A synthetic heparan sulfate oligosaccharide library reveals the novel enzymatic action of D-glucosaminyl 3-O-sulfotransferase-3a

Heparan sulfate (HS) glucosaminyl 3-O-sulfotranferases sulfate the C3-hydroxyl group of certain glucosamine residues on heparan sulfate. Six different 3-OST isoforms exist, each of which can sulfate very distinct glucosamine residues within the HS chain. Among these isoforms, 3-OST1 has been shown to play a role in generating ATIII-binding HS anticoagulants whereas 3-OST2, 3-OST3, 3-OST4 and 3OST-6 have been shown to play a vital role in generating gD-binding HS chains that permit the entry of herpes simplex virus type 1 into cells. 3-OST5 has been found to generate both ATIII- and gD-binding HS motifs. Previous studies have examined the substrate specificities of all the 3-OST isoforms using HS polysaccharides. However, very few studies have examined the contribution of the epimer configuration of neighboring uronic acid residues next to the target site to 3-OST action. In this study, we utilized a well-defined synthetic oligosaccharide library to examine the substrate specificity of 3-OST3a and compared it to 3-OST1. We found that both 3-OST1 and 3-OST3a preferentially sulfate the 6-O-sulfated, N-sulfoglucosamine when an adjacent iduronyl residue is located to its reducing side. On the other hand, 2-O-sulfation of this uronyl residue can inhibit the action of 3-OST3a on the target residue. The results reveal novel substrate sites for the enzyme actions of 3-OST3a. It is also evident that both these enzymes have promiscuous and overlapping actions that are differentially regulated by iduronyl 2-O-sulfation.     

New stochastic simulation capability applied to the GREET model

Background, Aims and Scope

In 1995, the Center for Transportation Research (CTR) of Argonne National Laboratory (ANL) began to develop a model, called GREET (Greenhouse gases, Regulated Emissions, and Energy use in Transportation), for estimating the full fuel-cycle energy and emissions impacts of alternative transportation fuels and advanced vehicle technologies. The parametric assumptions used in the GREET model involve uncertainties. A new stochastic simulation tool, developed by Vishwamitra Research Institute (VRI), is built into the GREET model to address uncertainties. This paper presents the methodology and features of this new stochastic simulation tool and evaluates the performance of the sampling techniques in the tool.

Methods

The new tool is interfaced through the graphical user interface (GUI) to perform the stochastic simulation. In general, five steps need to be followed to run a complete simulation: 1) Specify probability distribution functions; 2) Indicate the number of samples and the sampling technique; 3) Define the forecast variables; 4) Delete distribution functions (if necessary); and 5) Propagate the uncertainties and statistically analyze the outputs. The GREET model contains more than 700 default distribution functions for a wide variety of key parameters and as many as 3000 forecast variables. The stochastic simulation tool has been developed to incorporate 11 probability distribution function types for representing uncertain parameters and four sampling techniques (Monte Carlo sampling [MCS], Hammersley Sequence sampling [HSS], Latin Hypercube sampling [LHS] and Latin Hypercube Hammersley sampling [LHHS]) for stochastic simulation. To evaluate the performance of the four sampling techniques, 16 independent stochastic simulation runs were conducted in GREET and the output results were analyzed and compared.

Results and Discussion

With the same number of samples, the output distribution curve simulated by HSS is the smoothest corresponding to the highest level of uniformity. To achieve the same level of smoothness as HSS with 1,000 samples, LHHS needs to be simulated with ～1500 samples and LHS and MCS with ～3,000 samples. As a result, HSS can achieve more than 200% reduction in running time compared to LHS or MCS without compromising the accuracy and quality of the prediction curves. The simulated mean values are close enough to the actual mean value (within ±1%) despite the selection of sampling technique and the number of samples (between 1,000 and 4,000). The standard deviation values from each other are close enough as well (within ±5%). It shows the trend that the increasing number of samples makes the simulated mean value marginally closer to the actual mean value; however, the improvement effect is negligible. The simulation time is strictly positive-correlated with the number of samples; therefore, the trade-off between extending simulation time and improving the smoothness of the output distribution curve needs to be carefully assessed.

Conclusion

A new stochastic simulation tool has been developed to be built into Argonne’s GREET model to enhance its capability for addressing uncertainty. This new tool guides the user in each step of the process through the user-friendly GUI windows. According to the performance comparison among the four sampling techniques, HSS was found to be the most efficient technique. Therefore, HSS was set as the default technique in GREET.     

Comparative Analysis of the Orphan CRISPR2 Locus in 242 Enterococcus faecalis Strains

Clustered, Regularly Interspaced Short Palindromic Repeats and their associated Cas proteins (CRISPR-Cas) provide prokaryotes with a mechanism for defense against mobile genetic elements (MGEs). A CRISPR locus is a molecular memory of MGE encounters. It contains an array of short sequences, called spacers, that generally have sequence identity to MGEs. Three different CRISPR loci have been identified among strains of the opportunistic pathogen Enterococcus faecalis. CRISPR1 and CRISPR3 are associated with the cas genes necessary for blocking MGEs, but these loci are present in only a subset of E. faecalis strains. The orphan CRISPR2 lacks cas genes and is ubiquitous in E. faecalis, although its spacer content varies from strain to strain. Because CRISPR2 is a variable locus occurring in all E. faecalis, comparative analysis of CRISPR2 sequences may provide information about the clonality of E. faecalis strains. We examined CRISPR2 sequences from 228 E. faecalis genomes in relationship to subspecies phylogenetic lineages (sequence types; STs) determined by multilocus sequence typing (MLST), and to a genome phylogeny generated for a representative 71 genomes. We found that specific CRISPR2 sequences are associated with specific STs and with specific branches on the genome tree. To explore possible applications of CRISPR2 analysis, we evaluated 14 E. faecalis bloodstream isolates using CRISPR2 analysis and MLST. CRISPR2 analysis identified two groups of clonal strains among the 14 isolates, an assessment that was confirmed by MLST. CRISPR2 analysis was also used to accurately predict the ST of a subset of isolates. We conclude that CRISPR2 analysis, while not a replacement for MLST, is an inexpensive method to assess clonality among E. faecalis isolates, and can be used in conjunction with MLST to identify recombination events occurring between STs.     

Flexibility of KorA,a plasmid-encoded,global transcription regulator,in the presence and the absence of its operator

The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53.     

Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events

Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked beta1-->3 to a beta-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here.     

Resveratrol induces catalytic bioscavenger paraoxonase 1 expression and protects against chemical warfare nerve agent toxicity in human cell lines

Current advances in enzyme bioscavenger prophylactic therapy against chemical warfare nerve agent (CWNA) exposure are moving towards the identification of catalytic bioscavengers that can degrade large doses of organophosphate (OP) nerve agents without self destruction. This is a preferred method compared to therapy with the purified stoichiometric bioscavenger, butyrylcholinesterase, which binds OPs 1:1 and would thus require larger doses for treatment. Paraoxonase-1 (PON-1) is one such catalytic bioscavenger that has been shown to hydrolyze OP insecticides and contribute to detoxification in animals and humans. Here we investigated the effects of a common red wine ingredient, Resveratrol (RSV), to induce the expression of PON-1 in the human hepatic cell line HC04 and evaluated the protection against CWNA simulants. Dose-response curves showed that a concentration of 20 microM RSV was optimal in inducing PON-1 expression in HC04 cells. RSV at 20 microM increased the extracellular PON-1 activity approximately 150% without significantly affecting the cells. Higher doses of RSV were cytotoxic to the cells. Resveratrol also induced PON-1 in the human lung cell line A549. RSV pre-treatment significantly (P = 0.05) protected the hepatic cells against exposure to 2x LD(50) of soman and sarin simulants. However, lung cells were protected against soman simulant exposure but not against sarin simulant exposure following RSV treatment. In conclusion, these studies indicate that dietary inducers, such as RSV, can up-regulate PON-1, a catalytic bioscavenger, which can then hydrolyze and protect against CWNA-induced toxicity, providing a prospective new method to protect against CWNA exposure.     

Using least median of squares for structural superposition of flexible proteins

Background

The conventional superposition methods use an ordinary least squares (LS) fit for structural comparison of two different conformations of the same protein. The main problem of the LS fit that it is sensitive to outliers, i.e. large displacements of the original structures superimposed.

Results

To overcome this problem, we present a new algorithm to overlap two protein conformations by their atomic coordinates using a robust statistics technique: least median of squares (LMS). In order to effectively approximate the LMS optimization, the forward search technique is utilized. Our algorithm can automatically detect and superimpose the rigid core regions of two conformations with small or large displacements. In contrast, most existing superposition techniques strongly depend on the initial LS estimating for the entire atom sets of proteins. They may fail on structural superposition of two conformations with large displacements. The presented LMS fit can be considered as an alternative and complementary tool for structural superposition.

Conclusion

The proposed algorithm is robust and does not require any prior knowledge of the flexible regions. Furthermore, we show that the LMS fit can be extended to multiple level superposition between two conformations with several rigid domains. Our fit tool has produced successful superpositions when applied to proteins for which two conformations are known. The binary executable program for Windows platform, tested examples, and database are available from https://engineering.purdue.edu/PRECISE/LMSfit.     

Receptor Specificity and Transmission of H2N2 Subtype Viruses Isolated from the Pandemic of 1957

Influenza viruses of the H2N2 subtype have not circulated among humans in over 40 years. The occasional isolation of avian H2 strains from swine and avian species coupled with waning population immunity to H2 hemagglutinin (HA) warrants investigation of this subtype due to its pandemic potential. In this study we examined the transmissibility of representative human H2N2 viruses, A/Albany/6/58 (Alb/58) and A/El Salvador/2/57 (ElSalv/57), isolated during the 1957/58 pandemic, in the ferret model. The receptor binding properties of these H2N2 viruses was analyzed using dose-dependent direct glycan array-binding assays. Alb/58 virus, which contains the 226L/228S amino acid combination in the HA and displayed dual binding to both alpha 2,6 and alpha 2,3 glycan receptors, transmitted efficiently to naïve ferrets by respiratory droplets. Inefficient transmission was observed with ElSalv/57 virus, which contains the 226Q/228G amino acid combination and preferentially binds alpha 2,3 over alpha 2,6 glycan receptors. However, a unique transmission event with the ElSalv/57 virus occurred which produced a 226L/228G H2N2 natural variant virus that displayed an increase in binding specificity to alpha 2,6 glycan receptors and enhanced respiratory droplet transmissibility. Our studies provide a correlation between binding affinity to glycan receptors with terminal alpha 2,6-linked sialic acid and the efficiency of respiratory droplet transmission for pandemic H2N2 influenza viruses.