uPAR/cathepsin B overexpression reverse angiogenesis by rescuing FAK phosphorylation in uPAR/cathepsin B down regulated meningioma

Background

Meningiomas are the most commonly occurring intracranial tumors and account for approximately 15−20% of central nervous system tumors. Surgery and radiation therapy is a common treatment for brain tumors, however, patients whose tumors recur after such treatments have limited therapeutic options. Earlier studies have reported important roles of uPA, uPAR and cathepsin B in tumor progression.

Methodology/Principal Findings

In the present study, we examined the therapeutic significance of RNAi-mediated simultaneous down regulation of these proteolytic networks using two bicistronic siRNA constructs, pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) either alone or in combination with radiation in two different meningioma cell lines. Transfection of meningioma cells with pUC and pU2 significantly reduced angiogenesis as compared to control treatment both in vitro and in vivo nude mice model. This effect is mediated by inhibiting angiogenic molecules (Ang-1, Ang-2 and VEGF). Expression of focal adhesion kinase (FAK) is elevated in malignant meningioma, yet the role of intrinsic FAK activity in promoting tumor progression remains undefined. We found that pUC treatment reduced FAK phosphorylation at Y925 more efficiently compared to pU2 treatment. In immunoprecipitation assay, we found pronounced reduction of FAK (Y925) interaction with Grb2 in meningioma cells transfected with pUC with and without irradiation. Transient over-expression of uPAR and cathepsin B by full length uPAR/cathepsin B (FLpU/C) in pUC transfected meningioma cells promoted vascular phenotype, rescued expression of Ang-1, Ang-2, VEGF, FAK (Y925) and Grb2 both in vitro and in vivo mice model.

Conclusion/Significance

These studies provide the first direct proof that bicistronic siRNA construct for uPAR and cathepsin B (pUC) reduces Y925-FAK activity and this inhibition is rescued by overexpression of both uPAR and cathepsin B which clearly demonstrates that pUC could thus be a potential therapeutic approach as an anti-angiogenic agent in meningioma.     

T-helper cell-mediated proliferation and cytokine responses against recombinant Merkel cell polyomavirus-like particles

The newly discovered Merkel Cell Polyomavirus (MCPyV) resides in approximately 80% of Merkel cell carcinomas (MCC). Causal role of MCPyV for this rare and aggressive skin cancer is suggested by monoclonal integration and truncation of large T (LT) viral antigen in MCC cells. The mutated MCPyV has recently been found in highly purified leukemic cells from patients with chronic lymphocytic leukemia (CLL), suggesting a pathogenic role also in CLL. About 50-80% of adults display MCPyV-specific antibodies. The humoral immunity does not protect against the development of MCC, as neutralizing MCPyV antibodies occur in higher levels among MCC patients than healthy controls. Impaired T-cell immunity has been linked with aggressive MCC behavior. Therefore, cellular immunity appears to be important in MCPyV infection surveillance. In order to elucidate the role of MCPyV-specific Th-cell immunity, peripheral blood mononuclear cells (PBMC) of healthy adults were stimulated with MCPyV VP1 virus-like particles (VLPs), using human bocavirus (HBoV) VLPs and Candida albicans antigen as positive controls. Proliferation, IFN-γ, IL-13 and IL-10 responses were examined in 15 MCPyV-seropositive and 15 seronegative volunteers. With the MCPyV antigen, significantly stronger Th-cell responses were found in MCPyV-seropositive than MCPyV-seronegative subjects, whereas with the control antigens, the responses were statistically similar. The most readily detectable cytokine was IFN-γ. The MCPyV antigen tended to induce stronger IFN-γ responses than HBoV VLP antigen. Taken together, MCPyV-specific Th-cells elicit vigorous IFN-γ responses. IFN-γ being a cytokine with major antiviral and tumor suppressing functions, Th-cells are suggested to be important mediators of MCPyV-specific immune surveillance.     

PolysacDB: a database of microbial polysaccharide antigens and their antibodies

Vaccines based on microbial cell surface polysaccharides have long been considered as attractive means to control infectious diseases. To realize this goal, detailed systematic information about the antigenic polysaccharide is necessary. However, only a few databases that provide limited knowledge in this area are available. This paper describes PolysacDB, a manually curated database of antigenic polysaccharides. We collected and compiled comprehensive information from literature and web resources about antigenic polysaccharides of microbial origin. The current version of the database has 1,554 entries of 149 different antigenic polysaccharides from 347 different microbes. Each entry provides comprehensive information about an antigenic polysaccharide, i.e., its origin, function, protocols for its conjugation to carriers, antibodies produced, details of assay systems, specificities of antibodies, proposed epitopes involved and antibody utilities. For convenience to the user, we have integrated web interface for searching, advanced searching and browsing data in database. This database will be useful for researchers working on polysaccharide-based vaccines. It is freely available from the URL: http://crdd.osdd.net/raghava/polysacdb/.     

Aloe vera induced biomimetic assemblage of nucleobase into nanosized particles

Aim

Biomimetic nano-assembly formation offers a convenient and bio friendly approach to fabricate complex structures from simple components with sub-nanometer precision. Recently, biomimetic (employing microorganism/plants) synthesis of metal and inorganic materials nano-particles has emerged as a simple and viable strategy. In the present study, we have extended biological synthesis of nano-particles to organic molecules, namely the anticancer agent 5-fluorouracil (5-FU), using Aloe vera leaf extract.

Methodology

The 5-FU nano- particles synthesized by using Aloe vera leaf extract were characterized by UV, FT-IR and fluorescence spectroscopic techniques. The size and shape of the synthesized nanoparticles were determined by TEM, while crystalline nature of 5-FU particles was established by X-ray diffraction study. The cytotoxic effects of 5-FU nanoparticles were assessed against HT-29 and Caco-2 (human adenocarcinoma colorectal) cell lines.

Results

Transmission electron microscopy and atomic force microscopic techniques confirmed nano-size of the synthesized particles. Importantly, the nano-assembled 5-FU retained its anticancer action against various cancerous cell lines.

Conclusion

In the present study, we have explored the potential of biomimetic synthesis of nanoparticles employing organic molecules with the hope that such developments will be helpful to introduce novel nano-particle formulations that will not only be more effective but would also be devoid of nano-particle associated putative toxicity constraints.     

Aspergillus bertholletius sp. nov. from Brazil Nuts

During a study on the mycobiota of brazil nuts (Bertholletia excelsa) in Brazil, a new Aspergillus species, A. bertholletius, was found, and is described here. A polyphasic approach was applied using morphological characters, extrolite data as well as partial β-tubulin, calmodulin and ITS sequences to characterize this taxon. A. bertholletius is represented by nineteen isolates from samples of brazil nuts at various stages of production and soil close to Bertholletia excelsa trees. The following extrolites were produced by this species: aflavinin, cyclopiazonic acid, kojic acid, tenuazonic acid and ustilaginoidin C. Phylogenetic analysis using partial β-tubulin and camodulin gene sequences showed that A. bertholletius represents a new phylogenetic clade in Aspergillus section Flavi. The type strain of A. bertholletius is CCT 7615 ( = ITAL 270/06 = IBT 29228).     

Trichodysplasia spinulosa-Associated Polyomavirus (TSV) and Merkel Cell Polyomavirus: Correlation between Humoral and Cellular Immunity Stronger with TSV

Merkel Cell Polyomavirus (MCV) is a common infectious agent likely to be involved in the pathogenesis of most Merkel cell carcinomas (MCC). Trichodysplasia spinulosa-associated polyomavirus (TSV), which exhibit high seroprevalence in general population, has been detected in trichodysplasia spinulosa (TS) skin lesions suggesting an etiological role for this disease. Previous studies have shown strong MCV-specific T-cell responses, while no data exist on T-cell immunity against TSV. In order to characterize Th-cell immunity against TSV, and to allow comparisons with the MCV-specific Th-cell immunity, we studied TSV-specific proliferation, IFN-γ, IL-10 and IL-13, and MCV-specific IFN-γ and IL-10 responses in 51 healthy volunteers, and in one MCC patient. Recombinant TSV and MCV VP1 virus-like particles (VLPs) were used as antigens. A significant correlation was found between virus-specific Th-cell and antibody responses with TSV; with MCV it proved weaker. Despite significant homology in amino acid sequences, Th-cell crossreactivity was not evident between these viruses. Some subjects seronegative to both TSV and MCV exhibited Th-cell responses to both viruses. The agent initially priming these Th-cells remains an enigma. As CD8⁺ cells specific to MCV T-Ag oncoprotein clearly provide an important defense against established MCC, the MCV VP1-specific Th-cells may, by suppressing MCV replication with antiviral cytokines such as IFN-γ, significantly contribute to preventing the full process of oncogenesis.     

Chitin binding proteins act synergistically with chitinases in Serratia proteamaculans 568

Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to β-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of α- and β-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on β-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥ 0.2 μM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues.