Concurrent with the explosion in the number of publications reporting biomarker discovery by profiling technologies, such as proteomics and pattern recognition, has been the increase in evidence highlighting the susceptibility of these approaches to analytical and experimental bias. The work presented here addresses these timely issues by delivering a detailed characterization of the effect of common sources of bias in clinical studies on serum and plasma profiles generated by a key technology in metabonomics, NMR spectroscopy. Specifically, differences in composition when blood samples were collected onto and in the absence of ice, over a series of serum-clot contact times, the stability of NMR-prepared samples over time and the effect on the metabolic profile of freeze-thawing were examined. While differences between individuals were far greater than variation from any other experimental factor, each of the conditions examined did cause slight alterations to the NMR profile that could produce a systematic bias. Variation due to clotting time caused changes in energy metabolites, which were delayed by ice with no other spectral effects. Room-temperature stability and hence NMR spectral repeatability were high (<1% intrasample variation). Higher molecular weight species such as lipoproteins were more susceptible to the variations present in the examined factors. These observations have implications for profiling study design, and hence, our results form a new and valuable resource for those attempting clinical metabolic profiling, for regulatory agencies involved in the licensing of clinical tests and in the generation of international reporting standards for metabonomics.

We present a three-dimensional map of the clathrin coat of coated vesicles, generated from tilt series of electron micrographs of unstained specimens embedded in vitreous ice. We have examined native placental coated vesicles and coats reassembled from their purified constituents, namely clathrin triskelions and accessory proteins of approximate mol. wts 100 kd and 50 kd. Our results show that the accessory proteins contribute a further shell of density within the double shell of the clathrin cage, extending from the terminal domains of the clathrin to the membrane of the vesicle. The thickness of the complete coat is approximately 22 nm.

We previously reported that remission duration < 1 year, extranodal disease, and B symptoms before salvage chemotherapy (SLT) can stratify relapsed or refractory Hodgkin lymphoma (HL) patients into favorable and unfavorable cohorts. In addition, pre-autologous stem cell transplant (ASCT) (18)FDG-PET response to SLT predicts outcome. This phase 2 study uses both pre-SLT prognostic factors and post-SLT FDG-PET response in a risk-adapted approach to improve PFS after high-dose radio-chemotherapy (HDT) and ASCT. The first SLT uses 2 cycles of ICE in a standard or augmented dose (ICE/aICE), followed by restaging FDG-PET scan. Patients with a negative scan received a transplant. If the FDG-PET scan remained positive, patients received 4 biweekly doses of gemcitabine, vinorelbine, and liposomal doxorubicin. Patients without evidence of disease progression proceeded to HDT/ASCT; those with progressive disease were study failures. At a median follow-up of 51 months, EFS analyzed by intent to treat as well as for transplanted patients is 70% and 79%, respectively. Patients transplanted with negative FDG-PET, pre-HDT/ASCT after 1 or 2 SLT programs, had an EFS of>> 80%, versus 28.6% for patients with a positive scan (P < .001). This prospective study provides evidence that the goal of SLT in patients with Hodgkin lymphoma should be a negative FDG-PET scan before HDT/ASCT.

The springtime stratospheric ozone (O3) layer over the Antarctic is thinning by as much as 50 percent, resulting in increased midultraviolet (UVB) radiation reaching the surface of the Southern Ocean. There is concern that phytoplankton communities confined to near-surface waters of the marginal ice zone will be harmed by increased UVB irradiance penetrating the ocean surface, thereby altering the dynamics of Antarctic marine ecosystems. Results from a 6-week cruise (Icecolors) in the marginal ice zone of the Bellingshausen Sea in austral spring of 1990 indicated that as the O3 layer thinned: (i) sea surface- and depth-dependent ratios of UVB irradiance (280 to 320 nanometers) to total irradiance (280 to 700 nanometers) increased and (ii) UVB inhibition of photosynthesis increased. These and other Icecolors findings suggest that O3-dependent shifts of in-water spectral irradiances alter the balance of spectrally dependent phytoplankton processes, including photoinhibition, photoreactivation, photoprotection, and photosynthesis. A minimum 6 to 12 percent reduction in primary production associated with O3 depletion was estimated for the duration of the cruise.

On-field monitoring of head impacts, combined with finite element (FE) biomechanical simulation, allow for predictions of regional strain associated with a diagnosed concussion. However, attempts to correlate these predictions with in vivo measures of brain injury have not been published. This article reports an approach to and preliminary results from the correlation of subject-specific FE model-predicted regions of high strain associated with diagnosed concussion and diffusion tensor imaging to assess changes in white matter integrity in the corpus callosum (CC). Ten football and ice hockey players who wore instrumented helmets to record head impacts sustained during play completed high field magnetic resonance imaging preseason and within 10 days of a diagnosed concussion. The Dartmouth Subject-Specific FE Head model was used to generate regional predictions of strain and strain rate following each impact associated with concussion. Maps of change in fractional anisotropy (FA) and median diffusivity (MD) were generated for the CC of each athlete to correlate strain with change in FA and MD. Mean and maximum strain rate correlated with change in FA (Spearman ρ = 0.77, p = 0.01; 0.70, p = 0.031), and there was a similar trend for mean and maximum strain (0.56, p = 0.10; 0.6, p = 0.07), as well as for maximum strain with change in MD (-0.63, p = 0.07). Change in MD correlated with injury-to-imaging interval (ρ = -0.80, p = 0.006) but change in FA did not (ρ = 0.18, p = 0.62). These results provide preliminary confirmation that model-predicted strain and strain rate in the CC correlate with changes in indices of white matter integrity.

BACKGROUND

Gene expression signatures are typically identified by correlating gene expression patterns to a disease phenotype of interest. However, individual gene-based signatures usually suffer from low reproducibility and interpretability.

RESULTS

We have developed a novel algorithm Iterative Clique Enumeration (ICE) for identifying relatively independent maximal cliques as co-expression modules and a module-based approach to the analysis of gene expression data. Applying this approach on a public breast cancer dataset identified 19 modules whose expression levels were significantly correlated with tumor grade. The correlations were reproducible for 17 modules in an independent breast cancer dataset, and the reproducibility was considerably higher than that based on individual genes or modules identified by other algorithms. Sixteen out of the 17 modules showed significant enrichment in certain Gene Ontology (GO) categories. Specifically, modules related to cell proliferation and immune response were up-regulated in high-grade tumors while those related to cell adhesion was down-regulated. Further analyses showed that transcription factors NYFB, E2F1/E2F3, NRF1, and ELK1 were responsible for the up-regulation of the cell proliferation modules. IRF family and ETS family proteins were responsible for the up-regulation of the immune response modules. Moreover, inhibition of the PPARA signaling pathway may also play an important role in tumor progression. The module without GO enrichment was found to be associated with a potential genomic gain in 8q21-23 in high-grade tumors. The 17-module signature of breast tumor progression clustered patients into subgroups with significantly different relapse-free survival times. Namely, patients with lower cell proliferation and higher cell adhesion levels had significantly lower risk of recurrence, both for all patients (p = 0.004) and for those with grade 2 tumors (p = 0.017).

CONCLUSIONS

The ICE algorithm is effective in identifying relatively independent co-expression modules from gene co-expression networks and the module-based approach illustrated in this study provides a robust, interpretable, and mechanistic characterization of transcriptional changes.

OBJECTIVE

We sought to evaluate the efficacy and safety of a novel cryothermal balloon ablation system in creating pulmonary vein (PV) isolation.

BACKGROUND

Pulmonary vein isolation using standard radiofrequency ablation techniques is limited by procedure-related complications, such as thrombus formation and PV stenosis. Cryothermal ablation may reduce the risk of such complications.

METHODS

Eight dogs underwent circumferential ablation of both superior PVs for either 4 or 8 min using a cryothermal balloon catheter (CryoCath Technologies Inc., Kirkland, Canada). Both fluoroscopy and intracardiac ultrasound (ICE)-guided balloon and Lasso catheter positioning at the PV ostia assessed short-term PV integrity. In six additional dogs, long-term PV integrity was assessed by computed tomography at 16 weeks after ablation.

RESULTS

Successful electrical isolation was achieved acutely in 14 of 16 (87.5%) PVs and was confirmed in one-week survival studies in 10 of 12 (83%) PVs. Successful isolation was higher in the absence of any peri-balloon flow leak as seen by ICE (p = 0.015), and with balloon temperatures < or =-80 degrees C (p = 0.015). Cryolesions were located at the veno-atrial junction and were homogeneous, with intact endothelium and free of thrombus formation. Although limited angiographic PV narrowing was noted in the early follow-up period, no significant PV narrowing was seen long-term. Right phrenic nerve injury was seen in 50% of the animals studied at one week.

CONCLUSIONS

This novel cryothermal balloon ablation system is effective for isolating PVs, but injury to the right phrenic nerve was noted in this early experience. Further studies are needed to assess the long-term efficacy and safety of this technique.

Interleukin-1 beta converting enzyme (ICE) is a cytoplasmic cysteine protease required for generating the bioactive form of the interleukin-1 beta cytokine from its inactive precursor. We report the identification of ICH-2, a novel human gene encoding a member of the ICE cysteine protease family, and characterization of its protein product. ICH-2 mRNA is widely expressed in human tissues in a pattern similar to, but distinct from, that of ICE. Overexpression of ICH-2 in insect cells induces apoptosis. Purified ICH-2 is functional as a protease in vitro. A comparison of the inhibitor profiles and substrate cleavage by ICH-2 and ICE shows that the enzymes share catalytic properties but may differ in substrate specificities, suggesting that the two enzymes have different functions in vivo.

Sports injuries are one of the most common injuries in modern western societies. Treating sports injuries is often difficult, expensive and time consuming, and thus, preventive strategies and activities are justified on medical as well as economic grounds. A successful injury surveillance and prevention requires valid pre- and post-intervention data on the extent of the problem. The aetiology, risk factors and exact mechanisms of injuries need to be identified before initiating a measure or programme for preventing sports injuries, and measurement of the outcome (injury) must include a standardised definition of the injury and its severity, as well as a systematic method of collecting the information. Valid and reliable measurement of the exposure includes exact information about the population at risk and exposure time. The true efficacy of a preventive measure or programme can be best evaluated through a well-planned randomised trial. Until now, 16 randomised, controlled trials (RCT) have been published on prevention of sports injuries. According to these RCT, the general injury rate can be reduced by a multifactorial injury prevention programme in soccer (relative risk 0.25, p < 0.001, in the intervention group), or by ankle disk training, combined with a thorough warm-up, in European team handball [odds ratio 0.17; 95% confidence interval (CI) 0.09 to 0.32, p < 0.01]. Ankle sprains can be prevented by ankle supports (i.e. semirigid orthoses or air-cast braces) in high-risk sporting activities, such as soccer and basketball (Peto odds ratio 0.49; 95% CI 0.37 to 0.66), and stress fractures of the lower limb by the use of shock-absorbing insoles in footwear (Peto odds ratio 0.47; 95% CI 0.30 to 0.76). In future studies, it is extremely important for researches to seek consultation with epidemiologists and statisticians to be certain that the study hypothesis is appropriate and that the methodology can lead to reliable and valid information. Further well-designed randomised studies are needed on preventive actions and devices that are in common use, such as preseason medical screenings, warming up, proprioceptive training, stretching, muscle strengthening, taping, protective equipment, rehabilitation programmes and education interventions (such as increasing general injury awareness among a team). The effect of a planned rule change on the injury risk in a particular sport could be tested via a RCT before execution of the change. The most urgent needs are in commonly practised or high-risk sports, such as soccer, American football, rugby, ice hockey, European team handball, karate, floorball, basketball, downhill skiing and motor sports.

OBJECTIVE

We present the initial clinical human experience with the use of a robotic remote navigation system (Hansen Medical, Mountain View, California), to perform left and right atrial mapping and radiofrequency ablation of atrial fibrillation (AF) and atrial flutter (AFL).

BACKGROUND

Catheter ablation is an established curative modality for various arrhythmias. A robotic steerable sheath system (SSS) (Hansen Medical) allows better catheter stability and greater degrees of freedom of catheter movement.

METHODS

A total of 40 patients (mean age 57 years) with antiarrhythmic drug (AAD)-refractory AF (23 had also concomitant documented typical AFL) were studied. Three-dimensional reconstruction of the corresponding atrial chamber anatomy was performed with the CARTO electroanatomic mapping system (Biosense Webster, Diamond Bar, California or the EnSite NavX system (St. Jude Medical, Minneapolis, Minnesota) in combination with the Artisan catheter (Hansen Medical). In patients undergoing AF ablation, 2 transseptal punctures were performed under intracardiac ultrasound (ICE) guidance, with one of the punctures being performed using SSS. Pulmonary vein antrum isolation was performed with a 3.5-mm thermocool catheter manipulated with the use of the SSS and was verified by circular mapping. Patients were followed clinically for recurrence of arrhythmia with an event transmitter and ambulatory holter monitoring. Clinical recurrence of AF/AFL was defined as AF/AFL episodes >1 min in duration.

RESULTS

Pulmonary vein antrum isolation was performed in 40 patients, including 23 with concomitant typical AFL ablation. All pulmonary veins, including the superior vena cava, were successfully isolated. In 23 of 40 patients, cavotricuspid ablation was also performed with bidirectional block obtained. At 1-year follow-up, 34 patients (86%) and 5 patients were free from atrial arrhythmia off AADs and on AADs, respectively.

CONCLUSIONS

This preliminary human experience suggests that mapping and ablation of AFL and AF using this novel robotic catheter with remote control system is feasible with similar results to conventional approach.

The integration of linear retrovirus DNA by the viral integrase (IN) into the host chromosome occurs by a concerted mechanism (full-site reaction). IN purified from avian myeloblastosis virus and using retrovirus-like DNA restriction fragments (487 bp in length) as donors and circular DNA (pGEM-3) as the target can efficiently catalyze that reaction. Nonionic detergent lysates of purified human immunodeficiency virus type 1 (HIV-1) virions were also capable of catalyzing the concerted integration reaction. The donor substrates were restriction fragments (469 bp) containing either U3-U5 (H-2 donor) or U5-U5 (H-5 donor) long terminal repeat sequences at their ends. As was shown previously with bacterially expressed HIV-1 IN, the U5 terminus of H-2 was preferred over the U3 terminus by virion-associated IN. The reactions involving two donors per circular target by HIV-1 IN preferred Mg2+ over Mn2+. Both metal ions were equally effective for the circular half-site reaction involving only one donor molecule. The linear 3.8-kbp recombinant products produced from two donor insertions into pGEM were genetically selected, and the donor-target junctions of individual recombinants were sequenced. A total of 55% of the 87 sequenced recombinants had host site duplications of between 5 and 7 bp, with the HIV-1 5-bp-specific duplication predominating. The other recombinants that migrated at the linear 3.8-kbp position were mainly small deletions that were grouped into four sets of 17, 27, 40, and 47 bp, each having a periodicity mimicking a turn of the DNA helix. Aprotic solvents (dimethyl sulfoxide and 1,4-dioxane) enhanced both the half-site and the linear 3.8-kbp strand transfer reactions which favored low-salt conditions (30 mM NaCl). The order of addition of the donor and target during preincubation with HIV-1 IN on ice did not affect the quantity of linear 3.8-kbp recombinants relative to that of the circular half-site products that were produced; only the quantity of donor-donor versus donor-target recombinants was affected. The presence of Mg2+ in the preincubation mixtures containing donor and target substrates was not necessary for the stability of preintegration complexes on ice or at 22 degrees C. Comparisons of the avian and HIV-1 concerted integration reactions are discussed.

A replica plating method for rapid quantitation of ice nucleation-active (INA) bacteria was developed. Leaf washings of plant samples from California, Colorado, Florida, Louisiana, and Wisconsin were tested for the presence of INA bacteria. Of the 95 plant species sampled, 74 were found to harbor INA bacteria. Only the conifers were, as a group, unlikely to harbor INA bacteria. All of the INA bacteria isolated resembled either Pseudomonas syringae or Erwinia herbicola. Sufficient numbers of INA bacteria were present on the samples to account for the ice nuclei associated with leaves that are necessary for freezing injury to occur. Numbers of INA bacteria were large enough to suggest that plant surfaces may constitute a significant source of atmospheric ice nuclei.

The Bcl-2 and Bcl-x proteins suppress programmed cell death, whereas Bax promotes apoptosis. We investigated the pattern of expression of Bcl-2, Bax and Bcl-x during neuronal differentiation and development. All three proteins were widely expressed in neonatal rats but, in the adult, Bax levels were 20- to 140-fold lower in the cerebral cortex, cerebellum and heart muscle, whereas Bcl-x was not downregulated in any of the tissues examined. In the cerebral cortex and cerebellum, the decrease in Bax levels occurred after the period of developmental cell death. Further, microinjection of a Bax expression vector into cultured sympathetic neurons, which depend on nerve growth factor for survival, induced apoptosis in the presence of survival factor and increased the rate of cell death after nerve growth factor withdrawal. This effect could be blocked by co-injection of an expression vector for Bcl-xL or for the baculovirus p35 protein, an inhibitor of caspases (ICE-like proteases). These results suggest that, during development, the sensitivity of neurons to signals that induce apoptosis may be regulated by modulating Bax levels and that Bax-induced death requires caspase activity.

Avoidance of ice formation during cooling can be achieved by vitrification, which is defined as solidification in an amorphous glassy state that obviates ice nucleation and growth. We show that a vitrification approach to storing vascular tissue results in markedly improved tissue function compared with a standard method involving freezing. The maximum contractions achieved in vitrified vessels were >80% of fresh matched controls with similar drug sensitivities, whereas frozen vessels exhibited maximal contractions below 30% of controls and concomitant decreases in drug sensitivity. In vivo studies of vitrified vessel segments in an autologous transplant model showed no adverse effects of vitreous cryopreservation compared with fresh tissue grafts.

Images and diffraction patterns of frozen-hydrated thin crystals of phi 29 connectors were recorded using low dose microscopy and diffraction. The resolution obtained in two cases was 7 and 4 A, thus showing the suitability of this specimen for high-resolution studies. Seven of the best images and three of the best electron diffraction patterns were analysed semiquantitatively. The best image showed the presence of two connectors per unit cell (165 A x 165 A), each having an internal 4-fold axis and being related to one-another by 2-fold and 2-fold screw axes in the space group p42(1)2. Internal phase comparison within the best image showed good phase residuals for p42(1)2 and all its sub-groups. The other images were consistent with this interpretation taking into consideration the possible crystal tilt and specimen drift. We conclude that the most likely crystal space group is p42(1)2. Electron diffraction patterns were consistent with this assignment, but none of the patterns showed the precise 422 symmetry expected, almost certainly because the crystals were slightly tilted (2 to 5 degrees).

BACKGROUND

Concussions are potentially serious injuries. The few investigations of prevalence or incidence in youth ice hockey have typically relied on prospective reports from physicians or trainers and did not survey players, despite the knowledge that many athletes do not report probable concussions.

OBJECTIVE

This study sought to compare concussion rates in youth ice hockey that were estimated from a variety of reporting strategies.

METHODS

Rates were calculated from British Columbia Amateur Hockey Association (BCAHA) official injury reports, from direct game observation by minor hockey volunteers (such as coaches and managers), as well as from retrospective surveys of both elite and non-elite youth players. All research was conducted within the BCAHA.

RESULTS

Estimates from official injury reports for male players were between 0.25 and 0.61 concussions per 1000 player game hours (PGH). Concussion estimates from volunteer reports were between 4.44 and 7.94 per 1000 PGH. Player survey estimates were between 6.65 and 8.32 per 1000 PGH, and 9.72 and 24.30 per 1000 PGH for elite and non-elite male youth hockey, respectively.

CONCLUSIONS

It was found that concussions are considerably under-reported to the BCAHA by youth hockey players and team personnel.

The Earth's cryosphere comprises those regions that are cold enough for water to turn into ice. Recent findings show that the icy realms of polar oceans, glaciers and ice sheets are inhabited by microorganisms of all three domains of life, and that temperatures below 0 °C are an integral force in the diversification of microbial life. Cold-adapted microorganisms maintain key ecological functions in icy habitats: where sunlight penetrates the ice, photoautotrophy is the basis for complex food webs, whereas in dark subglacial habitats, chemoautotrophy reigns. This Review summarizes current knowledge of the microbial ecology of frozen waters, including the diversity of niches, the composition of microbial communities at these sites and their biogeochemical activities.

BACKGROUND

High-throughput proteomic methods for disease biomarker discovery in human serum are promising, but concerns exist regarding reproducibility of results and variability introduced by sample handling. This study investigated the influence of different preanalytic handling methods on surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) protein profiles of prefractionated serum. We investigated whether older collections with longer sample transit times yield useful protein profiles, and sought to establish the most feasible collection methods for future clinical proteomic studies.

METHODS

To examine the effect of tube type, clotting time, transport/incubation time, temperature, and storage method on protein profiles, we used 6 different handling methods to collect sera from 25 healthy volunteers. We used a high-throughput, prefractionation strategy to generate anion-exchange fractions and examined their protein profiles on CM10, IMAC30-Cu, and H50 arrays by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry.

RESULTS

Prolonged transport and incubation at room temperature generated low mass peaks, resulting in distinctions among the protocols. The most and least stringent methods gave the lowest overall peak variances, indicating that proteolysis in the latter may have been nearly complete. For samples transported on ice there was little effect of clotting time, storage method, or transit time. Certain proteins (TTR, ApoCI, and transferrin) were unaffected by handling, but others (ITIH4 and hemoglobin beta) displayed significant variability.

CONCLUSIONS

Changes in preanalytical handling variables affect profiles of serum proteins, including proposed disease biomarkers. Proteomic analysis of samples from serum banks collected using less stringent protocols is applicable if all samples are handled identically.

Current global fisheries production of approximately 160 million tons is rising as a result of increases in aquaculture production. A number of climate-related threats to both capture fisheries and aquaculture are identified, but we have low confidence in predictions of future fisheries production because of uncertainty over future global aquatic net primary production and the transfer of this production through the food chain to human consumption. Recent changes in the distribution and productivity of a number of fish species can be ascribed with high confidence to regional climate variability, such as the El Niño-Southern Oscillation. Future production may increase in some high-latitude regions because of warming and decreased ice cover, but the dynamics in low-latitude regions are governed by different processes, and production may decline as a result of reduced vertical mixing of the water column and, hence, reduced recycling of nutrients. There are strong interactions between the effects of fishing and the effects of climate because fishing reduces the age, size, and geographic diversity of populations and the biodiversity of marine ecosystems, making both more sensitive to additional stresses such as climate change. Inland fisheries are additionally threatened by changes in precipitation and water management. The frequency and intensity of extreme climate events is likely to have a major impact on future fisheries production in both inland and marine systems. Reducing fishing mortality in the majority of fisheries, which are currently fully exploited or overexploited, is the principal feasible means of reducing the impacts of climate change.

Accurate prediction of global sea-level rise requires that we understand the cause of recent, widespread and intensifying glacier acceleration along Antarctic ice-sheet coastal margins. Atmospheric and oceanic forcing have the potential to reduce the thickness and extent of floating ice shelves, potentially limiting their ability to buttress the flow of grounded tributary glaciers. Indeed, recent ice-shelf collapse led to retreat and acceleration of several glaciers on the Antarctic Peninsula. But the extent and magnitude of ice-shelf thickness change, the underlying causes of such change, and its link to glacier flow rate are so poorly understood that its future impact on the ice sheets cannot yet be predicted. Here we use satellite laser altimetry and modelling of the surface firn layer to reveal the circum-Antarctic pattern of ice-shelf thinning through increased basal melt. We deduce that this increased melt is the primary control of Antarctic ice-sheet loss, through a reduction in buttressing of the adjacent ice sheet leading to accelerated glacier flow. The highest thinning rates occur where warm water at depth can access thick ice shelves via submarine troughs crossing the continental shelf. Wind forcing could explain the dominant patterns of both basal melting and the surface melting and collapse of Antarctic ice shelves, through ocean upwelling in the Amundsen and Bellingshausen seas, and atmospheric warming on the Antarctic Peninsula. This implies that climate forcing through changing winds influences Antarctic ice-sheet mass balance, and hence global sea level, on annual to decadal timescales.

The cacti are one of the most celebrated radiations of succulent plants. There has been much speculation about their age, but progress in dating cactus origins has been hindered by the lack of fossil data for cacti or their close relatives. Using a hybrid phylogenomic approach, we estimated that the cactus lineage diverged from its closest relatives ≈35 million years ago (Ma). However, major diversification events in cacti were more recent, with most species-rich clades originating in the late Miocene, ≈10-5 Ma. Diversification rates of several cactus lineages rival other estimates of extremely rapid speciation in plants. Major cactus radiations were contemporaneous with those of South African ice plants and North American agaves, revealing a simultaneous diversification of several of the world's major succulent plant lineages across multiple continents. This short geological time period also harbored the majority of origins of C(4) photosynthesis and the global rise of C(4) grasslands. A global expansion of arid environments during this time could have provided new ecological opportunity for both succulent and C(4) plant syndromes. Alternatively, recent work has identified a substantial decline in atmospheric CO(2) ≈15-8 Ma, which would have strongly favored C(4) evolution and expansion of C(4)-dominated grasslands. Lowered atmospheric CO(2) would also substantially exacerbate plant water stress in marginally arid environments, providing preadapted succulent plants with a sharp advantage in a broader set of ecological conditions and promoting their rapid diversification across the landscape.

Hippocampal slices often have more synapses than perfusion-fixed hippocampus, but the cause of this synaptogenesis is unclear. Ultrastructural evidence for synaptogenic triggers during slice preparation was investigated in 21-day-old rats. Slices chopped under warm or chilled conditions and fixed after 0, 5, 25, 60, or 180 minutes of incubation in an interface chamber were compared with hippocampi fixed by perfusion or by immersion of the whole hippocampus. There was no significant synaptogenesis in these slices compared with perfusion-fixed hippocampus, but there were other structural changes during slice preparation and recovery in vitro. Whole hippocampus and slices prepared under warm conditions exhibited an increase in axonal coated vesicles, suggesting widespread neurotransmitter release. Glycogen granules were depleted from astrocytes and neurons in 0-min slices, began to reappear by 1 hour, and had fully recovered by 3 hours. Dendritic microtubules were initially disassembled in slices, but reassembled into normal axial arrays after 5 minutes. Microtubules were short at 5 minutes (12.3 +/- 1.1 microm) but had recovered normal lengths by 3 hours (84.6 +/- 20.0 microm) compared with perfusion-fixed hippocampus (91 +/- 22 microm). Microtubules appeared transiently in 15 +/- 3% and 9 +/- 4% of dendritic spines 5 and 25 minutes after incubation, respectively. Spine microtubules were absent from perfusion-fixed hippocampus and 3-hour slices. Ice-cold dissection and vibratomy in media that blocked activity initially produced less glycogen loss, coated vesicles, and microtubule disassembly. Submersing these slices in normal oxygenated media at 34 degrees C led to glycogen depletion, as well as increased coated vesicles and microtubule disassembly within 1 minute.

The Mesorhizobium loti strain R7A symbiosis island is an Integrative Conjugative Element (ICE), herein termed ICEMlSymR7A, which integrates into a phetRNA gene. Integration reconstructs the phetRNA gene at one junction with the core chromosome, and a direct repeat of the 3-prime 17 bp of the gene is formed at the other junction. We show that the ICEMlSymR7AintS gene, which encodes an integrase of the phage P4 family, is required for integration and excision of the island. Excision also depended on a novel recombination directionality factor encoded by msi109 (rdfS). Constitutive expression of rdfS resulted in curing of ICEMlSymR7A. The rdfS gene is part of an operon with genes required for conjugative transfer, allowing co-ordinate regulation of ICEMlSymR7A excision and transfer. The excised form of ICEMlSymR7A was detectable during exponential growth but occurred at higher frequency during stationary phase. ICEMlSymR7A encodes homologues of the traR and traI genes of Agrobacterium tumefaciens that regulate Ti plasmid transfer via quorum sensing. The presence of a plasmid with cloned island traR traI2 genes resulted in excision of ICEMlSymR7A in all cells regardless of culture density, indicating that excision may be similarly regulated. Maintenance of ICEMlSymR7A in these cells depended on msi106 (rlxS) that encodes a putative relaxase. Transfer of the island to non-symbiotic mesorhizobia required intS, rlxS and rdfS. The rdfS and rlxS genes are conserved across a diverse range of alpha-, beta- and gamma-proteobacteria and identify a large family of genomic islands with a common transfer mechanism.

BACKGROUND

Infective endocarditis caused by non-HACEK (species other than Haemophilus species, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, or Kingella species) gram-negative bacilli is rare, is poorly characterized, and is commonly considered to be primarily a disease of injection drug users.

OBJECTIVE

To describe the clinical characteristics and outcomes of patients with non-HACEK gram-negative bacillus endocarditis in a large, international, contemporary cohort of patients.

METHODS

Observations from the International Collaboration on Infective Endocarditis Prospective Cohort Study (ICE-PCS) database.

METHODS

61 hospitals in 28 countries.

METHODS

Hospitalized patients with definite endocarditis.

METHODS

Characteristics of non-HACEK gram-negative bacillus endocarditis cases were described and compared with those due to other pathogens.

RESULTS

Among the 2761 case-patients with definite endocarditis enrolled in ICE-PCS, 49 (1.8%) had endocarditis (20 native valve, 29 prosthetic valve or device) due to non-HACEK, gram-negative bacilli. Escherichia coli (14 patients [29%]) and Pseudomonas aeruginosa (11 patients [22%]) were the most common pathogens. Most patients (57%) with non-HACEK gram-negative bacillus endocarditis had health care-associated infection, whereas injection drug use was rare (4%). Implanted endovascular devices were frequently associated with non-HACEK gram-negative bacillus endocarditis compared with other causes of endocarditis (29% vs. 11%; P < 0.001). The in-hospital mortality rate of patients with endocarditis due to non-HACEK gram-negative bacilli was high (24%) despite high rates of cardiac surgery (51%).

CONCLUSIONS

Because of the small number of patients with non-HACEK gram-negative bacillus endocarditis in each treatment group and the lack of long-term follow-up, strong treatment recommendations are difficult to make.

CONCLUSIONS

In this large, prospective, multinational cohort, more than one half of all cases of non-HACEK gram-negative bacillus endocarditis were associated with health care contact. Non-HACEK gram-negative bacillus endocarditis is not primarily a disease of injection drug users.