Small-Angle Neutron Scattering Characterization of Monoclonal Antibody Conformations and Interactions at High Concentrations

Small-angle neutron scattering (SANS) is used to probe the solution structure of two protein therapeutics (monoclonal antibodies 1 and 2 (MAb1 and MAb2)) and their protein-protein interaction (PPI) at high concentrations. These MAbs differ by small sequence alterations in the complementarity-determining region but show very large differences in solution viscosity. The analyses of SANS patterns as a function of different solution conditions suggest that the average intramolecular structure of both MAbs in solution is not significantly altered over the studied protein concentrations and experimental conditions. Even though a strong repulsive interaction is expected for both MAbs due to their net charges and low solvent ionic strength, analysis of the SANS data shows that the effective PPI for MAb1 is dominated by a very strong attraction at small volume fraction that becomes negligible at large concentrations. The MAb1 PPI cannot be modeled simply by a spherically symmetric central forces model. It is proposed that an anisotropic attraction strongly affects the local interprotein structure and leads to an anomalously large viscosity of concentrated MAb1 solutions. Conversely, MAb2 displays a repulsive interaction potential throughout the concentration series probed and a comparatively small solution viscosity.     

Silver Protein Staining of Nerve Fibers in Celloidin Sections

Celloidin sections from formalin-fixed brain and spinal cord of primates are stored in 70% alcohol after cutting, soaked in 2% pyridine in 50% alcohol for 6-8 hr at 37 C, and transferred to 1% concentrated NH₄OH in 50% alcohol 15-18 hr at 20-25 C. After washing and flattening, the sections are transferred to 1% silver protein solution containing 30 ml of 0.2 M H₃BO₃/100 ml. Impregnation is accomplished in 50 ml screw-top jars, 50 mm in diameter, which are filled to a depth of 35 mm, and have 1 gm of copper foil, 0.002 inch thick added. The foil is folded in loose accordion-fashion, pierced and threaded, cleaned in 5% HNO₃, rinsed in distilled water, and suspended in the solution just above the sections by fastening the thread to the jar lid. The sections are impregnated for 24 hr at 37 C, rinsed in distilled water, reduced in a solution of 5% Na₂SO₃ and 1% hydroquinone for 10 min, washed in distilled water and toned in 0.2% gold chloride for 5 min. After rinsing in distilled water, the sections are transferred to 1% oxalic acid for 45-60 sec, washed in distilled water and placed in 5% Na₂S₂O₃ for 5 min. Sections are then washed, dehydrated to 95% alcohol, cleared in terpineol, followed by 3 changes in xylene, and mounted.     

Impact of Bythotrephes longimanus on zooplankton assemblages of Harp Lake, Canada: an assessment based on predator consumption and prey production

1. Following the 1993 invasion of Harp Lake by Bythotrephes longimanus a number of small-bodied zooplankton declined dramatically compared to pre-invasion densities, and some larger species increased. 2. To test whether these changes were caused by Bythotrephes, we measured its consumption of zooplankton in 1995 and compared this to production by the prey species with which it overlapped spatially and temporally. 3. On a seasonal basis Bythotrephes consumption was 199 mg dry mass (DM) m⁻², or 25% of zooplankton production which was 783 mg DW m⁻². However, for some species, such as the small copepod Tropocyclops extensus , consumption greatly exceeded production—this was one of the species noted to decline in Harp Lake following the appearance of Bythotrephes . By contrast, for the larger cladocerans Daphnia galeata mendotae and Holopedium gibberum , consumption was much less than production—these species were observed to increase following the invasion. 4. Our results thus support the hypothesis that zooplankton changes in Harp Lake were caused by Bythotrephes . We speculate that lower quality prey remaining in Harp Lake may lead to reduced densities of Bythotrephes , or impose strong selective forces that lead to new adaptations by this predator.     

Ignoring discards biases the assessment of fisheries' ecological fingerprint

Understanding the pressures of fisheries on the ecosystem is crucial for effective management. Fishery removals, or catch, are composed of both landings and discards. However, the use of discards data in studies investigating the effect of the fishing pressures is sparse. Here, we explore the individual contribution of both these catch components to the overall pressure of fisheries on the ecosystem metrics. Using Irish observer data, we compare the linear relationship between several ecological metrics calculated for landings and discards with those of catch. Our results show that in fisheries with high discarding rates, discards can drive the fisheries’ ecological fingerprint and highlight the need to rectify landings-based estimates to make them representative of those of catch in order to gain a robust picture of the impact of fisheries.     

Mechanical measures during maximal velocity knee extension exercise and their relation to fibre composition of the human vastus lateralis muscle

A method for measuring the maximal velocity of knee extension exercise is described using a very light lever arm. Instrumentation of the lever arm with a potentiometer and accelerometer also allows for the measurement of peak acceleration, time to peak acceleration, the average rate of development of acceleration (jerk) and peak torque. With this apparatus and surface electromyography, electromechanical delay (EMD) was also determined. This apparatus was tested using 17 female and 10 male subjects, and the measures obtained were related to the percentage of fast twitch fibres (% FT) and the relative area of fast twitch fibres (% FTA) in the vastus lateralis determined from duplicate muscle biopsy samples. Peak velocity of unloaded knee extension averaged 12.1 +/- 1.2 and 12.2 +/- 1.7 rad.s-1 for females and males, respectively, and were not significantly different. As well, peak acceleration, time to peak acceleration jerk and EMD values were not significantly different between the female and male subjects, but the mean peak torque for the female subjects (73.5 +/- 14.7 N.m) was significantly lower than that for the males (98.4 +/- 31.5 N.m). Peak acceleration was significantly correlated with %FT (r = 0.40, P = 0.04) for the total subject population. None of the other measures was significantly related to either %FT or %FTA for the male and female subjects or the combined population of subjects.     

Molecular phylogeny of the benthic shallow-water octopuses (Cephalopoda: Octopodinae)

Octopus has been regarded as a "catch all" genus, yet its monophyly is questionable and has been untested. We inferred a broad-scale phylogeny of the benthic shallow-water octopuses (subfamily Octopodinae) using amino acid sequences of two mitochondrial DNA genes: Cytochrome oxidase subunit III and Cytochrome b apoenzyme, and the nuclear DNA gene Elongation Factor-1alpha. Sequence data were obtained from 26 Octopus species and from four related genera. Maximum likelihood and Bayesian approaches were implemented to estimate the phylogeny, and non-parametric bootstrapping was used to verify confidence for Bayesian topologies. Phylogenetic relationships between closely related species were generally well resolved, and groups delineated, but the genes did not resolve deep divergences well. The phylogenies indicated strongly that Octopus is not monophyletic, but several monophyletic groups were identified within the genus. It is therefore clear that octopodid systematics requires major revision.     

Co-evolution of Human Leukocyte Antigen (HLA) Class I Ligands with Killer-Cell Immunoglobulin-Like Receptors (KIR) in a Genetically Diverse Population of Sub-Saharan Africans

Interactions between HLA class I molecules and killer-cell immunoglobulin-like receptors (KIR) control natural killer cell (NK) functions in immunity and reproduction. Encoded by genes on different chromosomes, these polymorphic ligands and receptors correlate highly with disease resistance and susceptibility. Although studied at low-resolution in many populations, high-resolution analysis of combinatorial diversity of HLA class I and KIR is limited to Asian and Amerindian populations with low genetic diversity. At the other end of the spectrum is the West African population investigated here: we studied 235 individuals, including 104 mother-child pairs, from the Ga-Adangbe of Ghana. This population has a rich diversity of 175 KIR variants forming 208 KIR haplotypes, and 81 HLA-A, -B and -C variants forming 190 HLA class I haplotypes. Each individual we studied has a unique compound genotype of HLA class I and KIR, forming 1–14 functional ligand-receptor interactions. Maintaining this exceptionally high polymorphism is balancing selection. The centromeric region of the KIR locus, encoding HLA-C receptors, is highly diverse whereas the telomeric region encoding Bw4-specific KIR3DL1, lacks diversity in Africans. Present in the Ga-Adangbe are high frequencies of Bw4-bearing HLA-B*53:01 and Bw4-lacking HLA-B*35:01, which otherwise are identical. Balancing selection at key residues maintains numerous HLA-B allotypes having and lacking Bw4, and also those of stronger and weaker interaction with LILRB1, a KIR-related receptor. Correspondingly, there is a balance at key residues of KIR3DL1 that modulate its level of cell-surface expression. Thus, capacity to interact with NK cells synergizes with peptide binding diversity to drive HLA-B allele frequency distribution. These features of KIR and HLA are consistent with ongoing co-evolution and selection imposed by a pathogen endemic to West Africa. Because of the prevalence of malaria in the Ga-Adangbe and previous associations of cerebral malaria with HLA-B*53:01 and KIR, Plasmodium falciparum is a candidate pathogen.