Bay-scale population structure in coastal Atlantic cod in Labrador and Newfoundland, Canada

Polymorphisms at five microsatellite DNA loci provide evidence that Atlantic cod Gadus morhua inhabiting Gilbert Bay, Labrador are genetically distinguishable from offshore cod on the north-east Newfoundland shelf and from inshore cod in Trinity Bay, Newfoundland. Antifreeze activity in the blood suggests that Gilbert Bay cod overwinter within the Bay. Gilbert Bay cod are also smaller (weight and length) for their age and consequently less fecund for their age, than cod elsewhere within the northern cod complex. The productivity and recruitment potential of coastal cod off Labrador may thus be much lower than that of offshore northern cod or of inshore cod farther south, implying that a more conservative management strategy may be required for cod from coastal Labrador than traditionally practised for northern cod inhabiting less harsh environments. Relatively high F _ST and R _ST measures of population structure suggest that important barriers to gene flow exist among five components that include two inshore (Gilbert and Trinity Bay) and three offshore cod aggregations on the north-east Newfoundland Shelf and the Grand Bank. D _A and D _SW estimates of genetic distance that involve Gilbert Bay cod are approximately three- and 10–fold larger, respectively, than estimates not involving Gilbert Bay cod. The differences between inshore cod from Gilbert Bay and Trinity Bay raise the possibility that other genetically distinguishable coastal populations may exist, or may have existed prior to the northern cod fishery collapse. Harvesting strategies for northern cod should recognize the existence of genetic diversity between inshore and offshore components as well as among coastal components.     

Isolation of dermatoxin from frog skin, an antibacterial peptide encoded by a novel member of the dermaseptin genes family.

A 32-residue peptide, named dermatoxin, has been extracted from the skin of a single specimen of the tree frog Phyllomedusa bicolor, and purified to homogeneity using a four-step protocol. Mass spectral analysis and sequencing of the purified peptide, as well as chemical synthesis and cDNA analysis were consistent with the structure: SLGSFLKGVGTTLASVGKVVSDQF GKLLQAGQ. This peptide proved to be bactericidal towards mollicutes (wall-less eubacteria) and Gram-positive eubacteria, and also, though to a lesser extent, towards Gram-negative eubacteria. Measurement of the bacterial membrane potential revealed that the plasma membrane is the primary target of dermatoxin. Observation of bacterial cells using reflected light fluorescence microscopy after DNA-staining was consistent with a mechanism of cell killing based upon the alteration of membrane permeability rather than membrane solubilization, very likely by forming ion-conducting channels through the plasma membrane. CD spectroscopy and secondary structure predictions indicated that dermatoxin assumes an amphipathic alpha-helical conformation in low polarity media which mimic the lipophilicity of the membrane of target microorganisms. PCR analysis coupled with cDNA cloning and sequencing revealed that dermatoxin is expressed in the skin, the intestine and the brain. Preprodermatoxin from the brain and the intestine have the same sequence as the skin preproform except for two amino-acid substitutions in the preproregion of the brain precursor. The dermatoxin precursor displayed the characteristic features of preprodermaseptins, a family of peptide precursors found in the skin of Phyllomedusa ssp. Precursors of this family have a common N-terminal preproregion followed by markedly different C-terminal domains that give rise to 19-34-residue peptide antibiotics named dermaseptins B and phylloxin, and to the D-amino-acid-containing opioid heptapeptides dermorphins and deltorphins. Because the structures and cidal mechanisms of dermatoxin, dermaseptins B and phylloxin are very different, dermatoxin extends the repertoire of structurally and functionally diverse peptides derived from the rapidly evolving C-terminal domains of precursors of the dermaseptins family.     

Balancing charge in the complementarity-determining regions of humanized mAbs without affecting pI reduces non-specific binding and improves the pharmacokinetics

Lowering the isoelectric point (pI) through engineering the variable region or framework of an IgG can improve its exposure and half-life via a reduction in clearance mediated through non-specific interactions. As such, net charge is a potentially important property to consider in developing therapeutic IgG molecules having favorable pharmaceutical characteristics. Frequently, it may not be possible to shift the pI of monoclonal antibodies (mAbs) dramatically without the introduction of other liabilities such as increased off-target interactions or reduced on-target binding properties. In this report, we explored the influence of more subtle modifications of molecular charge on the in vivo properties of an IgG1 and IgG4 monoclonal antibody. Molecular surface modeling was used to direct residue substitutions in the complementarity-determining regions (CDRs) to disrupt positive charge patch regions, resulting in a reduction in net positive charge without affecting the overall pI of the mAbs. The effect of balancing the net positive charge on non-specific binding was more significant for the IgG4 versus the IgG1 molecule that we examined. This differential effect was connected to the degree of influence on cellular degradation in vitro and in vivo clearance, distribution and metabolism in mice. In the more extreme case of the IgG4, balancing the charge yielded an ∼7-fold improvement in peripheral exposure, as well as significantly reduced tissue catabolism and subsequent excretion of proteolyzed products in urine. Balancing charge on the IgG1 molecule had a more subtle influence on non-specific binding and yielded only a modest alteration in clearance, distribution and elimination. These results suggest that balancing CDR charge without affecting the pI can lead to improved mAb pharmacokinetics, the magnitude of which is likely dependent on the relative influence of charge imbalance and other factors affecting the molecule''s disposition.     

Host‐induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce

Host‐induced gene silencing (HIGS) is an RNA interference‐based approach in which small interfering RNAs (siRNAs) are produced in the host plant and subsequently move into the pathogen to silence pathogen genes. As a proof‐of‐concept, we generated stable transgenic lettuce plants expressing siRNAs targeting potentially vital genes of Bremia lactucae, a biotrophic oomycete that causes downy mildew, the most important disease of lettuce worldwide. Transgenic plants, expressing inverted repeats of fragments of either the Highly Abundant Message #34 (HAM34) or Cellulose Synthase (CES1) genes of B. lactucae, specifically suppressed expression of these genes, resulting in greatly reduced growth and inhibition of sporulation of B. lactucae. This demonstrates that HIGS can provide effective control of B. lactucae in lettuce; such control does not rely on ephemeral resistance conferred by major resistance genes and therefore offers new opportunities for durable control of diverse diseases in numerous crops.     

Qualification of a free ligand assay in the presence of anti-ligand antibody Fab fragments

The aim of this work was to develop and characterize an ELISA to measure free ligand concentrations in rat serum in the presence of a Fab to the same ligand. A variety of experiments were conducted to understand optimal assay conditions and to verify that only free ligand was detected. The parameters explored included sample incubation time on plate, the initial concentrations of Fab and ligand, and the pre-incubation time required for the Fab-ligand complex concentrations to reach equilibrium. We found the optimal experimental conditions to include a 10-minute on-plate incubation of ligand-containing samples, with a 24-hour pre-incubation time for test samples of Fab and ligand to reach equilibrium. An alternative approach, involving removal of Fab-ligand complexes from the solution prior to measuring concentrations of the ligand, was also used to verify that the assay only measured free ligand. Rats were dosed subcutaneously with Fab and the assay was used to demonstrate dose-dependent suppression of endogenous free ligand levels in vivo.     

Potential biomarkers of an exaggerated response to endotoxemia

Serial plasma protein analysis was used to study the acute plasma proteome response to endotoxemia (presence of toxic bacterial products called endotoxins in the blood stream). Plasma samples from healthy volunteers before and multiple time points up to 24 h following administration of low-dose endotoxin were evaluated. Plasma protein profiles were obtained by rapid extraction of whole plasma followed by analysis with matrix-assisted laser desorption ionisation-time of flight mass spectrometry. The profiles were unique to each individual and stable over the time of the experiment. Administration of low-dose endotoxin caused profound change in six of 18 individuals. At 8 h many proteins showed quantitative oxidation, in addition to the appearance of new components and disappearance of common baseline components. An exceptionally intense new component at 4154 mass units was identified as the activation peptide of C1 esterase inhibitor. While recovery of baseline protein structure was nearly complete by 24 h, serum amyloid A, an acute-phase reactant, was still increasing and minor profile changes persisted. Clinical features did not distinguish these extreme responders from others, suggesting that plasma proteome changes offered unique insights into and potential biomarkers of subclinical events following endotoxin exposure.     

New Case of Intragroup Infanticide in the Chimpanzees of Gombe National Park

Researchers have reported a total of 31 infanticides in 4 different chimpanzee (Pan troglodytes) populations. Though infanticide is infrequent, low reproductive rates of females likely make it a strong selective pressure in the species. We report a new incident of intragroup infanticide in Gombe National Park, Tanzania, in which a community male attacked a 3.5-yr-old male. We then consider the infanticide in terms of adaptive and nonadaptive explanations for infanticide including the social pathology, by-product of male aggression, nutritive benefits, resource competition, and sexual selection hypotheses. The incident reported here is not well explained by any of them. While the infanticide is puzzling in terms of ultimate explanations for infanticide, it provides a good context in which to consider proximate mechanisms for offspring recognition. The incident provides some evidence that males may use their mating history with the mother to assess paternity likelihood.     

His-311 and Arg-559 are key residues involved in fatty acid oxygenation in pathogen-inducible oxygenase

Pathogen-inducible oxygenase (PIOX) oxygenates fatty acids into 2R-hydroperoxides. PIOX belongs to the fatty acid alpha-dioxygenase family, which exhibits homology to cyclooxygenase enzymes (COX-1 and COX-2). Although these enzymes share common catalytic features, including the use of a tyrosine radical during catalysis, little is known about other residues involved in the dioxygenase reaction of PIOX. We generated a model of linoleic acid (LA) bound to PIOX based on computational sequence alignment and secondary structure predictions with COX-1 and experimental observations that governed the placement of carbon-2 of LA below the catalytic Tyr-379. Examination of the model identified His-311, Arg-558, and Arg-559 as potential molecular determinants of the dioxygenase reaction. Substitutions at His-311 and Arg-559 resulted in mutant constructs that retained virtually no oxygenase activity, whereas substitutions of Arg-558 caused only moderate decreases in activity. Arg-559 mutant constructs exhibited increases of greater than 140-fold in K(m), whereas no substantial change in K(m) was observed for His-311 or Arg-558 mutant constructs. Thermal shift assays used to measure ligand binding affinity show that the binding of LA is significantly reduced in a Y379F/R559A mutant construct compared with that observed for Y379F/R558A construct. Although Oryza sativa PIOX exhibited oxygenase activity against a variety of 14-20-carbon fatty acids, the enzyme did not oxygenate substrates containing modifications at the carboxylate, carbon-1, or carbon-2. Taken together, these data suggest that Arg-559 is required for high affinity binding of substrates to PIOX, whereas His-311 is involved in optimally aligning carbon-2 below Tyr-379 for catalysis.