Significance
Lytic polysaccharide monooxygenases (LPMOs) have unique catalytic centers, at which a single copper catalyzes the oxidative cleavage of a glycosidic bond. The mechanism by which LPMOs ...activate molecular oxygen is key to understanding copper (bio)catalysis but remains poorly understood, largely because the insoluble and heterogeneous nature of LPMO substrates precludes the use of usual laboratory techniques. Using an integrated NMR/EPR approach, we have unraveled structural and electronic details of the interactions of an LPMO from
Bacillus licheniformis
and β-chitin. EPR spectroscopy on uniformly isotope
15
N-labeled
63
Cu(II)-LPMO provided insight into substrate-driven rearrangement of the copper coordination sphere that predisposes the enzyme for O
2
activation.
Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO–substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of
apo
-
Bl
LPMO10A from
Bacillus licheniformis
, along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and
15
N-HYSCORE spectroscopy study on the uniformly isotope-labeled
63
Cu(II)-bound
15
N-
Bl
LPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal–ligand covalency and an attendant increase in the d(x
2
−y
2
) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu–O
2
intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O
2
activation in chitin-active AA10 LPMOs.
T-cell activation requires stimulation of specific intracellular signaling pathways in which protein-tyrosine kinases, phosphatases, and adapter proteins interact to transmit signals from the T-cell ...receptor to the nucleus. Interactions of LCK proto-oncogene, SRC family tyrosine kinase (LCK), and the IL-2–inducible T cell kinase (ITK) with the T cell-specific adapter protein (TSAD) promotes LCK-mediated phosphorylation and thereby ITK activation. Both ITK and LCK interact with TSAD's proline-rich region (PRR) through their Src homology 3 (SH3) domains. Whereas LCK may also interact with TSAD through its SH2 domain, ITK interacts with TSAD only through its SH3 domain. To begin to understand on a molecular level how the LCK SH3 and ITK SH3 domains interact with TSAD in human HEK293T cells, here we combined biochemical analyses with NMR spectroscopy. We found that the ITK and LCK SH3 domains potentially have adjacent and overlapping binding sites within the TSAD PRR amino acids (aa) 239–274. Pulldown experiments and NMR spectroscopy revealed that both domains may bind to TSAD aa 239–256 and aa 257–274. Co-immunoprecipitation experiments further revealed that both domains may also bind simultaneously to TSAD aa 242–268. Accordingly, NMR spectroscopy indicated that the SH3 domains may compete for these two adjacent binding sites. We propose that once the associations of ITK and LCK with TSAD promote the ITK and LCK interaction, the interactions among TSAD, ITK, and LCK are dynamically altered by ITK phosphorylation status.
•Modern stratigraphic records successfully establish reference conditions.•Surrounding bedrock introduces naturally high concentrations of Ni.•Geochemical parameters and benthic foraminiferal indices ...showed no fish farm impact.•Benthic foraminifera are relevant to implement in environmental monitoring systems.•Macrofauna and living foraminifera seem to respond in a similar fashion.
Intensive fish farming is a major industry, but the extent of organic matter (OM) and heavy metal pollution by fish farms is debated. This study established in situ reference conditions using geochemical parameters and fossil benthic foraminiferal assemblages in dated sediment cores to identify potential impacts of fish farming in two basins of the inner Øksfjord, Northern Norway. Living (rose Bengal stained) benthic foraminifera were used to assess the present day environmental conditions. The fossil foraminiferal records were compared with the living foraminifera, which in turn were compared with macrofaunal data. Long-term (>100 yrs) sediment core records of the geochemical parameters (TOC63, C/N, δ13CVPDB TOC and heavy metals) and foraminiferal indices (Norwegian Quality Index (fNQI), AZTI’s Marine Biotic Index (fAMBI), fHlog2, ES100) did not indicate an impact from fish farming through time. Long-term changes in foraminiferal absolute abundances and relative abundances of ecological groups (EGs) reflecting organic matter (OM) tolerance suggest that the OM supply slightly increased compared to reference conditions. Relative abundances of Brizalina skagerrakensis and Epistominella vitrea, previously associated with phytodetrital input, suggest a minor increase in primary productivity compared to reference conditions. The Stainforthia group (S. fusiformis and S. feylingi), indicative of OM enrichment, in the living foraminiferal assemblages may indicate a response to fish farming activities, but foraminiferal seasonality could not be excluded as a potential cause. The indices of both fossil and living foraminifera, in addition to the macrofauna showed a good to high Ecological Quality Status (EcoQS) through time and at present. This indicates that environmental conditions have been and still are acceptable.
For the first time on the Norwegian Continental Shelf, deeply buried paleoregolith profiles have been identified as part of the petroleum reservoirs in recently discovered oil fields on the Utsira ...High, Norwegian North Sea. Reservoir properties (porosity and permeability) in the granitic basement on the Utsira High are mainly the result of physical and chemical alteration of the rock occurring in the near-surface environment during sub-aerial exposure of the high in the Mesozoic. Evaluating the reservoir potential of altered basement rocks requires a different approach than in conventional petroleum exploration. In this paper, macroscopic, mineralogical and micromorphological alteration features observed in two deeply buried paleoregolith profiles are compared with surface paleoregoliths from Ivö Klack, Sweden and Georgia, USA. The paleoregolith profiles are subdivided into specific weathering facies (altered coherent rock facies, saprock facies and saprolite facies) based on the rock fabric and mechanical strength. The reservoir potential of each weathering facies is controlled by the type and degree of alteration. In the altered coherent rock facies, porosity and permeability is mainly controlled by joints and microfractures that developed prior to subaerial exposure of the granitic pluton. In the saprock facies, intensified chemical dissolution of plagioclase enhanced porosity and the development of mesofractures improved the connectivity between pores. In the saprolite facies, progressive dissolution of plagioclase creates porosity, but the precipitation of clays within voids and mesofractures has a destructive effect on the overall reservoir properties.
The deeply buried paleoregolith profiles from the Utsira High display comparable macroscopic, mineralogical and micromorphological alteration features to what was observed in surface paleoregoliths from Ivö Klack and Georgia. Outcrop studies may therefore be an important tool when evaluating the reservoir potential in subsurface paleoregoliths.
•Deeply buried paleoregolith profiles are part of petroleum reservoirs in the Norwegian North Sea.•Paleoregolith profiles may be subdivided into specific weathering facies that display contrasting reservoir properties.•We compare deeply buried paleoregolith profiles with surface paleoweathering profiles from Southern Sweden and Georgia.
The receptor tyrosine kinase AXL is associated with epithelial plasticity in several solid tumors including breast cancer and AXL-targeting agents are currently in clinical trials. We hypothesized ...that AXL is a driver of stemness traits in cancer by co-option of a regulatory function normally reserved for stem cells. AXL-expressing cells in human mammary epithelial ducts co-expressed markers associated with multipotency, and AXL inhibition abolished colony formation and self-maintenance activities while promoting terminal differentiation in vitro. Axl-null mice did not exhibit a strong developmental phenotype, but enrichment of Axl+ cells was required for mouse mammary gland reconstitution upon transplantation, and Axl-null mice had reduced incidence of Wnt1-driven mammary tumors. An AXL-dependent gene signature is a feature of transcriptomes in basal breast cancers and reduced patient survival irrespective of subtype. Our interpretation is that AXL regulates access to epithelial plasticity programs in MaSCs and, when co-opted, maintains acquired stemness in breast cancer cells.
Display omitted
•AXL + mammary epithelial cells have multipotent activity conserved in women and mice•AXL allows accesses to epithelial-to-mesenchymal transition genes and prevents differentiation into luminal cells•Deletion of Axl reduced incidence of Wnt1-driven tumors in mice•Provides a rationale explaining the advantage to cancer cells that co-opt AXL signaling
Cell Biology; Stem Cells Research; Cancer
Following indications of recent warming trends in the Nordic Seas, we have studied the hydrography of these marginal seas from the summer of 2007 until the fall of 2008, using observations gathered ...by instrumented seals and Argo floats. The combined dataset shows that the upper ocean was indeed both warmer and saltier over much of the Nordic Seas in 2007–2008 compared to the average ocean state for the period 1956–2006 (based on the World Ocean Atlas 2009). There are also indications that the surface Polar Waters of the East Greenland Current were colder and fresher than the climatology, though the quality of the climatology is questionable for this region given the low number of historical observations. Dynamic height calculations suggest that the observed hydrographic changes were associated with enhanced northward upper-ocean thermal wind transport in the east and possibly also enhanced southward transport in the west.
•We study modern hydrographic data from the Nordic Seas collected by seals and Argo floats•The eastern Nordic Seas were anomalously warm and salty in 2007–2008.•There is indication that the East Greenland Current was colder and fresher in 2007–2008.•Dynamic height calculations also suggest baroclinic circulation changes.
Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of ...these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO–substrate interaction. Using NMR spectroscopy, we have elucidated the solutionphase structure of apo-BlLPMO10A from Bacillus licheniformis, along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and 15N-HYSCORE spectroscopy study on the uniformly isotope-labeled 63Cu(II)-bound 15N-BlLPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal–ligand covalency and an attendant increase in the d(x²–y²) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu–O₂ intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O₂ activation in chitin-active AA10 LPMOs.
Intensive fish farming is a major industry, but the extent of organic matter (OM) and heavy metal pollution by fish farms is debated. This study established in situ reference conditions using ...geochemical parameters and fossil benthic foraminiferal assemblages in dated sediment cores to identify potential impacts of fish farming in two basins of the inner Øksfjord, Northern Norway. Living (rose Bengal stained) benthic foraminifera were used to assess the present day environmental conditions. The fossil foraminiferal records were compared with the living foraminifera, which in turn were compared with macrofaunal data. Long-term (> 100 yrs) sediment core records of the geochemical parameters (TOC63, C/N, δ13CVPDB TOC and heavy metals) and foraminiferal indices (Norwegian Quality Index (fNQI), AZTI’s Marine Biotic Index (fAMBI), fHlog2, ES100) did not indicate an impact from fish farming through time. Long-term changes in foraminiferal absolute abundances and relative abun- dances of ecological groups (EGs) reflecting organic matter (OM) tolerance suggest that the OM supply slightly increased compared to reference conditions. Relative abundances of Brizalina skagerrakensis and Epistominella vitrea, previously associated with phytodetrital input, suggest a minor increase in primary productivity com- pared to reference conditions. The Stainforthia group (S. fusiformis and S. feylingi), indicative of OM enrichment, in the living foraminiferal assemblages may indicate a response to fish farming activities, but foraminiferal seasonality could not be excluded as a potential cause. The indices of both fossil and living foraminifera, in addition to the macrofauna showed a good to high Ecological Quality Status (EcoQS) through time and at present. This indicates that environmental conditions have been and still are acceptable.
Fibrin clots were induced in eyes of dogs by injection of autogenous citrated plasma into the anterior chamber. Twenty-four hours after clot formation, 0.01 ml of tissue plasminogen activator at a ...concentration of 1 microgram/100 microliter (group 1, n = 5) or 25 microgram/100 microliter (group 2, n = 5) was injected into 1 anterior chamber of each dog. The contralateral eye served as a nontreated control. Serial photographs were taken of the fibrin dots after intracameral injection of tissue plasminogen activator. Computerized morphometric analysis was then used to evaluate changes in cross-sectional surface area of the fibrin clot. Significant (P 0.001) fibrin-clot lysis was detected in treated eyes of group-2 dogs, but was not found in treated eyes of group-1 dogs. A mean decrease of 90% in clot surface area was detected by 120 minutes after injection in treated eyes of group-2 dogs
Contact wide-field specular microscopy was performed on eyes of 16 healthy dogs after tissue plasminogen activator at a concentration of 25 microgram/100 (group 1, n = 8) or 50 microgram/100 ...microliter (group 2, n = 8) was injected into 1 anterior chamber of each dog. The contralateral eye served as a nontreated control. Applanation tonometry was used to measure intraocular pressure in both eyes for up to 168 hours. By use of computerized morphometric analysis and pachymetry, changes from baseline values in endothelial cell density, cell morphologic features, and corneal thickness were evaluated at postinjection, hours 24, 48, and 168. Significant mean differences in intraocular pressure were not detected between treated eyes of group-1 dogs and those in group 2 at designated times, or between treated and nontreated eyes of dogs in either group. Mean corneal thickness of treated and nontreated eyes was similar in both groups through postinjection hour 168. Changes in mean percentage of endothelial cell sides were observed only in treated eyes of group-2 dogs, with the mean percentage of hexagons at postinjection hour 168 decreasing by 18%, a decrease that was significantly (P 0.05) greater than the decrease in nontreated eyes. The mean percentage of 6-sided cells in treated eyes of group-2 dogs was significantly (P 0.05) less than that in treated eyes of group-1 dogs at postinjection hour 168
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