Script-Free HTML: Preventing Cross-Site Scripting While Permitting HTML-Rich Content

When someone visits a web site, the site's server uses input from the person's web browser to dynamically generate the webpage returned to the user. If hackers can find a weakness in the site's code and control how webpages are generated, they can insert their own scripts into the webpage returned to visitors. These scripts run in the visitor's browser and can compromise the visitor's personal information. The injection of scripts into a webpage by means of evading input filtering is called a cross-site scripting (XSS) attack. Even popular websites, such as Google, Facebook, and YouTube, have been exploited by XSS attacks (KF & DP, 2012). In 2010, XSS attacks were ranked the 2nd-leading source of web security risk (OWASP, 2010). XSS attacks, by their very nature, are not detectable client-side (e.g., by web browsers or antivirus programs). Current methods to prevent XSS exploits are either ineffective (allowing some attacks to succeed) or overly prohibitive (preventing legitimate HTML-rich content). This project describes a new approach: The structure of safe input is rigorously defined and a server-side tool is implemented to detect the presence of a potential XSS attack. This tool prevents XSS attacks while still permitting HTML-rich content. We define a new context-free grammar (Script-Free HTML 4) that precisely characterizes safe input. Our approach is evaluated by applying it to a benchmark of known XSS vulnerabilities. We also consider the future evolution of this approach in the ever-changing world of web standards.College of Engineering Undergraduate Research ScholarshipNo embarg

A gold standard set of mechanistically diverse enzyme superfamilies

Superfamily and family analyses provide an effective tool for the functional classification of proteins, but must be automated for use on large datasets. We describe a 'gold standard' set of enzyme superfamilies, clustered according to specific sequence, structure, and functional criteria, for use in the validation of family and superfamily clustering methods. The gold standard set represents four fold classes and differing clustering difficulties, and includes five superfamilies, 91 families, 4,887 sequences and 282 structures

Protein Shape Sampled by Ion Mobility Mass Spectrometry Consistently Improves Protein Structure Prediction

Ion mobility (IM) mass spectrometry provides structural information about protein shape and size in the form of an orientationally-averaged collision cross-section (CCSIM). While IM data have been used with various computational methods, they have not yet been utilized to predict monomeric protein structure from sequence. Here, we show that IM data can significantly improve protein structure determination using the modelling suite Rosetta. We develop the Rosetta Projection Approximation using Rough Circular Shapes (PARCS) algorithm that allows for fast and accurate prediction of CCSIM from structure. Following successful testing of the PARCS algorithm, we use an integrative modelling approach to utilize IM data for protein structure prediction. Additionally, we propose a confidence metric that identifies near native models in the absence of a known structure. The results of this study demonstrate the ability of IM data to consistently improve protein structure prediction

Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes

AbstractBackground: Directed evolution by DNA shuffling has been used to modify physical and catalytic properties of biological systems. We have shuffled two highly homologous triazine hydrolases and conducted an exploration of the substrate specificities of the resulting enzymes to acquire a better understanding of the possible distributions of novel functions in sequence space.Results: Both parental enzymes and a library of 1600 variant triazine hydrolases were screened against a synthetic library of 15 triazines. The shuffled library contained enzymes with up to 150-fold greater transformation rates than either parent. It also contained enzymes that hydrolyzed five of eight triazines that were not substrates for either starting enzyme.Conclusions: Permutation of nine amino acid differences resulted in a set of enzymes with surprisingly diverse patterns of reactions catalyzed. The functional richness of this small area of sequence space may aid our understanding of both natural and artificial evolution

Intramolecular Epistasis and the Evolution of a New Enzymatic Function

Atrazine chlorohydrolase (AtzA) and its close relative melamine deaminase (TriA) differ by just nine amino acid substitutions but have distinct catalytic activities. Together, they offer an informative model system to study the molecular processes that underpin the emergence of new enzymatic function. Here we have constructed the potential evolutionary trajectories between AtzA and TriA, and characterized the catalytic activities and biophysical properties of the intermediates along those trajectories. The order in which the nine amino acid substitutions that separate the enzymes could be introduced to either enzyme, while maintaining significant catalytic activity, was dictated by epistatic interactions, principally between three amino acids within the active site: namely, S331C, N328D and F84L. The mechanistic basis for the epistatic relationships is consistent with a model for the catalytic mechanisms in which protonation is required for hydrolysis of melamine, but not atrazine

Caffeine Junkie: an Unprecedented Glutathione S-Transferase- Dependent Oxygenase Required for Caffeine Degradation by Pseudomonas putida CBB5 Downloaded from

c Caffeine and other N-methylated xanthines are natural products found in many foods, beverages, and pharmaceuticals. Therefore, it is not surprising that bacteria have evolved to live on caffeine as a sole carbon and nitrogen source. The caffeine degradation pathway of Pseudomonas putida CBB5 utilizes an unprecedented glutathione-S-transferase-dependent Rieske oxygenase for demethylation of 7-methylxanthine to xanthine, the final step in caffeine N-demethylation. The gene coding this function is unusual, in that the iron-sulfur and non-heme iron domains that compose the normally functional Rieske oxygenase (RO) are encoded by separate proteins. The non-heme iron domain is located in the monooxygenase, ndmC, while the Rieske [2Fe-2S] domain is fused to the RO reductase gene, ndmD. This fusion, however, does not interfere with the interaction of the reductase with N 1 -and N 3 -demethylase RO oxygenases, which are involved in the initial reactions of caffeine degradation. We demonstrate that the N 7 -demethylation reaction absolutely requires a unique, tightly bound protein complex composed of NdmC, NdmD, and NdmE, a novel glutathione-S-transferase (GST). NdmE is proposed to function as a noncatalytic subunit that serves a structural role in the complexation of the oxygenase (NdmC) and Rieske domains (NdmD). Genome analyses found this gene organization of a split RO and GST gene cluster to occur more broadly, implying a larger function for RO-GST protein partners. C affeine (1,3,7-trimethylxanthine) and other N-methylated xanthines are well known for applications in food and as pharmaceuticals that improve lung function for asthmatics and chronic obstructive pulmonary disease (COPD) sufferers. More recently, these compounds have been investigated for use as natural insecticides and in treatments for cancer, septic shock, and functional neutrophil disorders (1-3). Enzymatic methods for producing and degrading these N-methylated xanthines could have broader applications for health through both biosynthesis and environmental remediation of waste and by-products. Therefore, bacteria that have evolved to live on caffeine as the sole carbon and nitrogen source are of interest, as are their metabolic pathways toward N-methylated xanthines. Pseudomonas putida CBB5 degrades caffeine, theophylline (1,3-dimethylxanthine), and related methylxanthines via sequential N-demethylation to xanthine (4-6). The ordered N-demethylation of caffeine to xanthine occurs in three steps catalyzed by enzymes belonging to the Rieske oxygenase (RO) family (5, 6), which are encoded by the Alx operon. Initially, two Rieske, nonheme Fe(II) monooxygenases, NdmA and NdmB, remove the N 1 -and N 3 -methyl groups, respectively, from caffeine to form 7-methylxanthine. Both enzymes require an unusually large 65-kDa redox-dense RO reductase, NdmD, which transfers electrons from NADH to NdmA and NdmB for oxygen activation. The final step in the caffeine degradation pathway is N 7 -demethylation of 7-methylxanthine to xanthine. This N 7 -demethylation activity was inseparable from NdmD after four chromatographic steps (6). A highly enriched protein fraction containing this activity was comprised of NdmD and two additional major protein bands, as visualized by SDS-PAGE. These two additional peptides are encoded by two genes in the Alx operon, labeled orf7 and orf8, which flank ndmD on the CBB5 genome Here, we report that ndmE encodes a new type of GST that is absolutely required for N 7 -demethylation of 7-methylxanthine, the final step of caffeine degradation in P. putida CBB5. The N 7 -demethylase RO is unusual in itself because the iron-sulfur and non-heme iron domains that compose the normally functional oxygenase are encoded by two separate genes. The non-heme iron is contained in NdmC, while the iron-sulfur domain is fused to NdmD. NdmE is proposed to facilitate the formation of the NdmCDE complex, which catalyzes the N 7 -demethylation. This is the first report of a new class of GST-dependent ROs. Additional identification of similar uncharacterized gene clusters within genome databases suggests that there is a more generalized role for GSTs in oxygenation and/or biodegradation

Efficiency of Purine Utilization by Helicobacter pylori: Roles for Adenosine Deaminase and a NupC Homolog

The ability to synthesize and salvage purines is crucial for colonization by a variety of human bacterial pathogens. Helicobacter pylori colonizes the gastric epithelium of humans, yet its specific purine requirements are poorly understood, and the transport mechanisms underlying purine uptake remain unknown. Using a fully defined synthetic growth medium, we determined that H. pylori 26695 possesses a complete salvage pathway that allows for growth on any biological purine nucleobase or nucleoside with the exception of xanthosine. Doubling times in this medium varied between 7 and 14 hours depending on the purine source, with hypoxanthine, inosine and adenosine representing the purines utilized most efficiently for growth. The ability to grow on adenine or adenosine was studied using enzyme assays, revealing deamination of adenosine but not adenine by H. pylori 26695 cell lysates. Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines. This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides. Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens. In this study we define a more comprehensive model for purine acquisition and salvage in H. pylori that includes purine uptake by a NupC homolog and catabolism of adenosine via adenosine deaminase