Indirect and direct effects of salinity on the quantity and quality of total amino acids in Ulva ohnoi (Chlorophyta)

Salinity can affect the quantity and quality of total amino acids (TAAs) in seaweeds indirectly by altering growth rates and thereby diluting or concentrating the amino acid content of the biomass, or directly by altering the synthesis of specific amino acids and osmolytes. This study attempted to partition the indirect and direct effects of salinity on the quantity and quality of TAAs in the green seaweed Ulva ohnoi by culturing it under a range of salinities without nutrient limitation. Both the quantity and quality of TAAs varied across the salinity treatments. Quantity was most strongly related to the growth rate of the seaweed and was highest in the slowest growing seaweed. In contrast, the quality of TAAs (individual amino acids as a proportion of total content) was most strongly related to salinity for all amino acids, although this varied substantially among individual amino acids. Increases in salinity were positively correlated with the proportion of proline (46% increase), tyrosine (36% increase), and histidine (26% increase), whereas there was a negative correlation with alanine (29% decrease). The proportion of methionine, with strong links to the synthesis of the osmolyte dimethylsulfoniopropionate, did not correlate linearly with salinity and instead was moderately higher at the optimal salinities for growth. These results show that salinity simultaneously affects the quantity and quality of TAAs in seaweed through both indirect and direct mechanisms, with growth rates playing the overarching role in determining the quantity of TAAs.     

A Highlights from MBoC Selection: Spatial distribution of cell–cell and cell–ECM adhesions regulates force balance while main-taining E-cadherin molecular tension in cell pairs

Mechanical linkage between cell–cell and cell–extracellular matrix (ECM) adhesions regulates cell shape changes during embryonic development and tissue homoeostasis. We examined how the force balance between cell–cell and cell–ECM adhesions changes with cell spread area and aspect ratio in pairs of MDCK cells. We used ECM micropatterning to drive different cytoskeleton strain energy states and cell-generated traction forces and used a Förster resonance energy transfer tension biosensor to ask whether changes in forces across cell–cell junctions correlated with E-cadherin molecular tension. We found that continuous peripheral ECM adhesions resulted in increased cell–cell and cell–ECM forces with increasing spread area. In contrast, confining ECM adhesions to the distal ends of cell–cell pairs resulted in shorter junction lengths and constant cell–cell forces. Of interest, each cell within a cell pair generated higher strain energies than isolated single cells of the same spread area. Surprisingly, E-cadherin molecular tension remained constant regardless of changes in cell–cell forces and was evenly distributed along cell–cell junctions independent of cell spread area and total traction forces. Taken together, our results showed that cell pairs maintained constant E-cadherin molecular tension and regulated total forces relative to cell spread area and shape but independently of total focal adhesion area.     

Chemical and biological characterisation of a sensor surface for bioprocess monitoring

This paper describes the step-wise fabrication and characterisation of a multi-layer dual polarization interferometry (DPI) based biosensor utilising Protein G (ProG) as the bio-recognition layer for the detection of a fragment antibody (Fab'). The biosensor is capable of monitoring the concentration of Fab' product within the extracellular medium of a fed-batch fermentation after leakage from Escherichia coli (E.coli). The activity, stability and functionality of each sensor layer were analysed in situ using DPI, whilst the chemical identity and homogeneity of the chemical layers were assessed ex situ using X-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS). Two different biotin linkers were found to produce hugely differing surfaces after the capture of NeutrAvidin? (NA) and biotinylated Protein G (b-ProG). The hydrophilic (PEG)(4)-biotin linker resulted in a surface where the b-ProG layer was deposited and organised above the NA layer producing an active and stable surface, whilst the hydrophobic LC-biotin linker generated a surface where the b-ProG layer was buried within the NA layer leading to variable surfaces and poor binding of the Fab' target. The biosensor has a detection limit of 1.7 μg/ml with a dynamic range covering two orders of magnitude. The sensor can detect the onset of Fab' leakage as early as 2h following product induction, with high signal-to-noise ratios and little interference from extracellular components. Leakage of Fab' followed a biphasic profile, switching to a more rapid rate 20 h after induction, indicating accelerated product loss and the need for cultivation harvest.     

An integrated approach to assessing nitroso-redox balance in systemic inflammation

Most studies examining the metabolic fate of NO during systemic inflammation have focused on measuring the quantitatively predominating, stable anions nitrite and nitrate within the circulation. However, these are not necessarily the NO-related products that govern NO metabolism and signaling in tissues. We assessed all major NO derivatives temporally in blood and vital organs during inflammation and explored their relationship to insult severity and redox status. Male rats receiving intraperitoneal endotoxin or vehicle were sacrificed for organ and blood sampling between 0 and 24 h. Endotoxin induced transient and organ-specific changes in a variety of NO metabolites. Nitrite and nitrate increased, peaking at 8 and 12 h, respectively. S- and N-nitrosation and heme-nitrosylation products also peaked at 8 h; these posttranslational protein modifications were associated with decreased myocardial function (echocardiography). Evidence of oxidative stress and systemic inflammation was also obtained. The rise in most NO derivatives was proportional to insult severity. All metabolite levels normalized within 24 h, despite evidence of persisting myocardial dysfunction and clinical unwellness. Our findings point to a complex interplay between NO production, antioxidant defense, and redox status. Although the precise (patho)physiologic roles of specific NO derivatives and their diagnostic/prognostic utility await further investigation, nitroso species in erythrocytes are the most sensitive markers of NO in systemic inflammation, detectable before clinical symptoms manifest.     

Evolutionary convergence on sleep loss in cavefish populations

Patterns of sleep vary widely among species, but the functional and evolutionary principles responsible for this diversity remain unknown. The characin fish, Astyanax mexicanus, has eyed surface and numerous blind cave populations. The cave populations are largely independent in their origins, and the species is ideal for studying the genetic bases of convergent evolution. Here we show that this system is also uniquely valuable for the investigation of variability in patterns of sleep. We find that a clearly defined change in ecological conditions, from surface to cave, is correlated with a dramatic reduction in sleep in three independently derived cave populations of A. mexicanus. Analyses of surface × cave hybrids show that the alleles for reduced sleep in the Pachón and Tinaja cave populations are dominant in effect to the surface alleles. Genetic analysis of hybrids between surface and Pachón cavefish suggests that only a small number of loci with dominant effects are involved. Our results demonstrate that sleep is an evolutionarily labile phenotype, highly responsive to changes in ecological conditions. To our knowledge, this is the first example of a single species with a convergence on sleep loss exhibited by several independently evolved populations correlated with population-specific ecologies.     

The identification of substituted benzothiophene derivatives as PGE(2) subtype 4 receptor antagonists: From acid to non-acid

We disclose herein our preliminary SAR study on the identification of substituted benzothiophene derivatives as PGE₂ subtype 4 receptor antagonists. A potent EP₄ antagonist 6a (K_i = 1.4 nM with 10% HSA) was identified. Furthermore, we found that an acidic group was not essential for the EP₄ antagonizing activity in the series and neutral replacements were identified. This opens a new direction for future EP₄ antagonist design.     

Pyrazolopyridine inhibitors of B-RafV600E. Part 2: structure-activity relationships

Structure-activity relationships around a novel series of B-Raf(V600E) inhibitors are reported. The enzymatic and cellular potencies of inhibitors derived from two related hinge-binding groups were compared and3-methoxypyrazolopyridine proved to be superior. The 3-alkoxy group of lead B-Raf(V600E) inhibitor 1 was extended and minimally affected potency. The propyl sulfonamide tail of compound 1, which occupies the small lipophilic pocket formed by an outward shift of the αC-helix, was expanded to a series of arylsulfonamides. X-ray crystallography revealed that this lipophilic pocket unexpectedly enlarges to accommodate the bulkier aryl group.     

Dynamics of the phosphoinositide 3-kinase p110δ interaction with p85α and membranes reveals aspects of regulation distinct from p110α

Phosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110β and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes.