Comparative analysis of the methods used for finding surface energy to investigate protein interaction behavior on chromatographic supports

Hydrophobic interaction chromatography, an important and effective purification strategy, is generally used for the purification of variety of biomolecules. A basic understanding of the protein interaction behavior is required to effectively separate these biomolecules. A colloidal type extended Derjaguin, Landau, Verwey, and Overbeek calculations were utilized to study the interactions behavior of model proteins to commercially available hydrophobic chromatographic materials that is, Toyopearl Phenyl 650C and Toyopearl Butyl 650C. Physicochemical properties of selected model proteins were achieved by contact angle and zeta potential measurements. The contact angle of chromatographic materials used was achieved through sessile drop method on disrupted beads and capillary penetration method (CPM) on intact beads. The surface properties were further used to calculate the interactions of the proteins to chromatographic supports. The calculated secondary energy minimum of the proteins with the chromatographic materials (from the contact angle values determined through both methods can be correlated with the retention volumes from the real chromatography. The secondary energy minimum values are higher for each protein to the chromatographic materials calculated from the inputs derived through sessile drop method compared to CPM. For instance, immunoglobulin G has secondary energy minimum value of 0.17 kT compared to 0.11 kT, obtained through sessile drop method and CPM, respectively. Average relative values of the energy minimum calculated for all proteins are as 1.51 kT and 1.29 kT for Toyopearl Butyl 650C and Toyopearl Phenyl 650C, respectively, as a conversion factor for estimation of secondary energy minimum for both methods.     

Rapid degradation of pyrogenic carbon

Pyrogenic carbon (PC‐ charcoal, biochar or black carbon) represents a poorly understood component of the global carbon (C) cycle, but one that has considerable potential to mitigate climate change through provision of long‐term soil C sequestration. Mass balance calculations suggest global PC production and stocks are not in balance, indicating a major gap in our understanding of the processes by which PC is re‐mineralized. We collected PC samples derived from the same wood material and exposed to natural environmental conditions for 1 and 11 years. We subjected these materials to repeated laboratory incubation studies at temperatures of up to 60 °C, as ground surface temperatures above 30 °C and up to 60 °C occur regularly over a significant area of the tropics and sub‐tropics. Mineralization rates were not different for the two samples and followed an exponential Arrhenius function that suggest an average turnover time of 67 years for conditions typical of a tropical savannah environment. Microbial biomass as measured by chloroform fumigation and DNA extractions was the same for the two samples, but abiotic CO₂ production was lower for the fresh PC sample than that for the aged sample. Nuclear magnetic resonance spectroscopy, hydrogen pyrolysis and scanning electron microscopy demonstrate that the measured CO₂ production originates dominantly from polycyclic aromatic compounds rather than any minor labile components. Therefore, rapid, sub‐centennial rates of re‐mineralization of PC on the soil surface in tropical and sub‐tropical environments may represent a major and hitherto unidentified mechanism for balancing the PC production at the global scale.     

The Effects of Root-knot Nematode Infection and Mi-mediated Nematode Resistance in Tomato on Plant Fitness

The Mi-1.2 resistance gene in tomato (Solanum lycopersicum) confers resistance against several species of root-knot nematodes (Meloidogyne spp.). This study examined the impact of M. javanica on the reproductive fitness of near-isogenic tomato cultivars with and without Mi-1.2 under field and greenhouse conditions. Surprisingly, neither nematode inoculation or host plant resistance impacted the yield of mature fruits in field microplots (inoculum=8,000 eggs/plant), or fruit or seed production in a follow-up greenhouse bioassay conducted with a higher inoculum level (20,000 eggs/plant). However, under heavy nematode pressure (200,000 eggs/plant), greenhouse-grown plants carrying Mi-1.2 had more than ten-fold greater fruit production than susceptible plants and nearly forty-fold greater estimated lifetime seed production, confirming prior reports of the benefits of Mi-1.2. In all cases Mi-mediated resistance significantly reduced nematode reproduction. These results indicated that tomato can utilize tolerance mechanisms to compensate for moderate levels of nematode infection, but that the Mi-1.2 resistance gene confers a dramatic fitness benefit under heavy nematode pressure. No significant cost of resistance was detected in the absence of nematode infection.     

All-Atom Structural Models for Complexes of Insulin-Like Growth Factors IGF1 and IGF2 with Their Cognate Receptor

Type 1 insulin-like growth factor receptor (IGF1R) is a membrane-spanning glycoprotein of the insulin receptor family that has been implicated in a variety of cancers. The key questions related to molecular mechanisms governing ligand recognition by IGF1R remain unanswered, partly due to the lack of testable structural models of apo or ligand-bound receptor complexes. Using a homology model of the IGF1R ectodomain IGF1RΔβ, we present the first experimentally consistent all-atom structural models of IGF1/IGF1RΔβ and IGF2/IGF1RΔβ complexes. Our explicit-solvent molecular dynamics (MD) simulation of apo-IGF1RΔβ shows that it displays asymmetric flexibility mechanisms that result in one of two binding pockets accessible to growth factors IGF1 and IGF2, as demonstrated via an MD-assisted Monte Carlo docking procedure. Our MD-generated ensemble of structures of apo and IGF1-bound IGF1RΔβ agrees reasonably well with published small-angle X-ray scattering data. We observe simultaneous contacts of each growth factor with sites 1 and 2 of IGF1R, suggesting cross-linking of receptor subunits. Our models provide direct evidence in favor of suggested electrostatic complementarity between the C-domain (IGF1) and the cysteine-rich domain (IGF1R). Our IGF1/IGF1RΔβ model provides structural bases for the observation that a single IGF1 molecule binds to IGF1RΔβ at low concentrations in small-angle X-ray scattering studies. We also suggest new possible structural bases for differences in the affinities of insulin, IGF1, and IGF2 for their noncognate receptors.     

Fab is the most efficient format to express functional antibodies by yeast surface display

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.     

Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers

Ostreolysin, a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), recognizes and binds specifically to membrane domains enriched in cholesterol and sphingomyelin (or saturated phosphatidylcholine). These events, leading to permeabilization of the membrane, suggest that a cholesterol-rich liquid-ordered membrane phase, which is characteristic of lipid rafts, could be its possible binding site. In this work, we present effects of ostreolysin on membranes containing various steroids. Binding and membrane permeabilizing activity of ostreolysin was studied using lipid mono- and bilayers composed of sphingomyelin combined, in a 1/1 molar ratio, with natural and synthetic steroids (cholesterol, ergosterol, β-sitosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, cholesteryl acetate, and 5-cholesten-3-one). Binding to membranes and lytic activity of the protein are both shown to be dependent on the intact sterol 3β-OH group, and are decreased by introducing additional double bonds and methylation of the steroid skeleton or C17-isooctyl chain. The activity of ostreolysin mainly correlates with the ability of the steroids to promote formation of liquid-ordered membrane domains, and is the highest with cholesterol-containing membranes. Furthermore, increasing the cholesterol concentration enhanced ostreolysin binding in a highly cooperative manner, suggesting that the membrane lateral distribution and accessibility of the sterols are crucial for the activity of this new member of cholesterol-dependent cytolysins.     

Divergent selection drives the adaptive radiation of crossbills

Abstract Knowledge of how phenotype influences fitness is necessary if we are to understand the basis of natural selection and how natural selection contributes to adaptive radiations. Here I quantify selection on a wild population of red crossbills ( Loxia curvirostra complex) in the South Hills, Idaho. Bill depth is the target of selection and selection on bill depth is stabilizing. I then show how fitness is related to both bill depth and performance. I use these and previously published relationships to estimate a fitness surface for five species of red crossbills that are part of an ongoing adaptive radiation in western North America. The fitness surface for crossbills has distinct peaks and valleys, with each crossbill species residing on or very near the summits. This work strongly supports a key tenet of the ecological theory of adaptive radiations; namely, divergent selection for utilizing alternative resources is the ultimate cause of adaptive radiations.     

Enzyme synthesis of oligosaccharides using cashew apple juice as substrate

The use of agriculture substrates in industrial biotechnological processes has been increasing because of their low cost. In this work, the use of clarified cashew apple juice was investigated as substrate for enzyme synthesis of prebiotic oligosaccharide. The results showed that cashew apple juice is a good source of reducing sugars and can be used as substrate for the production of dextransucrase by Leuconostoc citreum B-742 for the synthesis of oligosaccharides using the crude enzyme. Optimal oligosaccharide yield (approximately 80%) was obtained for sucrose concentrations lower than 60 g/L and reducing sugar concentrations higher than 100 g/L.     

Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in plasma cholesterol regulation through modulation of low density lipoprotein receptor (LDLR) levels. Naturally occurring mutations can lead to hyper- or hypocholesterolemia in human. Recently, we reported that PCSK9 was also able to modulate CD81 in Huh7 cells. In the present study, several gain-of-function and loss-of-function mutants as well as engineered mutants of PCSK9 were compared for their ability to modulate the cell surface expression of LDLR and CD81. Although PCSK9 gain-of-function D374Y enhanced the degradation both receptors, D374H and D129N seemed to only reduce LDLR levels. In contrast, mutations in the C-terminal hinge-cysteine-histidine-rich domain segment primarily affected the PCSK9-induced CD81 degradation. Furthermore, when C-terminally fused to an ACE2 transmembrane anchor, the secretory N-terminal catalytic or hinge-cysteine-histidine-rich domain domains of PCSK9 were able to reduce CD81 and LDLR levels. These data confirm that PCSK9 reduces CD81 levels via an intracellular pathway as reported for LDLR. Using immunocytochemistry, a proximity ligation assay, and co-immunoprecipitation, we found that the cell surface level of PCSK9 was enhanced upon overexpression of CD81 and that both PCSK9 and LDLR interact with this tetraspanin protein. Interestingly, using CHO-A7 cells lacking LDLR expression, we revealed that LDLR was not required for the degradation of CD81 by PCSK9, but its presence strengthened the PCSK9 effect.