Vijay Gupta

Vacuolating cytotoxin and variants in Atg16L1 that disrupt autophagy promote helicobacter pylori infection in humans

BACKGROUND & AIMS The Helicobacter pylori toxin vacuolating cytotoxin (VacA) promotes gastric colonization, and its presence (VacA(+)) is associated with more-severe disease. The exact mechanisms by which VacA contributes to infection are unclear. We previously found that limited exposure to VacA induces autophagy of gastric cells, which eliminates the toxin; we investigated whether autophagy serves as a defense mechanism against H pylori infection. METHODS We investigated the effect of VacA on autophagy in human gastric epithelial cells and primary gastric cells from mice. Expression of p62, a marker of autophagy, was also assessed in gastric tissues from patients infected with toxigenic (VacA(+)) or nontoxigenic strains. We analyzed the effect of VacA on autophagy in peripheral blood monocytes obtained from subjects with different genotypes of ATG16L1, which regulates autophagy. We performed genotyping for ATG16L1 in 2 cohorts of infected and uninfected subjects. RESULTS Prolonged exposure of human gastric epithelial cells and mouse gastric cells to VacA disrupted induction of autophagy in response to the toxin, because the cells lacked cathepsin D in autophagosomes. Loss of autophagy resulted in the accumulation of p62 and reactive oxygen species. Gastric biopsy samples from patients infected with VacA(+), but not nontoxigenic strains of H pylori, had increased levels of p62. Peripheral blood monocytes isolated from individuals with polymorphisms in ATG16L1 that increase susceptibility to Crohns disease had reduced induction of autophagy in response to VacA(+) compared to cells from individuals that did not have these polymorphisms. The presence of the ATG16L1 Crohns disease risk variant increased susceptibility to H pylori infection in 2 separate cohorts. CONCLUSIONS Autophagy protects against infection with H pylori; the toxin VacA disrupts autophagy to promote infection, which could contribute to inflammation and eventual carcinogenesis.

Sphingomyelin Functions as a Novel Receptor for Helicobacter pylori VacA

The vacuolating cytotoxin (VacA) of the gastric pathogen Helicobacter pylori binds and enters epithelial cells, ultimately resulting in cellular vacuolation. Several host factors have been reported to be important for VacA function, but none of these have been demonstrated to be essential for toxin binding to the plasma membrane. Thus, the identity of cell surface receptors critical for both toxin binding and function has remained elusive. Here, we identify VacA as the first bacterial virulence factor that exploits the important plasma membrane sphingolipid, sphingomyelin (SM), as a cellular receptor. Depletion of plasma membrane SM with sphingomyelinase inhibited VacA-mediated vacuolation and significantly reduced the sensitivity of HeLa cells, as well as several other cell lines, to VacA. Further analysis revealed that SM is critical for VacA interactions with the plasma membrane. Restoring plasma membrane SM in cells previously depleted of SM was sufficient to rescue both toxin vacuolation activity and plasma membrane binding. VacA association with detergent-resistant membranes was inhibited in cells pretreated with SMase C, indicating the importance of SM for VacA association with lipid raft microdomains. Finally, VacA bound to SM in an in vitro ELISA assay in a manner competitively inhibited by lysenin, a known SM-binding protein. Our results suggest a model where VacA may exploit the capacity of SM to preferentially partition into lipid rafts in order to access the raft-associated cellular machinery previously shown to be required for toxin entry into host cells.

Molecular silicate and aluminate species in anhydrous and hydrated cements.

The compositions and molecular structures of anhydrous and hydrated cements are established by using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy methods to distinguish among different molecular species and changes that occur as a result of cement hydration and setting. One- and two-dimensional (2D) solid-state (29)Si and (27)Al magic-angle spinning NMR methodologies, including T(1)-relaxation-time- and chemical-shift-anisotropy-filtered measurements and the use of very high magnetic fields (19 T), allow resonances from different silicate and aluminate moieties to be resolved and assigned in complicated spectra. Single-pulse (29)Si and (27)Al NMR spectra are correlated with X-ray fluorescence results to quantify the different crystalline and disordered silicate and aluminate species in anhydrous and hydrated cements. 2D (29)Si{(1)H} and (27)Al{(1)H} heteronuclear correlation NMR spectra of hydrated cements establish interactions between water and hydroxyl moieties with distinct (27)Al and (29)Si species. The use of a (29)Si T(1)-filter allows anhydrous and hydrated silicate species associated with iron-containing components in the cements to be distinguished, showing that they segregate from calcium silicate and aluminate components during hydration. The different compositions of white Portland and gray oilwell cements are shown to have distinct molecular characteristics that are correlated with their hydration behaviors.

Sphingomyelin is important for the cellular entry and intracellular localization of Helicobacter pylori VacA.

Plasma membrane sphingomyelin (SM) binds the Helicobacter pylori vacuolating toxin (VacA) to the surface of epithelial cells. To evaluate the importance of SM for VacA cellular entry, we characterized toxin uptake and trafficking within cells enriched with synthetic variants of SM, whose intracellular trafficking properties are strictly dependent on the acyl chain lengths of their sphingolipid backbones. While toxin binding to the surface of cells was independent of acyl chain length, cells enriched with 12‐ or 18‐carbon acyl chain variants of SM (e.g. C12‐SM or C18‐SM) were more sensitive to VacA, as indicated by toxin‐induced cellular vacuolation, than those enriched with shorter 2‐ or 6‐carbon variants (e.g. C2‐SM or C6‐SM). In C18‐SM‐enriched cells, VacA was taken into cells by a previously described Cdc42‐dependent pinocytic mechanism, localized initially to GPI‐enriched vesicles, and ultimately trafficked to Rab7/Lamp1 compartments. In contrast, within C2‐SM‐enriched cells, VacA was taken up at a slower rate by a Cdc42‐independent mechanism and trafficked to Rab11 compartments. VacA‐associated predominantly with detergent‐resistant membranes (DRMs) in cells enriched with C18‐SM, but predominantly with non‐DRMs in C2‐SM‐enriched cells. These results suggest that SM is required for targeting VacA to membrane rafts important for subsequent Cdc42‐dependent pinocytic cellular entry.

Internet search engine freshness by Web server help

We study how to keep the Internet search engines up-to-date with the changes occurring at the various Web servers in the Internet. Currently, Web search engines poll the Web servers on a per-URL basis for obtaining update information. We advocate an approach in which Web servers themselves track the changes happening to their content files for propagating updates to search engines. We propose an algorithm which uses both freshness and popularity of data at the Web servers for deciding the discrepancy between a Web site and a search engine. This algorithm batches the push of updates from the Web server to the search engine. We prove that this algorithm is competitive with an optimal algorithm.

Reactions and surface interactions of saccharides in cement slurries.

Glucose, maltodextrin, and sucrose exhibit significant differences in their alkaline reaction properties and interactions in aluminate/silicate cement slurries that result in diverse hydration behaviors of cements. Using 1D solution- and solid-state (13)C nuclear magnetic resonance (NMR), the structures of these closely related saccharides are identified in aqueous cement slurry solutions and as adsorbed on inorganic oxide cement surfaces during the early stages of hydration. Solid-state 1D (29)Si and 2D (27)Al{(1)H} and (13)C{(1)H} NMR techniques, including the use of very high magnetic fields (18.8 T), allow the characterization of the hydrating silicate and aluminate surfaces, where interactions with adsorbed organic species influence hydration. These measurements establish the molecular features of the different saccharides that account for their different adsorption behaviors in hydrating cements. Specifically, sucrose is stable in alkaline cement slurries and exhibits selective adsorption at hydrating silicate surfaces but not at aluminate surfaces in cements. In contrast, glucose degrades into linear saccharinic or other carboxylic acids that adsorb relatively weakly and nonselectively on nonhydrated and hydrated cement particle surfaces. Maltodextrin exhibits intermediate reaction and sorption properties because of its oligomeric glucosidic structure that yields linear carboxylic acids and stable ring-containing degradation products that are similar to those of the glucose degradation products and sucrose, respectively. Such different reaction and adsorption behaviors provide insight into the factors responsible for the large differences in the rates at which aluminate and silicate cement species hydrate in the presence of otherwise closely related saccharides.

Origins of saccharide-dependent hydration at aluminate, silicate, and aluminosilicate surfaces

Sugar molecules adsorbed at hydrated inorganic oxide surfaces occur ubiquitously in nature and in technologically important materials and processes, including marine biomineralization, cement hydration, corrosion inhibition, bioadhesion, and bone resorption. Among these examples, surprisingly diverse hydration behaviors are observed for oxides in the presence of saccharides with closely related compositions and structures. Glucose, sucrose, and maltodextrin, for example, exhibit significant differences in their adsorption selectivities and alkaline reaction properties on hydrating aluminate, silicate, and aluminosilicate surfaces that are shown to be due to the molecular architectures of the saccharides. Solid-state 1H, 13C, 29Si, and 27Al nuclear magnetic resonance (NMR) spectroscopy measurements, including at very high magnetic fields (19 T), distinguish and quantify the different molecular species, their chemical transformations, and their site-specific adsorption on different aluminate and silicate moieties. Two-dimensional NMR results establish nonselective adsorption of glucose degradation products containing carboxylic acids on both hydrated silicates and aluminates. In contrast, sucrose adsorbs intact at hydrated silicate sites and selectively at anhydrous, but not hydrated, aluminate moieties. Quantitative surface force measurements establish that sucrose adsorbs strongly as multilayers on hydrated aluminosilicate surfaces. The molecular structures and physicochemical properties of the saccharides and their degradation species correlate well with their adsorption behaviors. The results explain the dramatically different effects that small amounts of different types of sugars have on the rates at which aluminate, silicate, and aluminosilicate species hydrate, with important implications for diverse materials and applications.

Activation of the abundant nuclear factor poly(ADP-ribose) polymerase-1 by Helicobacter pylori

Modification of eukaryotic proteins is a powerful strategy used by pathogenic bacteria to modulate host cells during infection. Previously, we demonstrated that Helicobacter pylori modify an unidentified protein within mammalian cell lysates in a manner consistent with the action of a bacterial ADP-ribosylating toxin. Here, we identified the modified eukaryotic factor as the abundant nuclear factor poly(ADP-ribose) polymerase-1 (PARP-1), which is important in the pathologies of several disease states typically associated with chronic H. pylori infection. However, rather than being ADP-ribosylated by an H. pylori toxin, the intrinsic poly(ADP-ribosyl) polymerase activity of PARP-1 is activated by a heat- and protease-sensitive H. pylori factor, resulting in automodification of PARP-1 with polymers of poly(ADP-ribose) (PAR). Moreover, during infection of gastric epithelial cells, H. pylori induce intracellular PAR-production by a PARP-1-dependent mechanism. Activation of PARP-1 by a pathogenic bacterium represents a previously unrecognized strategy for modulating host cell signaling during infection.

A disstributed backoff algorithm to support real-time traffic on ethernet

Ethernets are very popular in LANs because of their ease of administration. With the growing trend towards distributed multimedia applications, it has become essential for the underlying systems to provide resource guarantees. Traditionally, Ethernet uses the Truncated Binary Exponential Backoff (BEB) algorithm for medium access. BEB has very good characteristics for reducing the average latency of packets, but is not designed to support low delay jitter. We propose an IEEE 802.3-compliant distributed backoff algorithm which gives good bounds on the delay jitter and average delay for the transmission of real-time traffic over shared 100 Mbit/sec Ethernet medium. Simulation results show that for a mixture of data and constant bit rate (CBR) video traffic, our backoff algorithm has an order of magnitude less delay jitter than BEB for offered loads below 76%. The delay jitter of our algorithm is around 4 times lower than BEB for simulation of a mixture of data and variable bit rate (VBR) video traffic.

Reliable sender-initiated multicast for improved QoS

Network support for reliable sender-initiated multicast can aid software distribution, server-pushing of web-pages as well as pushing of prerecorded audio and video. Currently, bandwidth availability and propagation delays between the sender and the different recipients differ by many orders of magnitude. Hence, an important problem which needs to be addressed for facilitating reliable sender-initiated multicast is this problem of network heterogeneity. Prior work in reliable multicast either presents solutions to transmit at the rate of the bottleneck link of the entire multicast tree, or assumes knowledge of static bandwidth to each of the recipients. We propose an algorithm which does not make these assumptions but partitions the set of recipients on the basis of the available bandwidths at the time when the multicast is started; it then transmits separately to each set of recipients sharing a common quality of service (QoS). To the best of our knowledge, this is a first such solution. To achieve these goals, we propose (i) an algorithm to divide the set of receivers into classes with similar QoS, and (ii) callbacks for error recovery in reliable multicast. Our algorithm makes use of L4 switching at the routers but assumes the state at the routers to be soft state. Using analysis, we show our algorithm to be scalable for certain restricted network characteristics.