particles. Sample was obtained from a very clean area of Delhi at a height of 16 m from ground with the help of a five stage cascade impactor in the winters of 2006. Impactor collects particles in five different size ranges (i.e. ≥10.9, 10.9 - 5.4, 5.4 - 1.6, 1.6 - 0.7 and ≤0.7 μm). In the present investigation only the particles collected in the size range 1.6 - 0.7 μm (D50 = 0.980 μm) have been considered. It has clearly been observed that the soot particles tend to grow or rather agglomerate in a fractal like structure. During this process they incorporate other chemically and structurally different particles (crystal silicate in the present investigation) to make multi phase and multi chemical amorphous aggregates. These aggregates are formed during/after its collection on the sampling substrate and may be as many as hundred times more than the expected size interval (D50 or cut off range).

Pristimerin enhances recombinant adeno-associated virus vector-mediated transgene expression in human cell lines in vitro and murine hepatocytes in vivo

OBJECTIVE： In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus （rAAV） serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo. METHODS： Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma （NSG） mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time. RESULTS： We observed that treatment with pristimerin, at as low as 1 IJmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co- administration with pristimerin at 4 mg/（kg.d） for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes. CONCLUSION： These studies suggest the potential beneficial use of pristimerin and pristimerincontaining herb extract in future liver-targeted gene therapy with rAAV vectors.

Cytotoxic genes from traditional Chinese medicine inhibit tumor growth both in vitro and in vivo

OBJECTIVE： Little effort has been made to study the protein-encoding genes isolated from traditional Chinese medicine（TCM） drugs, and the delivery of these genes into malignant cells through recombinant adeno-associated virus（r AAV） vectors has not been attempted. METHODS： We synthesized the c DNAs of five known cytotoxic proteins isolated from TCM drugs and the FLAG epitope-tagged c DNAs were subcloned into a r AAV plasmid vector. The protein expression was confi rmed by Western blot assay. Various cancer cell lines were transfected with the above plasmids and cell growth was monitored both in vitro and in vivo. The best cytotoxic gene was further packaged into r AAV vectors, under the control of a liver cancer-specifi c promoter. The liver tumor growth was then monitored following intratumor administration of the r AAV vectors.RESULTS： The expression plasmids, encoding individual potential cytotoxic genes tagged with FLAG epitope, were successfully generated and sequenced. Among these genes, trichosanthin（TCS） gene yielded the most promising results for the inhibition of cancer cell growth in vitro. The over-expressed TCS functioned as a type I ribosome-inactivating protein, followed by inducing apoptosis that is associated with the Bcl-PARP signaling pathway. Furthermore, intratumor injection of r AAV vectors containing the TCS gene signifi cantly inhibited the growth of human hepatocellular carcinoma tumors in a murine xenograft model.CONCLUSION： Our studies suggest that the use of TCM cytotoxic genes is a useful therapeutic strategy for treating human cancers in general, and liver tumors in particular.

Prevalence of neutralizing antibodies against liver-tropic adeno-associated virus serotype vectors in 100 healthy Chinese and its potential relation to body constitutions

Recombinant adeno-associated virus （rAAV） serotype 2, 3 and 8 vectors are the most promising liver- tropic AAV serotype vectors. Liver diseases are significant problems in China. However, to date, few studies on AAV neutralizing antibodies （Nabs） were working with the Chinese population or with the rAAV3 vectors. The present study aimed to determine the prevalence of Nabs in Chinese population against wild-type AAV2, AAV3 and AAV8 capsids as well as additional two AAV3 variants. In addition, we performed a preliminary analysis to investigate the potential influence of traditional Chinese medicine body constitutions on AAV Nabs. Our work demonstrated that the majority of healthy Chinese subjects were positive for AAV Nabs, with the order of AAV2 〉 AAV3 = AAVLK03 〉 AAV8. There was no difference between： 1）AAV3 and its variants; 2） male and female subjects; and 3） different age cohorts （〈 35, 36- 50, and 〉 51 years old）. People in the Qi-deficiency constitution had significantly increased AAV8 Nabs than people in the Gentleness constitution. Our studies may have impact on the future clinical design of AAV-based gene therapy in the Chinese population.