Benzyl isothiocyanate suppresses pancreatic tumor angiogenesis and invasion by inhibiting HIF-α/VEGF/Rho-GTPases: pivotal role of STAT-3

Our previous studies have shown that benzyl isothiocyanate (BITC) suppresses pancreatic tumor growth by inhibiting STAT-3; however, the exact mechanism of tumor growth suppression was not clear. Here we evaluated the effects and mechanism of BITC on pancreatic tumor angiogenesis. Our results reveal that BITC significantly inhibits neovasularization on rat aorta and Chicken-Chorioallantoic membrane. Furthermore, BITC blocks the migration and invasion of BxPC-3 and PanC-1 pancreatic cancer cells in a dose dependant manner. Moreover, secretion of VEGF and MMP-2 in normoxic and hypoxic BxPC-3 and PanC-1 cells was significantly suppressed by BITC. Both VEGF and MMP-2 play a critical role in angiogenesis and metastasis. Our results reveal that BITC significantly suppresses the phosphorylation of VEGFR-2 (Tyr-1175), and expression of HIF-α. Rho-GTPases, which are regulated by VEGF play a crucial role in pancreatic cancer progression. BITC treatment reduced the expression of RhoC whereas up-regulated the expression of tumor suppressor RhoB. STAT-3 over-expression or IL-6 treatment significantly induced HIF-1α and VEGF expression; however, BITC substantially suppressed STAT-3 as well as STAT-3-induced HIF-1α and VEGF expression. Finally, in vivo tumor growth and matrigel-plug assay show reduced tumor growth and substantial reduction of hemoglobin content in the matrigel plugs and tumors of mice treated orally with 12 μmol BITC, indicating reduced tumor angiogenesis. Immunoblotting of BITC treated tumors show reduced expression of STAT-3 phosphorylation (Tyr-705), HIF-α, VEGFR-2, VEGF, MMP-2, CD31 and RhoC. Taken together, our results suggest that BITC suppresses pancreatic tumor growth by inhibiting tumor angiogenesis through STAT-3-dependant pathway.     

Trends in Resource Utilization by Children with Neurological Impairment in the United States Inpatient Health Care System: A Repeat Cross-Sectional Study

Background

Care advances in the United States (US) have led to improved survival of children with neurological impairment (NI). Children with NI may account for an increasing proportion of hospital resources. However, this assumption has not been tested at a national level.

Methods and Findings

We conducted a study of 25,747,016 US hospitalizations of children recorded in the Kids'' Inpatient Database (years 1997, 2000, 2003, and 2006). Children with NI were identified with International Classification of Diseases, 9th Revision, Clinical Modification diagnoses resulting in functional and/or intellectual impairment. We assessed trends in inpatient resource utilization for children with NI with a Mantel-Haenszel chi-square test using all 4 y of data combined. Across the 4 y combined, children with NI accounted for 5.2% (1,338,590) of all hospitalizations. Epilepsy (52.2% [n = 538,978]) and cerebral palsy (15.9% [n = 164,665]) were the most prevalent NI diagnoses. The proportion of hospitalizations attributable to children with NI did not change significantly (p = 0.32) over time. In 2006, children with NI accounted for 5.3% (n = 345,621) of all hospitalizations, 13.9% (n = 3.4 million) of bed days, and 21.6% (US$17.7 billion) of all hospital charges within all hospitals. Over time, the proportion of hospitalizations attributable to children with NI decreased within non-children''s hospitals (3.0% [n = 146,324] in 1997 to 2.5% [n = 113,097] in 2006, p<.001) and increased within children''s hospitals (11.7% [n = 179,324] in 1997 to 13.5% [n = 209,708] in 2006, p<0.001). In 2006, children with NI accounted for 24.7% (2.1 million) of bed days and 29.0% (US$12.0 billion) of hospital charges within children''s hospitals.

Conclusions

Children with NI account for a substantial proportion of inpatient resources utilized in the US. Their impact is growing within children''s hospitals. We must ensure that the current health care system is staffed, educated, and equipped to serve this growing segment of vulnerable children. Please see later in the article for the Editors'' Summary     

Thermostability of In Vitro Evolved Bacillus subtilis Lipase A: A Network and Dynamics Perspective

Proteins in thermophilic organisms remain stable and function optimally at high temperatures. Owing to their important applicability in many industrial processes, such thermostable proteins have been studied extensively, and several structural factors attributed to their enhanced stability. How these factors render the emergent property of thermostability to proteins, even in situations where no significant changes occur in their three-dimensional structures in comparison to their mesophilic counter-parts, has remained an intriguing question. In this study we treat Lipase A from Bacillus subtilis and its six thermostable mutants in a unified manner and address the problem with a combined complex network-based analysis and molecular dynamic studies to find commonality in their properties. The Protein Contact Networks (PCN) of the wild-type and six mutant Lipase A structures developed at a mesoscopic scale were analyzed at global network and local node (residue) level using network parameters and community structure analysis. The comparative PCN analysis of all proteins pointed towards important role of specific residues in the enhanced thermostability. Network analysis results were corroborated with finer-scale molecular dynamics simulations at both room and high temperatures. Our results show that this combined approach at two scales can uncover small but important changes in the local conformations that add up to stabilize the protein structure in thermostable mutants, even when overall conformation differences among them are negligible. Our analysis not only supports the experimentally determined stabilizing factors, but also unveils the important role of contacts, distributed throughout the protein, that lead to thermostability. We propose that this combined mesoscopic-network and fine-grained molecular dynamics approach is a convenient and useful scheme not only to study allosteric changes leading to protein stability in the face of negligible over-all conformational changes due to mutations, but also in other molecular networks where change in function does not accompany significant change in the network structure.     

The absence of invariant chain in MHC II cancer vaccines enhances the activation of tumor-reactive type 1 CD4<Superscript>+</Superscript> T lymphocytes

Activation of tumor-reactive T lymphocytes is a promising approach for the prevention and treatment of patients with metastatic cancers. Strategies that activate CD8⁺ T cells are particularly promising because of the cytotoxicity and specificity of CD8⁺ T cells for tumor cells. Optimal CD8⁺ T cell activity requires the co-activation of CD4⁺ T cells, which are critical for immune memory and protection against latent metastatic disease. Therefore, we are developing “MHC II” vaccines that activate tumor-reactive CD4⁺ T cells. MHC II vaccines are MHC class I⁺ tumor cells that are transduced with costimulatory molecules and MHC II alleles syngeneic to the prospective recipient. Because the vaccine cells do not express the MHC II-associated invariant chain (Ii), we hypothesized that they will present endogenously synthesized tumor peptides that are not presented by professional Ii⁺ antigen presenting cells (APC) and will therefore overcome tolerance to activate CD4⁺ T cells. We now report that MHC II vaccines prepared from human MCF10 mammary carcinoma cells are more efficient than Ii⁺ APC for priming and boosting Type 1 CD4⁺ T cells. MHC II vaccines consistently induce greater expansion of CD4⁺ T cells which secrete more IFNγ and they activate an overlapping, but distinct repertoire of CD4⁺ T cells as measured by T cell receptor Vβ usage, compared to Ii⁺ APC. Therefore, the absence of Ii facilitates a robust CD4⁺ T cell response that includes the presentation of peptides that are presented by traditional APC, as well as peptides that are uniquely presented by the Ii⁻ vaccine cells.     

Vibrio cholerae persistence in aquatic environments and colonization of intestinal cells: involvement of a common adhesion mechanism

Forty-one Tnpho A mutants of Vibrio cholerae O1 classical strain CD81 were analyzed for their ability to interact with chitin particles, Tigriopus fulvus copepods and the Intestine 407 cell line compared to the parent strain. Thirteen mutants were less adhesive than CD81; in particular, T21, T33 and T87 were less adhesive towards all substrates and insensitive to inhibition by N-acetyl glucosamine (GlcNAc). By SDS-PAGE analysis of sarkosyl-insoluble membrane proteins (siMPs) isolated from mutants and parent, it was found that a 53 kDa siMP is missing in T21, T33 and T87 mutants. It is hypothesized that this protein might have the function to mediate adherence to GlcNAc-containing substrates both in the aquatic environment and in human intestine.     

Arsenic affects mineral nutrients in grains of various Indian rice (Oryza sativa L.) genotypes grown on arsenic-contaminated soils of West Bengal

The exposure of paddy fields to arsenic (As) through groundwater irrigation is a serious concern that may not only lead to As accumulation to unacceptable levels but also interfere with mineral nutrients in rice grains. In the present field study, profiling of the mineral nutrients (iron (Fe), phosphorous, zinc, and selenium (Se)) was done in various rice genotypes with respect to As accumulation. A significant genotypic variation was observed in elemental retention on root Fe plaque and their accumulation in various plant parts including grains, specific As uptake (29–167 mg kg^?1 dw), as well as As transfer factor (4–45%). Grains retained the least level of As (0.7–3%) with inorganic As species being the dominant forms, while organic As species, viz., dimethylarsinic acid and monomethylarsonic acid, were non-detectable. In all tested varieties, the level of Se was low (0.05–0.12 mg kg^?1 dw), whereas that of As was high (0.4–1.68 mg kg^?1 dw), considering their safe/recommended daily intake limits, which may not warrant their human consumption. Hence, their utilization may increase the risk of arsenicosis, when grown in As-contaminated areas.     

Solution conformation of a rationally designed nonapeptide

Partial 'turn-helix' type modules comprised of LD and DL chiral beta-turns serving as potential helix nucleators have been connected with a view to designing a nascent 'helix-turn-helix' type structure. Conformation of the resultant peptide Boc-(D)Glu-Ala-Aib-Lys-Val-Pro-(D)Asp-Leu-Leu-NHMe has been described in both DMSO and water.     

Beyond the proton abstracting role of Glu-376 in medium-chain acyl-CoA dehydrogenase: influence of Glu-376-->Gln substitution on ligand binding and catalysis

The active site residue, Glu-376, of medium-chain acyl-CoA dehydrogenase (MCAD) has been known to abstract the alpha-proton from acyl-CoA substrates during the course of the reductive half-reaction. The site-specific mutation of Glu-376-->Gln(E376Q) slows down the octanoyl-CoA-dependent reductive half-reaction of the enzyme by about 5 orders of magnitude due to impairment in the proton-transfer step. To test whether the carboxyl group of Glu-376 exclusively serves as the active site base (for abstracting the alpha-proton) during the enzyme catalysis, we undertook a detailed kinetic investigation of the enzyme-ligand interaction and enzyme catalysis, utilizing octanoyl-CoA/octenoyl-CoA as a physiological substrate/product pair and the wild-type and E376Q mutant enzymes as the catalysts. The transient kinetic data revealed that the E376Q mutation not only impaired the rate of octanoyl-CoA-dependent reduction of the enzyme-bound FAD, but also impaired the association and dissociation rates for the binding of the reaction product, octenoyl-CoA. Besides, the E376Q mutation correspondingly impaired the kinetic profiles for the quenching of the intrinsic protein fluorescence during the course of the above diverse (i.e., "chemistry" versus "physical interaction") processes. A cumulative account of the experimental data led to the suggestion that the carboxyl group of Glu-376 of MCAD is intimately involved in modulating the microscopic environment (protein conformation) of the enzyme's active site during the course of ligand binding and catalysis. Arguments are presented that the electrostatic interactions among Glu-376, FAD, and CoA-ligands are responsible for structuring the enzyme's active site cavity in the ground and transition states of the enzyme during the above physicochemical processes.