High-dose adenosine overcomes the attenuation of myocardial perfusion reserve caused by caffeine.

Objectives:
We studied whether an increase in adenosine dose overcomes caffeine antagonism on adenosine-mediated coronary vasodilation.

Background:
Caffeine is a competitive antagonist at the adenosine receptors, but it is unclear whether caffeine in coffee alters the actions of exogenous adenosine, and whether the antagonism can be surmounted by increasing the adenosine dose.

Methods:
Myocardial perfusion scintigraphy (MPS) was used to assess adenosine-induced hyperemia in 30 patients before (baseline) and after coffee ingestion (caffeine). At baseline, patients received 140 µg/kg/min of adenosine combined with low-level exercise. For the caffeine study, 12 patients received 140 µg/kg/min of adenosine (standard) and 18 patients received 210 µg/kg/min (high dose) after caffeine intake (200 mg). Myocardial perfusion was assessed semiquantitatively and quantitatively, and perfusion defect was characterized according to the presence of reversibility.

Results:
Caffeine reduced the magnitude of perfusion abnormality induced by standard adenosine as measured by the summed difference score (SDS) (12.0 ± 4.4 at baseline vs. 4.1 ± 2.1 after caffeine, p < 0.001) as well as defect size (18% [3% to 38%] vs. 8% [0% to 22%], p < 0.01), whereas it had no effect on the abnormalities caused by high-dose adenosine (SDS, 7.7 ± 4.0 at baseline vs. 7.8 ± 4.2 after caffeine, p = 0.7). There was good agreement between baseline and caffeine studies for segmental defect category (kappa = 0.72, 95% confidence interval: 0.65 to 0.79) in the high-dose group. An increase in adenosine after caffeine intake was well tolerated.

Conclusions:
Caffeine in coffee attenuates adenosine-induced coronary hyperemia and, consequently, the detection of perfusion abnormality by adenosine MPS. This can be overcome by increasing the adenosine dose without compromising test tolerability.

The Type VI secretion system of Burkholderia cenocepacia affects multiple Rho family GTPases disrupting the actin cytoskeleton and the assembly of NADPH oxidase complex in macrophages

Burkholderia cenocepacia is a Gram-negative opportunistic pathogen of patients with cystic fibrosis and chronic granulomatous disease. The bacterium survives intracellularly in macrophages within a membrane-bound vacuole (BcCV) that precludes the fusion with lysosomes. The underlying cellular mechanisms and bacterial molecules mediating these phenotypes are unknown. Here, we show that intracellular B. cenocepacia expressing a type VI secretion system (T6SS) affects the activation of the Rac1 and Cdc42 RhoGTPase by reducing the cellular pool of GTP-bound Rac1 and Cdc42. The T6SS also increases the cellular pool of GTP-bound RhoA and decreases cofilin activity. These effects lead to abnormal actin polymerization causing collapse of lamellipodia and failure to retract the uropod. The T6SS also prevents the recruitment of soluble subunits of the NADPH oxidase complex including Rac1 to the BcCV membrane, but is not involved in the BcCV maturation arrest. Therefore, T6SS-mediated deregulation of Rho family GTPases is a common mechanism linking disruption of the actin cytoskeleton and delayed NADPH oxidase activation in macrophages infected with B. cenocepacia.

Autophagy stimulation by rapamycin suppresses lung inflammation and infection by Burkholderia cenocepacia in a model of cystic fibrosis

Cystic fibrosis (CF) is the most common inherited lethal disease in Caucasians which results in multiorgan dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In CFTR Delta F508 (Delta F508) mouse macrophages, B. cepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of IL-1 beta. It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Here we show for the first time that in wild-type but not in Delta F508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cepacia are higher in CFTR-Delta F508 macrophages than in WT macrophages. An autophagosome is a compartment that engulfs nonfunctional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Furthermore, we show that B. cepacia downregulates autophagy genes in WT and Delta F508 macrophages. However, autophagy dysfunction is more pronounced in Delta F508 macrophages since they already have compromised autophagy activity. We demonstrate that the autophagy-stimulating agent, rapamycin markedly decreases B. cepacia infection in vitro by enhancing the clearance of B. cepacia via induced autophagy. In vivo, rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. Together, our studies reveal that if efficiently activated, autophagy can control B. cepacia infection and ameliorate the associated inflammation. Therefore, autophagy is a novel target for new drug development for CF patients to control B. cepacia infection and accompanying inflammation.

Burkholderia cenocepacia Type VI Secretion System Mediates Escape of Type II Secreted Proteins into the Cytoplasm of Infected Macrophages

Burkholderia cenocepacia is an opportunistic pathogen that survives intracellularly in macrophages and causes serious respiratory infections in patients with cystic fibrosis. We have previously shown that bacterial survival occurs in bacteria-containing membrane vacuoles (BcCVs) resembling arrested autophagosomes. Intracellular bacteria stimulate IL-1ß secretion in a caspase-1-dependent manner and induce dramatic changes to the actin cytoskeleton and the assembly of the NADPH oxidase complex onto the BcCV membrane. A Type 6 secretion system (T6SS) is required for these phenotypes but surprisingly it is not required for the maturation arrest of the BcCV. Here, we show that macrophages infected with B. cenocepacia employ the NLRP3 inflammasome to induce IL-1ß secretion and pyroptosis. Moreover, IL-1ß secretion by B. cenocepacia-infected macrophages is suppressed in deletion mutants unable to produce functional Type VI, Type IV, and Type 2 secretion systems (SS). We provide evidence that the T6SS mediates the disruption of the BcCV membrane, which allows the escape of proteins secreted by the T2SS into the macrophage cytoplasm. This was demonstrated by the activity of fusion derivatives of the T2SS-secreted metalloproteases ZmpA and ZmpB with adenylcyclase. Supporting this notion, ZmpA and ZmpB are required for efficient IL-1ß secretion in a T6SS dependent manner. ZmpA and ZmpB are also required for the maturation arrest of the BcCVs and bacterial intra-macrophage survival in a T6SS-independent fashion. Our results uncover a novel mechanism for inflammasome activation that involves cooperation between two bacterial secretory pathways, and an unanticipated role for T2SS-secreted proteins in intracellular bacterial survival.

The suhB gene of Burkholderia cenocepacia is required for protein secretion, biofilm formation, motility and polymyxin B resistance

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex (Bcc), a group of Gram-negative opportunistic pathogens that cause severe lung infections in patients with cystic fibrosis and display extreme intrinsic resistance to antibiotics including antimicrobial peptides. B. cenocepacia BCAL2157 encodes a protein homologous to SuhB, an inositol-1-monophosphatase from Escherichia coli, which was suggested to participate in posttranscriptional control of gene expression. In this work we show that a deletion of the suhB-like gene in B. cenocepacia (?suhBBc) was associated with pleiotropic phenotypes. The ?suhBBc mutant had a growth defect manifested by an almost 2-fold increase in the generation time relative to the parental strain. The mutant also had a general defect in protein secretion, motility and biofilm formation. Further analysis of the Type-2 and the Type-6 secretion systems activities revealed that these secretion systems were inactive in the ?suhBBc mutant. In addition, the mutant exhibited increased susceptibility to polymyxin B but not to aminoglycosides like gentamicin and kanamycin. Together, our results demonstrate that suhBBc deletion compromises general protein secretion including the activity of T2SS and T6SS, and affects polymyxin B resistance, motility, and biofilm formation. The pleiotropic effects observed upon suhBBc deletion demonstrate that suhBBc plays a critical role in the physiology of B. cenocepacia.

The Klondike goldfields and Pleistocene environments of Beringia

The Klondike goldfields of Yukon, Canada, contain a key record of Pleistocene Beringia, the region of Alaska, Siberia, and Yukon that remained largely unglaciated during the late Cenozoic. A concentration of mining exposures, with relict permafrost that is locally more than 700,000 years old, provides exceptional preservation of paleoenvironmental archives and a new perspective on the nature of paleoenvironments during the Pleistocene. A critical feature is the stratigraphic association of distal tephra beds with these paleoenvironmental archives, which facilitates their regional correlation and, in many cases, provides independent ages for the paleoenvironmental assemblages. Paleoenvironmental analyses of fossil arctic ground-squirrel middens and buried vegetation indicate the presence of cryoxerophilous ("steppe-tundra") vegetation growing on well-drained substrates with deep active layers (seasonal thaw depths) during cold intervals of the Pleistocene. Studies of full-glacial paleosols and cryostratigraphic relations of associated ground ice indicate the importance of active loess deposition and surface vegetation cover in maintaining the functionally distinct mammoth-steppe biome, which supported grazing mega-fauna populations, including mammoth, horse, and bison.

Rapid changes in the level of Kluane Lake in Yukon Territory over the last millennium

The level of Kluane Lake, the largest lake in Yukon Territory, was lower than at present during most of the Holocene. The lake rose rapidly in the late seventeenth century to a level 12 m above present, drowning forest and stranding driftwood on a conspicuous high-stand beach, remnants of which are preserved at the south end of the lake. Kluane Lake fell back to near its present level by the end of the eighteenth century and has fluctuated within a range of about 3 m over the last 50 yr. The primary control on historic fluctuations in lake level is the discharge of Slims River, the largest source of water to the lake. We use tree ring and radiocarbon ages, stratigraphy and sub-bottom acoustic data to evaluate two explanations for the dramatic changes in the level of Kluane Lake. Our data support the hypothesis of Hugh Bostock, who suggested in 1969 that the maximum Little Ice Age advance of Kaskawulsh Glacier deposited large amounts of sediment in the Slims River valley and established the present course of Slims River into Kluane Lake. Bostock argued that these events caused the lake to rise and eventually overflow to the north. The overflowing waters incised the Duke River fan at the north end of Kluane Lake and lowered the lake to its present level. This study highlights the potentially dramatic impacts of climate change on regional hydrology during the Little Ice Age in glacierised mountains. © 2006 University of Washington.

Late Quaternary tephrostratigraphy, Ahklun Mountains, SW Alaska

Radiocarbon-dated sediment cores from six lakes in the Ahklun Mountains, south-western Alaska, were used to interpolate the ages of late Quaternary tephra beds ranging in age from 25.4 to 0.4ka. The lakes are located downwind of the Aleutian Arc and Alaska Peninsula volcanoes in the northern Bristol Bay area between 159° and 161°W at around 60°N. Sedimentation-rate age models for each lake were based on a published spline-fit procedure that uses Monte Carlo simulation to determine age model uncertainty. In all, 62 C ages were used to construct the six age models, including 23 ages presented here for the first time. The age model from Lone Spruce Pond is based on 18 ages, and is currently the best-resolved Holocene age model available from the region, with an average 2s age uncertainty of about±109 years over the past 14.5ka. The sedimentary sequence from Lone Spruce Pond contains seven tephra beds, more than previously found in any other lake in the area. Of the 26 radiocarbon-dated tephra beds at the six lakes and from a soil pit, seven are correlated between two or more sites based on their ages. The major-element geochemistry of glass shards from most of these tephra beds supports the age-based correlations. The remaining tephra beds appear to be present at only one site based on their unique geochemistry or age. The 5.8ka tephra is similar to the widespread Aniakchak tephra [3.7±0.2 (1s) ka], but can be distinguished conclusively based on its trace-element geochemistry. The 3.1 and 0.4ka tephras have glass major- and trace-element geochemical compositions indistinguishable from prominent Aniakchak tephra, and might represent redeposited beds. Only two tephra beds are found in all lakes: the Aniakchak tephra (3.7±0.2ka) and Tephra B (6.1±0.3ka). The tephra beds can be used as chronostratigraphic markers for other sedimentary sequences in the region, including cores from Cascade and Sunday lakes, which were previously undated and were analyzed in this study to correlate with the new regional tephrostratigraphy. © 2012 John Wiley & Sons, Ltd.

Pristine Early Eocene Wood Buried Deeply in Kimberlite from Northern Canada

We report exceptional preservation of fossil wood buried deeply in a kimberlite pipe that intruded northwestern Canada's Slave Province 53.3±0.6 million years ago (Ma), revealed during excavation of diamond source rock. The wood originated from forest surrounding the eruption zone and collapsed into the diatreme before resettling in volcaniclastic kimberlite to depths >300 m, where it was mummified in a sterile environment. Anatomy of the unpermineralized wood permits conclusive identification to the genus Metasequoia (Cupressaceae). The wood yields genuine cellulose and occluded amber, both of which have been characterized spectroscopically and isotopically. From cellulose d O and d H measurements, we infer that Early Eocene paleoclimates in the western Canadian subarctic were 12-17°C warmer and four times wetter than present. Canadian kimberlites offer Lagerstätte-quality preservation of wood from a region with limited alternate sources of paleobotanical information. © 2012 Wolfe et al.

Permafrost response to last interglacial warming: field evidence from non-glaciated Yukon and Alaska

We present stratigraphic observations from three sites in eastern Beringia - Ch'ijee's Bluff in northern Yukon and nearby exposures on the Old Crow River, the Palisades on the Yukon River in Alaska, and placer mining exposures at Thistle Creek in west-central Yukon - which provide insight into the response of permafrost to regional warming during the last interglaciation. Chronology is based on the presence of Old Crow tephra, an important regional stratigraphic marker that dates to late Marine Isotope Stage 6, supplemented by paleoecology and non-finite C ages on wood-rich organic silts. Old Crow tephra overlies several relict ice wedges at the Palisades and Thistle Creek, indicating that permafrost at these sites did not thaw completely during the last interglaciation. Prominent deposits of last interglacial wood-rich organic silt are present at multiple sites in eastern Beringia, and probably represent accumulations of reworked forest vegetation due to thaw slumping or deposition into thermokarst ponds or depressions. Consistent stratigraphic relations between these deposits, Old Crow tephra, and ice wedge pseudomorphs at our three study sites, and at least six other sites in eastern Beringia, suggest that thaw of shallow permafrost was widespread during the last interglaciation. Limited stratigraphic evidence suggests that thaw was probably on the order of meters, rather than 10s of meters. The ubiquity of shallow permafrost degradation during the last interglaciation suggests that current ground warming may foreshadow widespread near-surface thaw under even modest future warming scenarios. However, the persistence of relict pre-last interglacial ice wedges highlights the potential for the regional antiquity of discontinuous permafrost, and provides compelling field evidence for the long-term resilience of deep permafrost during sustained periods of warmer-than-present climate.

A late-Middle Pleistocene (Marine Isotope Stage 6) vegetated surface buried by Old Crow tephra at the Palisades, interior Alaska

A 40 cm thick primary bed of Old Crow tephra (131 ± 11 ka), an important stratigraphic marker in eastern Beringia, directly overlies a vegetated surface at Palisades West, on the Yukon River in central Alaska. Analyses of insect, bryophyte, and vascular plant macrofossils from the buried surface and underlying organic-rich silt suggest the local presence of an aquatic environment and mesic shrub-tundra at the time of tephra deposition. Autochthonous plant and insect macrofossils from peat directly overlying Old Crow tephra suggest similar aquatic habitats and hydric to mesic tundra environments, though pollen counts indicate a substantial herbaceous component to the regional tundra vegetation. Trace amounts of arboreal pollen in sediments associated with the tephra probably reflect reworking from older deposits, rather than the local presence of trees. The revised glass fission-track age for Old Crow tephra places its deposition closer to the time of the last interglaciation than earlier age determinations, but stratigraphy and paleoecology of sites with Old Crow tephra indicate a late Marine Isotope Stage 6 age. Regional permafrost degradation and associated thaw slumping are responsible for the close stratigraphic and paleoecological relations between Old Crow tephra and last interglacial deposits at some sites in eastern Beringia. © 2009 Elsevier Ltd.