Solution structure of the lymphocyte receptor Nkrp1a reveals a distinct conformation of the long loop region as compared to in the crystal structure

Mouse Nkrp1a receptor is a C‐type lectin‐like receptor expressed on the surface of natural killer cells that play an important role against virally infected and tumor cells. The recently solved crystal structure of Nkrp1a raises questions about a long loop region which was uniquely extended from the central region in the crystal. To understand the functional significance of the loop, the solution structure of Nkrp1a using nuclear magnetic resonance (NMR) spectroscopy was determined. A notable difference between the crystal and NMR structure of Nkrp1a appears in the conformation of the long loop region. While the extended loop points away from the central core and mediates formation of a domain swapped dimer in the crystal, the solution structure is monomeric with the loop tightly anchored to the central region. The findings described the first solution structure in the Nkrp1 family and revealed intriguing similarities and differences to the crystal structure. Proteins 2016; 84:1304–1311. © 2016 Wiley Periodicals, Inc.     

The drivers of avian‐haemosporidian prevalence in tropical lowland forests of New Guinea in three dimensions

Haemosporidians are among the most common parasites of birds and often negatively impact host fitness. A multitude of biotic and abiotic factors influence these associations, but the magnitude of these factors can differ by spatial scales (i.e., local, regional and global). Consequently, to better understand global and regional drivers of avian‐haemosporidian associations, it is key to investigate these associations at smaller (local) spatial scales. Thus, here, we explore the effect of abiotic variables (e.g., temperature, forest structure, and anthropogenic disturbances) on haemosporidian prevalence and host–parasite networks on a horizontal spatial scale, comparing four fragmented forests and five localities within a continuous forest in Papua New Guinea. Additionally, we investigate if prevalence and host–parasite networks differ between the canopy and the understory (vertical stratification) in one forest patch. We found that the majority of Haemosporidian infections were caused by the genus Haemoproteus and that avian‐haemosporidian networks were more specialized in continuous forests. At the community level, only forest greenness was negatively associated with Haemoproteus infections, while the effects of abiotic variables on parasite prevalence differed between bird species. Haemoproteus prevalence levels were significantly higher in the canopy, and an opposite trend was observed for Plasmodium. This implies that birds experience distinct parasite pressures depending on the stratum they inhabit, likely driven by vector community differences. These three‐dimensional spatial analyses of avian‐haemosporidians at horizontal and vertical scales suggest that the effect of abiotic variables on haemosporidian infections are species specific, so that factors influencing community‐level infections are primarily driven by host community composition.     

Decorated surfaces by biofunctionalized gold beads: application to cell adhesion studies

We describe a simple but versatile method to decorate solid surfaces randomly with colloidal gold particles to which ligands of cell receptors can be coupled to generate local attraction sites for the control of cell adhesion. A self-assembled monolayer of (3-mercaptopropyl)trimethoxysilane was deposited on glass slides. Gold beads were anchored to the functionalized surface through the sulfur group. We characterized the gold bead distribution on the functionalized surface with reflection interference contrast microscopy. The gold beads were functionalized with a disulfide-coupled cyclic pentapeptide containing an arginine-glycine-aspartic acid (RGD) tripeptide sequence which is selectively recognized by integrin receptors alpha(V)beta(3) of endothelial cells. A blocking layer of bovine serum albumin was adsorbed onto the surface to prevent non-specific binding of the cells. We demonstrate that the RGD-functionalized colloidal gold beads act as local attraction centers, mediating rapid cell anchoring on a substrate impeding cell adhesion in the absence of attraction centers. Surprisingly, microinterferometry shows that after a time delay of about 1 h, the regions of the cell surface between the gold beads form close contacts with the substrate, which is attributed to strong van der Waals attraction after escape of repeller molecules from the contact surface.     

Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

Enantioselective metabolism of primaquine by human CYP2D6

Given the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chloroquine (CQ) has been the most widely used anti-malarial, and new analogs (CQAns) presenting alkynes and side chain variations with high antiplasmodial activity were evaluated. Six diaminealkyne and diaminedialkyne CQAns were evaluated against CQ-resistant (CQ-R) (W2) and CQ-sensitive (CQ-S) (3D7) Plasmodium falciparum parasites in culture. Drug cytotoxicity to a human hepatoma cell line (HepG2) evaluated, allowed to calculate the drug selectivity index (SI), a ratio of drug toxicity to activity in vitro. The CQAns were re-evaluated against CQ-resistant and -sensitive P. berghei parasites in mice using the suppressive test. Docking studies with the CQAns and the human (Hss LDH) or plasmodial lactate dehydrogenase (Pf LDH) enzymes, and, a β-haematin formation assay were performed using a lipid as a catalyst to promote crystallization in vitro. All tested CQAns were highly active against CQ-R P. falciparum parasites, exhibiting half-maximal inhibitory concentration (IC50) values below 1 μΜ. CQAn33 and CQAn37 had the highest SIs. Docking studies revealed the best conformation of CQAn33 inside the binding pocket of Pf LDH; specificity between the residues involved in H-bonds of the Pf LDH with CQAn37. CQAn33 and CQAn37 were also shown to be weak inhibitors of Pf LDH. CQAn33 and CQAn37 inhibited β-haematin formation with either a similar or a 2-fold higher IC50 value, respectively, compared with CQ. CQAn37 was active in mice with P. berghei, reducing parasitaemia by 100%. CQAn33, -39 and -45 also inhibited CQ-resistant P. berghei parasites in mice, whereas high doses of CQ were inactive. The presence of an alkyne group and the size of the side chain affected anti-P. falciparum activity in vitro. Docking studies suggested a mechanism of action other than Pf LDH inhibition. The β-haematin assay suggested the presence of an additional mechanism of action of CQAn33 and CQAn37. Tests with CQAn34, CQAn37, CQAn39 and CQAn45 confirmed previous results against P. berghei malaria in mice, and CQAn33, 39 and 45 were active against CQ-resistant parasites, but CQAn28 and CQAn34 were not. The result likely reflects structure-activity relationships related to the resistant phenotype.     

Light scattering corrections to linear dichroism spectroscopy for liposomes in shear flow using calcein fluorescence and modified Rayleigh-Gans-Debye-Mie scattering

The interpretation of data from absorbance spectroscopy experiments of liposomes in flow systems is often complicated by the fact that there is currently no easy way to account for scattering artefacts. This has proved particularly problematic for linear dichroism (LD) spectroscopy, which may be used to determine binding modes of small molecules, peptides and proteins to liposomes if we can extract the absorbance signal from the combined absorbance/scattering experiment. Equations for a modified Rayleigh-Gans-Debye (RGD) approximation to the turbidity (scattering) LD spectrum are available in the literature though have not been implemented. This review summarises the literature and shows how it can be implemented. The implementation proceeds by first determining volume loss that occurs when a spherical liposome is subjected to flow. Calcein fluorescence can be used for this purpose since at high concentrations (>?60 mM) it has low intensity fluorescence with maxima at 525 and 563 nm whereas at low concentrations (<1 mM) the fluorescence intensity is enhanced and the band shifts to 536 nm. The scattering calculation process yields the average axis ratios of the distorted liposome ellipsoids and extent of orientation of the liposomes in flow. The scattering calculations require methods to estimate liposome integrity, volume loss, and orientation when subjected to shear stresses under flow.     

Predation on artificial and natural nests in the lowland rainforest of Papua New Guinea

Capsule: Although survival of nests was similar between forest fragments and continuous forest, the range of predators differed. Artificial nests provide an under-estimate of nest predation by snakes.

Aims: To estimate the natural nest predation rate in continuous primary forest, compare it with predation rates in forest fragments. To assess the reliability of nest survival rates determined by the use of artificial nests with clay eggs and identify the main nest predators.

Methods: We observed survival of natural nests during the incubation period in continuous primary forest in Papua New Guinea. Some nests were monitored with infrared cameras. We also used artificial nests deployed with clay eggs to identify predators.

Results: There was a predation rate of 50% for natural nests and snakes were major predators of nest contents. Clutch daily survival rates (DSRs) differed among nest types. The DSR of artificial nests (0.977) was not significantly different to that of natural cup nests (0.969). Survival rates of artificial nests were similar in forest fragments and continuous forest. Forest fragments had, however, a higher proportion of avian predators than continuous forest.

Conclusion: Although, we observed similar survival rates in artificial and natural nests, the composition of nest predators was different between natural and artificial nests. Artificial nests were not suitable for estimating the real predation caused by reptiles. Nevertheless, we find that participation of avian nest predators can be estimated correctly with the use of artificial nests.     

Infrared absorbance spectroscopy of aqueous proteins: Comparison of transmission and ATR data collection and analysis for secondary structure fitting

Attenuated total reflectance (ATR) infrared absorbance spectroscopy of proteins in aqueous solution is much easier to perform than transmission spectroscopy, where short path‐length cells need to be assembled reproducibly. However, the shape of the resulting ATR infrared spectrum varies with the refractive index of the sample and the instrument configuration. Refractive index in turn depends on the absorbance of the sample. In this work, it is shown that a room temperature triglycine sulfate detector and a ZnSe ATR unit can be used to collect reproducible spectra of proteins. A simple method for transforming the protein ATR spectrum into the shape of the transmission spectrum is also given, which proceeds by approximating a Kramers‐Krönig–determined refractive index of water as a sum of four linear components across the amide I and II regions. The light intensity at the crystal surface (with 45° incidence) and its rate of decay away from the surface is determined as a function of the wave number–dependent refractive index as well as the decay of the evanescent wave from the surface. The result is a single correction factor at each wave number. The spectra were normalized to a maximum of 1 between 1600 cm^?1 and 1700 cm^?1 and a self‐organizing map secondary structure fitting algorithm, SOMSpec, applied using the BioTools reference set. The resulting secondary structure estimates are encouraging for the future of ATR spectroscopy for biopharmaceutical characterization and quality control applications.     

Hay-bait traps are a useful tool for sampling of soil dwelling millipedes and centipedes

Some species of centipedes and millipedes inhabit upper soil layers exclusively and are not recorded by pitfall trapping. Because of their sensitivity to soil conditions, they can be sampled quantitatively for evaluation of soil conditions. Soil samples are heavy to transport and their processing is time consuming, and such sampling leads to disturbance of the soil surface which land-owners do not like. We evaluated the use of hay-bait traps to sample soil dwelling millipedes and centipedes. The effectiveness of this method was found to be similar to the effectiveness of soil sampling. Hay-bait traps installed for 8–10 weeks can substitute for direct soil sampling in ecological and inventory studies.