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Time is the crucial factor in the "chain of survival" treatment concept for out-of-hospital cardiac arrest (OHCA). We aimed to measure different response time intervals by comparing emergency medical system (EMS), fire fighters and smartphone aided volunteer responders.In two large Swedish regions, volunteer responders were timed from the alert until they arrived at the scene of the suspected OHCA. The first arriving volunteer responders who tried to fetch an automated external defibrillator (AED-responder) and who ran to perform bystander cardiopulmonary resuscitation (CPR-responder) were compared to both the first arriving EMS and fire fighters. Three-time intervals were measured, from call to dispatch, the unit response time (from dispatch to arrival) and the total response time.During 22 months, 2631 suspected OHCAs were included. The median time from call to dispatch was in minutes 1.8 (95% CI = 1.7-1.8) for EMS, 2.9 (95% CI = 2.8-3.0) for fire-fighters and 3.0 (95% CI = 2.9-3.1) for volunteer responders. The median unit response time was 8.3 (95% CI = 8.1-8.5) for EMS, 6.8 (95% CI = 6.7-6.9) for fire fighters and 6.0 (95% CI = 5.7-6.2) for AED-responders and 4.6 (95% CI = 4.5-4.8) for CPR-responders. The total response time was 10.4 (95% CI = 10.1-10.6) for EMS, 10.2 (95% CI = 9.9-10.4) for fire fighters, 9.6 (95% CI = 9.1-9.8) for AED-responders and 8.2 (95% CI = 8.0-8.3) for CPR-responders.First arriving volunteer responders had the shortest unit response time when compared to both fire fighters and EMS, however this advantage was reduced by delays introduced at the dispatch center. Earlier automatic dispatch should be considered in further studies.

Supplementary Figure Legends

Supplemental Figure 2. A. Quantitative real time RT-PCR was used to determine expression levels of NOD1 in AGS cells stably transfected with a non-targeting shRNA or NOD1-specific shRNA. Three different single cell colonies (col) transfected with each shRNA were tested. Data are represented as fold NOD1 mRNA expression relative to the mean expression level in Non-targeting control cells. B. Quantitative real time RT-PCR was used to determine expression levels of TRAF3 in AGS cells transiently transfected with either non-targeting shRNA or TRAF3-specific shRNA. Bars represent the mean{plus minus}SEM of TRAF3 transcripts in cell transfected with TRAF3-specific shRNA relative to the mean expression level in cells transfected with non-targeting shRNA. Data are represented as mean{plus minus}SEM from 2 independent experiments.

Supplemental Methods

Helicobacter pylori (H. pylori) is the strongest known risk factor for gastric carcinogenesis. One cancer-linked locus is the cag pathogenicity island, which translocates components of peptidoglycan into host cells. NOD1 is an intracellular immune receptor that senses peptidoglycan from Gram-negative bacteria and responds by inducing autophagy and activating NF-κB, leading to inflammation-mediated bacterial clearance; however chronic pathogens can evade NOD1-mediated clearance by altering peptidoglycan structure. We previously demonstrated that the H. pylori cag+ strain 7.13 rapidly induces gastric cancer in Mongolian gerbils. Using 2D-DIGE and mass spectrometry, we identified a novel mutation within the gene encoding the peptidoglycan deacetylase PgdA; therefore, we sought to define the role of H. pylori PgdA in NOD1-dependent activation of NF-κB, inflammation, and cancer. Coculture of H. pylori strain 7.13 or its pgdA− isogenic mutant with AGS gastric epithelial cells or HEK293 epithelial cells expressing a NF-κB reporter revealed that pgdA inactivation significantly decreased NOD1-dependent NF-κB activation and autophagy. Infection of Mongolian gerbils with an H. pylori pgdA− mutant strain led to significantly decreased levels of inflammation and malignant lesions in the stomach; however, preactivation of NOD1 before bacterial challenge reciprocally suppressed inflammation and cancer in response to wild-type H. pylori. Expression of NOD1 differs in human gastric cancer specimens compared with noncancer samples harvested from the same patients. These results indicate that peptidoglycan deacetylation plays an important role in modulating host inflammatory responses to H. pylori, allowing the bacteria to persist and induce carcinogenic consequences in the gastric niche. Cancer Res; 75(8); 1749–59. ©2015 AACR.

Supplemental Figure 3. Cell viability as determined by MTT assays of HEK293 cells transfected with empty vector (Control) or a NOD1-expression construct (pUNO1-hNOD1, Invivogen). Cells were challenged with C12-iE-DAP, PMA, or wild type H. pylori strain 7.13 at MOIs of 10 and 50 for 4 hours. Data are represented as mean{plus minus}SEM from 2 independent experiments.

Background Early defibrillation is critical for the chance of survival in in out-of-hospital cardiac arrest (OHCA). Drones, used to deliver automated external defibrillators (AEDs), may shorten time to defibrillation, but this has never been evaluated in real-life emergencies. Purpose The aim of this study was to investigate the feasibility of AED-delivery by drones in real-life cases of OHCA. Methods In this prospective clinical trial, three AED-equipped drones were placed within controlled airspace in Sweden, covering approximately 80,000 inhabitants (125km2). Drones were integrated in the emergency medical services for automated deployment in beyond-visual-line-of-sight flights in; a) consecutive real-life suspected OHCAs b) test-flights from 06–01–20 to 09–30–20. Primary outcome was the proportion of successful AED-deliveries when drones were dispatched in cases of suspected OHCA. Among secondary outcomes were the proportion of cases where AED-drones arrived prior to ambulance and time benefit vs. ambulance. Results Totally 14 cases were eligible for dispatch during the study period in which AED-drones took off in 12 alerts to suspected OHCA; with a median distance to location 3,1 km (IQR:2,8–3,4). AED-delivery was feasible within 9 meters (IQR:7,5–10,5) from the location and successful in 11 alerts, 92%. AED-drones arrived prior to ambulances in 64%, with a median time benefit of 01:52 minutes (IQR:01:35–04:54). In an additional 61 test-flights the AED-delivery success rate was 90% (55/61). Conclusion In this pilot study, we have shown that AED-delivery by drones in real-life cases of OHCA is feasible with a substantial time benefit and a successful delivery rate of 92%. Further technological improvements are needed to increase dispatch rate and time gains. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish heart-lung foundation

The exopolysaccharide (EPS) produced by probiotic Leuconostoc mesenteroides subsp. mesenteroides strain NTM048 has been reported to be an immunostimulant that enhances mucosal IgA production. In this study, we found that intranasal administration of mice with the EPS and an antigen (ovalbumin) resulted in secretion of antigen-specific IgA and IgG in the airway mucosa and the serum, suggesting that the EPS has the adjuvant activity for use with mucosal vaccination. Methylation analysis coupled to GC-MS, and 1D and 2D NMR spectroscopy revealed that 94% of the EPS consists of an α-(1 → 6) glucan containing 4% of 1→3-linked α-glucose branches. To determine structures of minor components, we enzymatically digested the glucan with dextranase and used 2D NMR spectroscopy to identify the remaining polymer as a fructan (or fructans), containing both β-(2 → 6)- and β-(2 → 1)-linked fructofuranose residues. These residues may either enter into separate polymers of each linkage type or form a mixed fructan containing both linkage types.

Move Over, TLR4 The innate immune system senses bacterial lipopolysaccharide (LPS) through Toll-like receptor 4 (TLR4) (see the Perspective by Kagan ). However, Kayagaki et al. (p 1246 , published online 25 July) and Hagar et al. (p. 1250 ) report that the hexa-acyl lipid A component of LPS from Gramnegative bacteria is able to access the cytoplasm and activate caspase-11 to signal immune responses independently of TLR4. Mice that lack caspase-11 are resistant to LPS-induced lethality, even in the presence of TLR4.

Streptococcus pyogenes (Group A Streptococcus or GAS) is a hemolytic human pathogen associated with a wide variety of infections ranging from minor skin and throat infections to life-threatening invasive diseases. The cell wall of GAS consists of peptidoglycan sacculus decorated with a carbohydrate comprising a polyrhamnose backbone with immunodominant N-acetylglucosamine side-chains. All GAS genomes contain the spyBA operon, which encodes a 35-amino-acid membrane protein SpyB, and a membrane-bound C3-like ADP-ribosyltransferase SpyA. In this study, we addressed the function of SpyB in GAS. Phenotypic analysis of a spyB deletion mutant revealed increased bacterial aggregation, and reduced sensitivity to β-lactams of the cephalosporin class and peptidoglycan hydrolase PlyC. Glycosyl composition analysis of cell wall isolated from the spyB mutant suggested an altered carbohydrate structure compared with the wild-type strain. Furthermore, we found that SpyB associates with heme and protoporphyrin IX. Heme binding induces SpyB dimerization, which involves disulfide bond formation between the subunits. Thus, our data suggest the possibility that SpyB activity is regulated by heme.

Recent advances in computer visualization have allowed us to develop new tools for analyzing the data gathered during planetary missions, which is important, since these data sets have grown exponentially in recent years to tens of terabytes in size. As part of the Advanced Visualization in Solar System Exploration and Research (ADVISER) project, we utilize several advanced visualization techniques created specifically with planetary image data in mind. The Geoviewer application allows real-time active stereo display of images, which in aggregate have billions of pixels. The ADVISER desktop application platform allows fast three-dimensional visualization of planetary images overlain on digital terrain models. Both applications include tools for easy data ingest and real-time analysis in a programmatic manner. Incorporation of these tools into our everyday scientific workflow has proved important for scientific analysis, discussion, and publication, and enabled effective and exciting educational activities for students from high school through graduate school.

In a user study comparing four visualization methods for three-dimensional vector data, participants used visualizations from each method to perform five simple but representative tasks: 1) determining whether a given point was a critical point, 2) determining the type of a critical point, 3) determining whether an integral curve would advect through two points, 4) determining whether swirling movement is present at a point, and 5) determining whether the vector field is moving faster at one point than another. The visualization methods were line and tube representations of integral curves with both monoscopic and stereoscopic viewing. While participants reported a preference for stereo lines, quantitative results showed performance among the tasks varied by method. Users performed all tasks better with methods that: 1) gave a clear representation with no perceived occlusion, 2) clearly visualized curve speed and direction information, and 3) provided fewer rich 3D cues (e.g., shading, polygonal arrows, overlap cues, and surface textures). These results provide quantitative support for anecdotal evidence on visualization methods. The tasks and testing framework also give a basis for comparing other visualization methods, for creating more effective methods, and for defining additional tasks to explore further the tradeoffs among the methods.

The current work reports protein extraction from Spirulina platensis cyanobacterial biomass in order to simultaneously generate a potential co-product and a biofuel feedstock with reduced nitrogen content. S. platensis cells were subjected to cell disruption by high pressure homogenization and subsequent protein isolation by solubilisation at alkaline pH followed by precipitation at acidic pH. Response surface methodology (RSM) was used to optimize the process parameters - pH, extraction (solubilisation/precipitation) time and biomass concentration for obtaining maximum protein yield. The optimized process conditions were found to be pH 11.38, solubilisation time of 35 min and biomass concentration of 3.6 % (w/w) solids for the solubilisation step, and pH 4.01 and precipitation time of 60 min for the precipitation step. At the optimized conditions, a high protein yield of 60.7 % (w/w) was obtained. The protein isolate (co-product) had a higher protein content (80.6 % (w/w)), lower ash (1.9 % (w/w)) and mineral content and was enriched in essential amino acids, the nutritious γ-lenolenic acid and other high-value unsaturated fatty acids compared to the original biomass. The residual biomass obtained after protein extraction had lower nitrogen content and higher total non-protein content than the original biomass. The loss of about 50 % of the total lipids from this fraction did not impact its composition significantly owing to the low lipid content of S.platensis (8.03 %).

Legumes establish mutualistic associations with nitrogen-fixing bacteria, commonly referred to as rhizobia. Most rhizobia enter roots of host plants via root hairs, where they induce the formation of infection threads. Bacterial invasion and formation of nodules containing nitrogen-fixing bacteroids (the Fix+ phenotype) depend on a molecular dialogue between the symbiotic partners. Host plants secrete flavonoids that induce the synthesis of rhizobial lipo-chito-oligosaccharides called Nod factors (24, 26) and perceive them via a specific signal transduction pathway that culminates in expression of symbiosis-specific plant genes required for nodule development (34). In many symbiotic associations, nodule formation also depends on rhizobial macromolecules that include exopolysaccharides (EPS), lipopolysaccharides, K-antigens, and cyclic β-glucans. These extracellular polysaccharides (or the oligosaccharides derived from them) are crucial in the early stages of nodule formation that begins after rhizobia have entered the host plant (3, 8, 17, 19, 25, 33, 36). In addition to a protective role, rhizobial EPS have vital but poorly understood roles in infection of legume roots (2). The EPS structures of various rhizobia, including Rhizobium sp. strain NGR234 (also called Sinorhizobium fredii NGR234), which nodulates more than 112 genera of legumes (30), have been described. The repeating subunit of the acidic EPS of NGR234 is a nonasaccharide consisting of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues (9) (Fig. ​(Fig.1A).1A). Mutants of NGR234 deficient in EPS synthesis formed nonmucoid (“dry”) colonies on agar plates and lost the capacity to induce the formation of nitrogen-fixing nodules on Leucaena leucocephala (Fix− phenotype). Instead, small empty nodules (pseudonodules) were formed (5). A number of genes of NGR234 involved in synthesis of EPS have been identified in an exo cluster (6, 15, 45), which has been sequenced (38) (Fig. ​(Fig.1B).1B). This cluster is located on the pNGR234b megaplasmid and is similar to the exo cluster of Sinorhizobium meliloti strain 1021 that is required for synthesis of succinoglycan (also called EPS I) (13). Based on known or assumed functions of exo genes in S. meliloti (2, 32), we predict that EPS synthesis in NGR234 depends on the glycosyl transferases (ExoY, ExoA, ExoL, ExoM, ExoO, and ExoU) that form an undecaprenol diphosphate-linked repeating subunit at the cytoplasmic face of the inner membrane. Probably, the lipid-linked subunits are then flipped across the inner membrane and finally assembled by an ExoQ/ExoP/ExoF-dependent polymerization system that is similar to the Wzy polymerization pathway of group 1 capsular polysaccharides in Escherichia coli (41). In this model, ExoQ is a putative polymerase and ExoP is a copolymerase belonging to the MPA1 (cytoplasmic membrane-periplasmic auxiliary protein 1) family. ExoF is probably an outer membrane auxiliary protein required for EPS export. Related genes required for secretion and export of EPS (pssL, pssT, pssP, and pssN) have been identified in Rhizobium leguminosarum bv. trifolii (reference 22 and references therein). FIG. 1. (A) Repeating subunit of the acidic EPS from NGR234 (9). Additional acetyl groups are present at unidentified positions. (B) Genetic map of the exo cluster of pNGR234b (38). The exs genes and neighboring exoB gene are not shown. BamHI fragments were cloned ... Low-molecular-weight (LMW) forms of EPS (called exo-oligosaccharides [EOSs]) have been identified in supernatants of rhizobial cultures. Nodulation experiments with purified LMW EPS applied to roots indicated that EOSs could complement the symbiotic defects of exo mutants under certain growth conditions (1, 10, 14, 40). Similar complementation effects were observed when exo mutants were coinoculated with noninvasive strains that synthesize EPS (17, 18). Although not always conclusive, the results of such complementation experiments suggest that EOSs are symbiotically active molecules in certain plants. Synthesis of EOSs seems to be controlled either by enzymes involved in export and polymerization of the lipid-linked repeating EPS subunit or by extracellular glycanases that release EOSs from high-molecular-weight (HMW) forms (14). Glycanases that cleave EPS have been identified in S. meliloti and R. leguminosarum bv. viciae. Corresponding mutants produced lower levels of EOSs without altering the symbiotic phenotype (12, 43). The previously characterized glycanase ExoK secreted by S. meliloti (43, 44) exhibits a high level of sequence similarity [e−129] to the putative ExoK protein of NGR234. In this study, we mutated exo genes of NGR234 involved in synthesis of the repeating subunit (exoL, exoY, and exoZ), polymerization (exoF, exoP, and exoQ), and production of EOSs (exoK). We provide evidence that EOSs of NGR234 are required for symbiosis with certain plants.

Particle Flurries is an interactive approach to 3D flow visualization. The approach produces a "synoptic visualization" and is used to examine both internal and external flows.

A broad-host-range endosymbiont, Sinorhizobium sp. NGR234 is a component of several legume-symbiont model systems; however, there is little structural information on the cell surface glycoconjugates. NGR234 cells in free-living culture produce a major rough lipopolysaccharide (LPS, lacking O-chain) and a minor smooth LPS (containing O-chain), and the structure of the lipid A components was investigated by chemical analyses, mass spectrometry, and NMR spectroscopy of the underivatized lipids A. The lipid A from rough LPS is heterogeneous and consists of six major bisphosphorylated species that differ in acylation. Pentaacyl species (52%) are acylated at positions 2, 3, 2', and 3', and tetraacyl species (46%) lack an acyl group at C-3 of the proximal glucosamine. In contrast to Rhizobium etli and Rhizobium leguminosarum, the NGR234 lipid A contains a bisphosphorylated beta-(1' --> 6)-glucosamine disaccharide, typical of enterobacterial lipid A. However, NGR234 lipid A retains the unusual acylation pattern of R. etli lipid A, including the presence of a distal, amide-linked acyloxyacyl residue containing a long chain fatty acid (LCFA) (e.g. 29-hydroxytriacontanoate) attached as the secondary fatty acid. As in R. etli, a 4-carbon fatty acid, beta-hydroxybutyrate, is esterified to (omega - 1) of the LCFA forming an acyloxyacyl residue at that location. The NGR234 lipid A lacks all other ester-linked acyloxyacyl residues and shows extensive heterogeneity of the amide-linked fatty acids. The N-acyl heterogeneity, including unsaturation, is localized mainly to the proximal glucosamine. The lipid A from smooth LPS contains unique triacyl species (20%) that lack ester-linked fatty acids but retain bisphosphorylation and the LCFA-acyloxyacyl moiety. The unusual structural features shared with R. etli/R. leguminosarum lipid A may be essential for symbiosis.

Bacillus anthracis (Ba) and human infection-associated Bacillus cereus (Bc) strains Bc G9241 and Bc 03BB87 have secondary cell wall polysaccharides (SCWPs) comprising an aminoglycosyl trisaccharide repeat: →4)-β-d-ManpNAc-(1→4)-β-d-GlcpNAc-(1→6)-α-d-GlcpNAc-(1→, substituted at GlcNAc residues with both α- and β-Galp. In Bc G9241 and Bc 03BB87, an additional α-Galp is attached to O-3 of ManNAc. Using NMR spectroscopy, mass spectrometry and immunochemical methods, we compared these structures to SCWPs from Bc biovar anthracis strains isolated from great apes displaying “anthrax-like” symptoms in Cameroon (Bc CA) and Côte d’Ivoire (Bc CI). The SCWPs of Bc CA/CI contained the identical HexNAc trisaccharide backbone and Gal modifications found in Ba, together with the α-Gal-(1→3) substitution observed previously at ManNAc residues only in Bc G9241/03BB87. Interestingly, the great ape derived strains displayed a unique α-Gal-(1→3)-α-Gal-(1→3) disaccharide substitution at some ManNAc residues, a modification not found in any previously examined Ba or Bc strain. Immuno-analysis with specific polyclonal anti-Ba SCWP antiserum demonstrated a reactivity hierarchy: high reactivity with SCWPs from Ba 7702 and Ba Sterne 34F2, and Bc G9241 and Bc 03BB87; intermediate reactivity with SCWPs from Bc CI/CA; and low reactivity with the SCWPs from structurally distinct Ba CDC684 (a unique strain producing an SCWP lacking all Gal substitutions) and non-infection-associated Bc ATCC10987 and Bc 14579 SCWPs. Ba-specific monoclonal antibody EAII-6G6-2-3 demonstrated a 10–20 fold reduced reactivity to Bc G9241 and Bc 03BB87 SCWPs compared to Ba 7702/34F2, and low/undetectable reactivity to SCWPs from Bc CI, Bc CA, Ba CDC684, and non-infection-associated Bc strains. Our data indicate that the HexNAc motif is conserved among infection-associated Ba and Bc isolates (regardless of human or great ape origin), and that the number, positions and structures of Gal substitutions confer unique antigenic properties. The conservation of this structural motif could open a new diagnostic route in detection of pathogenic Bc strains.

Short-time exposure to swine dust causes an intense inflammation of upper and lower airways and induces increased bronchial responsiveness to methacholine in previously non-exposed healthy volunteers. The objective to this study was to investigate the nasal inflammatory response and mucosal reactivity to swine dust exposure and whether nitric oxide metabolism is involved in the inflammatory process. Nitric oxide in expired air, nasal histamine test (NH), nasal lavage (NAL) and bronchial histamine challenges were studied before and after a 3 h exposure to swine dust in a swine confinement building in 17 non-smoking healthy subjects not previously exposed to farm dust. To detect any interference between NAL and NH, the subjects were divided into two groups: in group 1, NAL was performed after NH and in group 2, NAL preceded NH. Nasal histamine response increased significantly in group 1, but not in group 2 ( P =0·012). Albumin levels in NAL were higher before as well as after dust exposure in group 1 compared to group 2 ( P =0·036 and 0·015 respectively). Bronchial histamine responsiveness increased following exposure ( P =0·045). Nitric oxide in expired air decreased following bronchial histamine challenge at baseline ( P =0·013) but was otherwise unaltered. Short-time exposure to swine dust increases non-specific reactivity of both nose and bronchi. Nasal lavage procedure interferes with nasal histamine test when performed with connection to each other. The inflammatory reaction may involve NO metabolism.

Judged by migration of its lipopolysaccharide (LPS) in gel electrophoresis, the O antigen of Rhizobium etli mutant strain CE166 was apparently of normal size. However, its LPS sugar composition and staining of the LPS bands after electrophoresis indicated that the proportion of its LPS molecules that possessed O antigen was only 40% of the wild-type value. Its LPS also differed from the wild type by lacking quinovosamine (2-amino-2,6-dideoxyglucose). Both of these defects were due to a single genetic locus carrying a Tn 5 insertion. The deficiency in O-antigen amount, but not the absence of quinovosamine, was suppressed by transferring into this strain recombinant plasmids that shared a 7.8-kb stretch of the R. etli CE3 lps genetic region α, even though this suppressing DNA did not carry the genetic region mutated in strain CE166. Strain CE166 gave rise to pseudonodules on legume host Phaseolus vulgaris , whereas the mutant suppressed by DNA from lps region α elicited nitrogen-fixing nodules. However, the nodules in the latter case developed slowly and were widely dispersed. Two other R. etli mutants that had one-half or less of the normal amount of O antigen also gave rise to pseudonodules on P. vulgaris . The latter strains were mutated in lps region α and could be restored to normal LPS content and normal symbiosis by complementation with wild-type DNA from this region. Hence, the symbiotic role of LPS requires near-normal abundance of O antigen and may require a structural feature conferred by quinovosamine.

The O-antigenic polysaccharide of theRhizobium etli CE3 lipopolysaccharide (LPS) was structurally characterized using chemical degradations (Smith degradation and β-elimination of uronosyl residues) in combination with alkylation analysis, electrospray, and matrix-assisted laser desorption ionization-time of flight mass spectrometry, tandem mass spectrometry, and 1H COSY and TOCSY nuclear magnetic resonance spectroscopy analyses of the native polysaccharide and the derived oligosaccharides. The polysaccharide was found to be a unique, relatively low molecular weight glycan having a fairly discrete size, with surprisingly little variation in the number of repeating units (degree of polymerization = 5). The polysaccharide isO-acetylated and contains a variety ofO-methylated glycosyl residues, rendering the native glycan somewhat hydrophobic. The molecular mass of the major de-O-acetylated species, including the reducing end 3-deoxy-d-manno-2-octulosonic acid (Kdo) residue, is 3330 Da. The polysaccharide is comprised of a trisaccharide repeating unit having the structure →4)-α-d-GlcpA-(1→4)-[α-3-O-Me-6-deoxy-Talp-(1→3)]-α-l-Fucp-(1→. The nonreducing end of the glycan is terminated with the capping sequence α-2,3,4-tri-O-Me-Fucp-(1→4)-α-d-GlcpA-(1→, and the reducing end of the molecule consists of the non-repeating sequence →3)-α-l-Fucp-(1→3)-β-d-Manp-(1→3)-β-QuiNAcp-(1→4)-α-Kdop-(2→, where QuiNAc is N-acetylquinovosamine (2-N-acetamido-2,6-dideoxyglucose). The reducing end Kdo residue links the O-chain polysaccharide to the core region oligosaccharide, resulting in a unique location for a Kdo residue in LPS, removed four residues distally from the lipid A moiety. Structural heterogeneity in the O-chain arises mainly from theO-acetyl and O-methyl substitution. Methylation analysis using trideuteriomethyl iodide indicates that a portion of the 2,3,4-tri-O-methylfucosyl capping residues, typically 15%, are replaced with 2-O-methyl- and/or 2,3-di-O-methylfucosyl residues. In addition, approximately 25% of the 3,4-linked branching fucosyl residues and 10% of the 3-linked fucosyl residues are 2-O-methylated. A majority of the glucuronosyl residues are methyl-esterified at C-6. These unique structural features may be significant in the infection process.

Immersive virtual reality (IVR) has the potential to be a powerful tool for the visualization of burgeoning scientific data sets and models. We sketch a research agenda for the hardware and software technology underlying IVR for scientific visualization. In contrast to Brooks' (1999) excellent survey which reported on the state of IVR and provided concrete examples of its production use, this article is somewhat speculative. It does not present solutions but rather a progress report, a hope, and a call to action, to help scientists cope with a major crisis that threatens to impede their progress.

BACKGROUND Nitric oxide [NOexp] is present in exhaled air in many species. During experiments on pressure-controlled inverse ratio ventilation (PCIRV) in rabbits, increased [NOexp] was observed during PCIRV. The present study was undertaken to clarify which component of PCIRV increased [NOexp]. METHODS Three groups of six New Zealand White rabbits were anaesthetised and mechanically ventilated. Exhaled nitric oxide, lung mechanics and gas exchange were measured using an experimental protocol designed to assess the effects of variations in 1) flow profile, 2) inspiratory time and 3) time-weighted tidal volume. Ventilator settings used were volume and pressure control ventilation at I:E ratios of 1:2 and 4:1. RESULTS Constant and decelerating flow gave comparable [NOexp] levels (20.0 +/- 6.4 vs. 21.9 +/- 7.7 ppb, n.s.) when time-weighted tidal volume was kept constant. Using conventional (I:E 1:2) or inverted (I:E 4:1) I:E ratios in combination with decelerating flow and constant time-weighted tidal volumes did not alter [NOexp] (26.0 +/- 3.6 vs. 24.0 +/- 5.8 ppb, n.s.). An increased time-weighted tidal volume produced by pressure control with an I:E ratio of 4:1 increased [NOexp] (29.6 +/- 7.4) in comparison to constant (19.3 +/- 4.1, P < 0.05) and decelerating flow ventilation (19.6 +/- 3.6, P < 0.05) with I:E ratios of 1:2. CONCLUSION The exhaled NO concentration was affected by ventilator setting. Increased levels of [NOexp] were observed with increases in time-weighted tidal volume, whereas changes in flow pattern and inspiratory time did not seem to influence airway NO production or release.

The structural arrangement of oligosaccharides comprising the core region of Rhizobium etli CE3 lipopolysaccharide (LPS) has been elucidated through the characterization of the LPSs from two R. etli mutants. One mutant, CE358, completely lacks the O-chain polysaccharide, while the second mutant, CE359, contains a truncated portion of this polysaccharide. This structural arrangement of the core oligosaccharides in these LPSs was determined using electrospray ionization mass spectrometry, tandem mass spectrometry, and methylation analysis. Mild acid hydrolysis of the CE359 LPS produces two major core oligosaccharides: a tetrasaccharide (1) with the structure α-d-Galp-(1→6)-[α-d-GalpA-(1→4)]-α-d-Manp-(1→5)-Kdop(where Kdo represents 3-deoxy-d-manno-2-octulosonic acid) and a trisaccharide (2) having the structure α-d-GalpA-(1→4)-[α-d-GalpA-(1→5)]-Kdop. Structure 1 in CE358 LPS lacks the galacturonosyl residue. Glycosyl linkage and tandem mass spectrometry analyses show that the intact LPS core region consists of trisaccharide (2) attached to O-4 of the Kdo residue in tetrasaccharide1, and that an additional Kdo residue is attached to O-6 of the galactosyl residue of 1. The additional terminally linked Kdo residue is not in close proximity to the lipid A moiety, a unique location for a core Kdo residue. The mutant LPS preparations also contain minor LPS species, one of which lacks the Kdo linked to O-6 of the galactosyl residue, another that lacks the galacturonic acid attached to O-5 of Kdo, and a third that lacks two galacturonosyl residues and one Kdo residue. Thus, in addition to lacking both heptose and phosphate, the R. etliLPS core region differs substantially from the typical enterobacterial cores. The abundance of galacturonosyl residues in the R. etli core might serve as a suitable functional replacement for phosphate, such as would be predicted for Ca2+ binding.

Rhizobium fredii participates in a nitrogen-fixing symbiosis with soybeans, in a strain-cultivar-specific interaction, and past studies have shown that the cell surface and extracellular polysaccharides of rhizobia function in the infection process that leads to symbiosis. The structural analysis of the capsular polysaccharides (K antigens) from strain USDA257 was performed in this study. The K antigens were extracted from cultured cells with hot phenol-water and purified by size exclusion chromatography. We isolated two structurally distinct K antigens, both containing a high proportion of 3-deoxy-D-manno-2-octulosonic acid (Kdo). The polysaccharides were characterized by matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry, nuclear magnetic resonance spectrometry, and gas chromatography-mass spectrometry analyses. The primary polysaccharide, which constituted about 60% of the K-antigen preparation, consisted of repeating units of mannose (Man) and Kdo, [-->)3-beta-D-Manp-(1-->5)-beta-D-Kdop-(2-->], and a second polysaccharide consisted of 2-O-MeMan and Kdo, [-->)3-beta-D-2-O-MeManp-(1-->5)-beta-D-Kdop-(2-->]. These structures are similar to yet distinct from those of other strains of R. fredii and R. meliloti, and this finding provides further evidence that the K antigens of rhizobia are strain-specific antigens which are produced within a conserved motif.

To find some prognostic factors for the outcome of frozen-thawed cycles, we have retrospectively analysed all frozen pre-embryos that were thawed during 1993 and 1994 at two in-vitro fertilization (IVF) units in Sweden. Supernumerary pre-embryos were frozen from 551 oocyte retrievals and these resulted in 660 frozen-thawed cycles which lead to 623 thawed embryo transfers. The outcome of these transfers was 137 clinical pregnancies with a pregnancy rate of 22% per frozen-thawed embryo transfers. Women40 years of age had a higher birth rate than thoseor =40 years, 19 and 5% respectively (P0.01). Transfers with two and three pre-embryos resulted in pregnancy rates of 23 and 27%, respectively, compared with 14% for transfer of one embryo. A pregnancy resulting from the initial embryo transfers had a predictive value for results of the subsequent frozen-thawed cycle. Embryo grade and cleavage stage at the time of freezing was important for the survival of the frozen-thawed pre-embryos. The pregnancy rate was not influenced by the cleavage stage, but a tendency toward a lower pregnancy rate was seen for the embryos with lower grading. To conclude, cryopreservation seems to be beneficial in women40 years of age, who have supernumerary pre-embryos of good quality for freezing and of which at least two can be transferred.

Jot is a novel research interface for virtual reality modeling. This system seamlessly integrates and applies a variety of virtual and physical tools, each customized for specific tasks. The Jot interface not only moves smoothly from one tool to another but also physically and cognitively matches individual tools to the tasks they perform. In particular, we exploit the notion that gestural interaction is more direct, in many cases, than traditional widget based interaction. We also respect the time tested observation that some operations-even conceptually three dimensional ones-are better performed with 1D or 2D input devices, whereas other operations are more naturally performed using stereoscopic views, higher DOF input devices, or both. Ultimately we strive for a 3D modeling system with an interface as transparent as the interaction afforded by a pencil and a sheet of paper. For example, the system should facilitate the tasks of drawing and erasing and provide an easy transition between the two. Jot emerged from our previous work on a mouse based system, called Sketch, for gesturally creating imprecise 3D models. Jot extends Sketch's functionality to a wider spectrum of modeling, from concept design to detailed feature based parametric parts. Jot also extends the interaction in Sketch to better support individual modeling tasks. We extended Sketch's gestural framework to integrate interface components ranging from traditional desktop interface widgets to context sensitive gestures to direct manipulation techniques originally designed for immersive VR.

The prominent host muramidase lysozyme cleaves bacterial peptidoglycan (PG), and the enzyme is abundant in mucosal secretions. The lytic enzyme susceptibility of Gram-negative bacteria and mechanisms they use to thwart lytic enzyme activity are poorly studied. We previously characterized a Helicobacter pylori PG modification enzyme, an N-deacetylase (PgdA) involved in lysozyme resistance. In this study, another PG modification enzyme, a putative PG O-acetyltransferase (PatA), was identified. Mass spectral analysis of the purified PG demonstrated that a patA strain contained a greatly reduced amount of acetylated muropeptides, indicating a role for PatA in H. pylori PG O-acetylation. The PG modification mutant strains (pgdA, patA, or pgdA patA) were more susceptible to lysozyme killing than the parent, but this assay required high lysozyme levels (up to 50 mg/ml). However, addition of host lactoferrin conferred lysozyme sensitivity to H. pylori, at physiologically relevant concentrations of both host components (3 mg/ml lactoferrin plus 0.3 mg/ml lysozyme). The pgdA patA double mutant strain was far more susceptible to lysozyme/lactoferrin killing than the parent. Peptidoglycan purified from a pgdA patA mutant was five times more sensitive to lysozyme than PG from the parent strain, while PG from both single mutants displayed intermediate sensitivity. Both sensitivity assays for whole cells and for purified PGs indicated that the modifications mediated by PgdA and PatA have a synergistic effect, conferring lysozyme tolerance. In a mouse infection model, significant colonization deficiency was observed for the double mutant at 3 weeks postinoculation. The results show that PG modifications affect the survival of a Gram-negative pathogen. Importance Pathogenic bacteria evade host antibacterial enzymes by a variety of mechanisms, which include resisting lytic enzymes abundant in the host. Enzymatic modifications to peptidoglycan (PG, the site of action of lysozyme) are a known mechanism used by Gram-positive bacteria to protect against host lysozyme attack. However, Gram-negative bacteria contain a thin layer of PG and a recalcitrant outer membrane permeability barrier to resist lysis, so molecular modifications to cell wall structure in order to combat lysis remain largely unstudied. Here we show that two Helicobacter pylori PG modification enzymes (PgdA and PatA) confer a clear protective advantage to a Gram-negative bacterium. They protect the bacterium from lytic enzyme degradation, albeit via different PG modification activities. Many pathogens are Gram negative, so some would be expected to have a similar cell wall-modifying strategy. Understanding such strategies may be useful for combating pathogen growth., Importance Pathogenic bacteria evade host antibacterial enzymes by a variety of mechanisms, which include resisting lytic enzymes abundant in the host. Enzymatic modifications to peptidoglycan (PG, the site of action of lysozyme) are a known mechanism used by Gram-positive bacteria to protect against host lysozyme attack. However, Gram-negative bacteria contain a thin layer of PG and a recalcitrant outer membrane permeability barrier to resist lysis, so molecular modifications to cell wall structure in order to combat lysis remain largely unstudied. Here we show that two Helicobacter pylori PG modification enzymes (PgdA and PatA) confer a clear protective advantage to a Gram-negative bacterium. They protect the bacterium from lytic enzyme degradation, albeit via different PG modification activities. Many pathogens are Gram negative, so some would be expected to have a similar cell wall-modifying strategy. Understanding such strategies may be useful for combating pathogen growth.

[1] Ferrihydrite is an important iron oxyhydroxide for earth and environmental sciences, biology, and technology. Nevertheless, its mineral structure remains a matter of debate. The stumbling block is whether a significant amount of tetrahedrally coordinated iron is present. Here we present the first X-ray magnetic circular dichroism (XMCD) measurements performed on a well characterized synthetic sample of 6-line ferrihydrite, at both K and L2,3energy edges of iron. XMCD results demonstrate unambiguously the presence of tetrahedrally coordinated Fe(III) in the mineral structure, in quantities compatible with the latest extended X-ray absorption fine structure (EXAFS) analyses suggesting a concentration of 20–30%. Moreover, we find an antiferromagnetic coupling between tetrahedral and octahedral sublattices, with the octahedral sublattice parallel to the external magnetic field.

The structure of lipid A from the lipopolysaccharide of Rhizobium leguminosarum bv. phaseoli (wild type strain CE3) was investigated by alkylation analysis, nuclear magnetic resonance spectroscopy, and electrospray and fast atom bombardment mass spectrometry of the de-O-acylated lipid A. The lipid A carbohydrate backbone was shown to be a trisaccharide containing galacturonic acid, glucosamine, and the unique sugar 2-amino-2-deoxygluconic acid, previously unreported in lipopolysaccharides. Nuclear magnetic resonance spectroscopy and ethylation analyses revealed that the galacturonic acid is alpha-1,4-linked to the glucosamine, while the amino aldonic acid residue, which may exist as the 1,5-lactone, is attached as an aglycone to the glucosamine and, thus, occupies the reducing end of the molecule. The resulting backbone is hydrophilic and analogous to the commonly observed bisphosphorylated glucosamine disaccharide from enteric bacterial lipopolysaccharides in that both the nonreducing and reducing ends carry negatively charged substituents. The fatty acids of the R. leguminosarum lipid A are attached both as O- and N-acyl substituents to glucosamine and 2-aminogluconate. All fatty acids are hydroxylated consisting of 3-hydroxymyristate (3-OH-C14.0), 3-hydroxypentadecanoate (3-OH-C15.0), 3-hydroxypalmitate (3-OH-C16.0), 3-hydroxystearate (3-OH-C18.0), and 27-hydroxyoctacosanoate (27-OH-C28.0) in the approximate mole ratio 3:0.2:1:0.6:1. Unlike lipid As from enteric bacteria, the R. leguminosarum lipid A lacks 3-acyloxyacyl substituents; however, the long chain 27-hydroxy fatty acid carries ester-linked beta-hydroxybutyrate at the 27-hydroxy position. Fast atom bombardment mass spectrometry of the de-O-acylated lipid A demonstrated the presence of 2 molecular species that differ by 28 mass units due to fatty acid heterogeneity at the two amide linkages. One species carries amide-linked 3-OH-C14.0 and 3-OH-C16.0; the second species carries 3-OH-C14.0 and 3-OH-C18.0. Each molecular species also exists as the aldonolactone, yielding molecular ions at ((M+H)+)-18. The heterogeneity in the amide-linked fatty acids further distinguishes the Rhizobium lipid A from enteric lipid As.

Under conditions of nitrogen stress, leguminous plants form symbioses with soil bacteria called rhizobia. This partnership results in the development of structures called root nodules, in which differentiated endosymbiotic bacteria reduce molecular dinitrogen for the host. The establishment of rhizobium-legume symbioses requires the bacterial synthesis of oligosaccharides, exopolysaccharides, and capsular polysaccharides. Previous studies suggested that the 3-deoxy- d -manno-oct-2-ulopyranosonic acid (Kdo) homopolymeric capsular polysaccharide produced by strain Sinorhizobium meliloti Rm1021 contributes to symbiosis with Medicago sativa under some conditions. However, a conclusive symbiotic role for this polysaccharide could not be determined due to a lack of mutants affecting its synthesis. In this study, we have further characterized the synthesis, secretion, and symbiotic function of the Kdo homopolymeric capsule. We showed that mutants lacking the enigmatic rkp-1 gene cluster fail to display the Kdo capsule on the cell surface but accumulate an intracellular polysaccharide of unusually high M r . In addition, we have demonstrated that mutations in kdsB2 , smb20804, and smb20805 affect the polymerization of the Kdo homopolymeric capsule. Our studies also suggest a role for the capsular polysaccharide in symbiosis. Previous reports have shown that the overexpression of rkpZ from strain Rm41 allows for the symbiosis of exoY mutants of Rm1021 that are unable to produce the exopolysaccharide succinoglycan. Our results demonstrate that mutations in the rkp-1 cluster prevent this phenotypic suppression of exoY mutants, although mutations in kdsB2 , smb20804, and smb20805 have no effect.

Structural modification of peptidoglycan (PG) is one of the mechanisms that pathogenic bacteria use to evade the host innate immune system. For the noninvasive human gastric pathogen Helicobacter pylori, PG delivery to the host cells is one trigger of the immune response. H. pylori HP310 was markedly up-expressed upon cell exposure to oxidative stress. However, disruption of HP310 did not produce a phenotype distinguishable from the parent, including oxidative stress resistance characteristics. HP310 shows very weak homology to a known gene pgdA encoding PG deacetylase in Streptococcous pneumoniae. PGs from wild type H. pylori and the HP310 mutant were purified and analyzed by matrix-assisted laser desorption ionization time-of-flight and high pressure liquid chromatography. The parent strain PG is partially deacetylated, whereas several major PG-deacetylated muropeptides are absent or significantly reduced in the HP310 mutant. PG deacetylase activity was directly demonstrated by use of pure PG and HP310 protein by measuring the release of acetic acid. The Gram-negative bacterium H. pylori is highly resistant to lysozyme (up to 50 mg/ml), but the HP310 mutant is less resistant to lysozyme compared with the parent strain. Complementation of an hp310 strain with the wild type gene restored lysozyme resistance. The purified PG from the mutant is more susceptible to lysozyme (0.3 mg/ml) digestion than the wild type PG. The PG deacetylation appears to confer lysozyme resistance to escape immune detection. HP310 is representative of a new subfamily of bacterial PG deacetylases.