Formation of virus-like clusters is an intrinsic property of the tumor necrosis factor family member BAFF (B cell activating factor)

The oligomeric state of BAFF (B cell activing factor), a tumor necrosis factor (TNF) family cytokine that plays a critical role in B cell development and survival, has been the subject of recent debate. Myc-tagged BAFF starting at residue Gln136 was previously reported to crystallize as trimers at pH 4.5, whereas a histidine-tagged construct of BAFF, starting at residue Ala134, formed a virus-like cluster containing 60 monomers when crystallized at pH 9.0. The formation of the BAFF 60-mer was pH dependent, requiring pH >or= 7.0. More recently, 60-mer formation was suggested to be artificially induced by the histidine tag, and it was proposed that BAFF, like all other TNF family members, is trimeric. We report here that a construct of BAFF with no amino-terminal tag (Ala134-BAFF) can form a 60-mer in solution. Using size exclusion chromatography and static light scattering to monitor trimer to 60-mer ratios in BAFF preparations, we find that 60-mer formation is pH-dependent and requires histidine 218 within the DE loop of BAFF. Biacore measurements established that the affinity of Ala134-BAFF for the BAFF receptor BAFFR/BR3 is similar to that of myc-Gln136-BAFF, which is exclusively trimeric in solution. However, Ala134-BAFF is more efficacious than myc-Gln136-BAFF in inducing B cell proliferation in vitro. We additionally show that BAFF that is processed and secreted by 293T cells transfected with full-length BAFF, or by a histiocytic lymphoma cell line (U937) that expresses BAFF endogenously, forms a pH-dependent 60-mer in solution. Our results indicate that the formation of the 60-mer in solution by the BAFF extracellular domain is an intrinsic property of the protein, and therefore that this more active form of BAFF may be physiologically relevant.     

Affinity enhancement of an in vivo matured therapeutic antibody using structure-based computational design

Improving the affinity of a high-affinity protein-protein interaction is a challenging problem that has practical applications in the development of therapeutic biomolecules. We used a combination of structure-based computational methods to optimize the binding affinity of an antibody fragment to the I-domain of the integrin VLA1. Despite the already high affinity of the antibody (Kd approximately 7 nM) and the moderate resolution (2.8 A) of the starting crystal structure, the affinity was increased by an order of magnitude primarily through a decrease in the dissociation rate. We determined the crystal structure of a high-affinity quadruple mutant complex at 2.2 A. The structure shows that the design makes the predicted contacts. Structural evidence and mutagenesis experiments that probe a hydrogen bond network illustrate the importance of satisfying hydrogen bonding requirements while seeking higher-affinity mutations. The large and diverse set of interface mutations allowed refinement of the mutant binding affinity prediction protocol and improvement of the single-mutant success rate. Our results indicate that structure-based computational design can be successfully applied to further improve the binding of high-affinity antibodies.     

Experimenting with Transmutation: Darwin, the Beagle, and Evolution

Detailed analysis of Darwin’s scientific notes and other writings from the Beagle voyage reveals a focus on endemism and replacement of allied taxa in time and in space that began early in the journey. Though it is impossible to determine exactly when Darwin became a transmutationist, the evidence suggests that he was conversant with the transmutational ideas of Lamarck and others and testing (“experimenting” with) them—before he received a copy of Lyell’s Principles of Geology, vol. 2, in November 1832, in which Lyell describes and disputes Lamarck’s theory. To the two rhea species of Patagonia and the four mockingbird species of the Galapagos, we can now add the living Patagonian cavy (rodent) species, and its extinct putatively related species that Darwin collected at Monte Hermoso (Bahia Blanca) in the Fall of 1832, as a replacement pattern absolutely critical to the development of Darwin’s transmutational thinking. Darwin developed his first transmutational theory by adopting “Brocchi’s analogy” (Rudwick 2008)—i.e. that births and deaths of species are analogous to the births and deaths of individuals. Births and deaths of species, as of individuals, are thus explicable in terms of natural causes. Darwin explored these themes and the replacement of the extinct cavy by the modern species explicitly in his February 1835 essay (Darwin 1835a).

A computational study of the flow through a vitreous cutter

Vitrectomy is an ophthalmic microsurgical procedure that removes part or all of the vitreous humor from the eye. The procedure uses a vitreous cutter consisting of a narrow shaft with a small orifice at the end through which the humor is aspirated by an applied suction. An internal guillotine oscillates back and forth across the orifice to alter the local shear response of the humor. In this work, a computational study of the flow in a vitreous cutter is conducted in order to gain better understanding of the vitreous behavior and provide guidelines for a new vitreous cutter design. The flow of a Newtonian surrogate of vitreous in a two-dimensional analog geometry is investigated using a finite difference-based immersed boundary method with an algebraically formulated fractional-step method. A series of numerical experiments is performed to evaluate the impact of cutting rate, aspiration pressure, and opening/closing transition on the vitreous cutter flow rate and transorifice pressure variation during vitrectomy. The mean flow rate is observed to increase approximately linearly with aspiration pressure and also increase nearly linearly with duty cycle. A study of time-varying flow rate, velocity field, and vorticity illuminates the flow behavior during each phase of the cutting cycle and shows that the opening/closing transition plays a key role in improving the vitreous cutter's efficacy and minimizing the potential damage to surrounding tissue. The numerical results show similar trend in flow rate as previous in vitro experiments using water and balanced saline solution and also demonstrate that high duty cycle and slow opening/closing phases lead to high flow rate and minor disturbance to the eye during vitrectomy, which are the design requirements of an ideal vitreous cutter.     

Numerical simulations of undulatory swimming at moderate Reynolds number

We perform numerical simulations of the swimming of a three-linkage articulated system in a moderately viscous regime. The computational methodology focuses on the creation, diffusion and transport of vorticity from the surface of the bodies into the fluid. The simulations are dynamically coupled, in that the motion of the three-linkage swimmer is computed simultaneously with the dynamics of the fluid. The novel coupling scheme presented in this work is the first to exploit the relationship between vorticity creation and body dynamics. The locomotion of the system, when subject to undulatory inputs of the hinges, is computed at Reynolds numbers of 200 and 1000. It is found that the forward swimming speed increases with the Reynolds number, and that in both cases the swimming is slower than in an inviscid medium. The vortex shedding is examined, and found to exhibit behavior consistent with experimental flow visualizations of fish.     

Genetic evidence of frequent long-distance recruitment in a vertebrate-dispersed tree

The importance of dispersal for the maintenance of biodiversity, while long-recognized, has remained unresolved. We used molecular markers to measure effective dispersal in a natural population of the vertebrate-dispersed Neotropical tree, Simarouba amara (Simaroubaceae) by comparing the distances between maternal parents and their offspring and comparing gene movement via seed and pollen in the 50 ha plot of the Barro Colorado Island forest, Central Panama. In all cases (parent-pair, mother-offspring, father-offspring, sib-sib) distances between related pairs were significantly greater than distances to nearest possible neighbours within each category. Long-distance seedling establishment was frequent: 74% of assigned seedlings established > 100 m from the maternal parent [mean = 392 +/- 234.6 m (SD), range = 9.3-1000.5 m] and pollen-mediated gene flow was comparable to that of seed [mean = 345.0 +/- 157.7 m (SD), range 57.6-739.7 m]. For S. amara we found approximately a 10-fold difference between distances estimated by inverse modelling and mean seedling recruitment distances (39 m vs. 392 m). Our findings have important implications for future studies in forest demography and regeneration, with most seedlings establishing at distances far exceeding those demonstrated by negative density-dependent effects.     

Mitochondrial restriction fragment length polymorphism (RFLP) and sequence variation among closely related avian species and the genetic characterization of hybrid Dendroica warblers

To address several interconnected goals, we used mitochondrial DNA (mtDNA) sequences to explore evolutionary relationships among four potentially hybridizing taxa in a North American avian superspecies (Dendroica occidentalis, D. townsendi, D. virens, and D. nigrescens). We first compared the results of a previous restriction fragment length polymorphism (RFLP)-based study with 1453 nucleotides from the mitochondrial cytochrome oxidase subunit I (COI), ATP-synthase 6 (ATPase 6), and ATP-synthase 8 (ATPase 8) genes. Separate phylogenetic analyses of the RFLP and sequence data provided identical and well-supported hierarchical species-level reconstructions that grouped occidentalis and townsendi as sister taxa. We then explored several general features of mitochondrial evolution via a comparison of the RFLP and sequence data sets. Qualitative rate differences that seemed evident in highly autocorrelated comparisons of RFLP vs. sequence pairwise distances were not supported when autocorrelation was removed. We also noted a high variance in corresponding RFLP and sequence distances after the removal of autocorrelation effects. This variance suggests that caution should be used when combining RFLP and sequence-based data in studies that require the large-scale synthesis of divergence estimates drawn from sources employing different molecular techniques. Finally, we used our parallel RFLP and sequence data to design and validate a rapid and inexpensive polymerase chain reaction-RFLP (PCR-RFLP) protocol for determining species-specific mitochondrial haplotypes. This PCR-RFLP technique will be applied in ongoing studies of the occidentalis/townsendi hybrid zone, where the historic and geographical complexity of the interbreeding populations necessitates the genotyping of thousands of individual warblers.     

Molecular phylogenetics and ecological diversification of the transisthmian fish genus Centropomus (Perciformes: Centropomidae).

Phylogenetic relationships among the 12 recognized fish species in the New World genus Centropomus (Pisces, Centropomidae) were analyzed using allozyme electrophoresis and 618 bp of the mitochondrial DNA 16S ribosomal RNA (rRNA) gene. Molecular phylogenetic trees were generally consistent with previously published partial hypotheses based on morphological evidence. However, previously undefined sister group relationships between major species groups were resolved using molecular data, and phylogenetic hypotheses for Centropomus based on 16S rRNA sequences were better supported than were allozyme-based hypotheses. The high level of congruence among the trees inferred from the nuclear and mitochondrial characters provided a firm phylogenetic basis for analysis of ecological diversification and molecular evolution in the genus. Compared to basal Centropomus species, members of the most nested species group were significantly larger in body size and occupied a marine niche only peripherally utilized by their congeners. We also observed substitution rate heterogeneity among 16S rRNA lineages; in contrast to expectations based on "metabolic rate" and "generation interval" models, relative substitution rates were faster than expected for the group of large-bodied snooks. Using the Pliocene rise of the Central American isthmian marine barrier to calibrate rates of 16S ribosomal gene evolution in Centropomus, we found that the rates for the genus were similar to those reported for higher vertebrates. Analysis of the three sets of transisthmian geminate taxa in Centropomus indicated that two of the pairs were probably formed during the Pliocene rise of the isthmus while the third pair diverged several million years earlier.     

Genetic structure of Mesoamerican populations of Big-leaf mahogany (Swietenia macrophylla) inferred from microsatellite analysis

While microsatellites have been used to examine genetic structure in local populations of Neotropical trees, genetic studies based on such high-resolution markers have not been carried out for Mesoamerica as a whole. Here we assess the genetic structure of the Mesoamerican mahogany Swietenia macrophylla King (big-leaf mahogany), a Neotropical tree species recently listed as endangered in CITES which is commercially extinct through much of its native range. We used seven variable microsatellite loci to assess genetic diversity and population structure in eight naturally established mahogany populations from six Mesoamerican countries. Measures of genetic differentiation (FST and RST) indicated significant differences between most populations. Unrooted dendrograms based on genetic distances between populations provide evidence of strong phylogeographic structure in Mesoamerican mahogany. The two populations on the Pacific coasts of Costa Rica and Panama were genetically distant from all the others, and from one another. The remaining populations formed two clusters, one comprised of the northern populations of Mexico, Belize and Guatemala and the other containing the southern Atlantic populations of Nicaragua and Costa Rica. Significant correlation was found between geographical distance and all pairwise measures of genetic divergence, suggesting the importance of regional biogeography and isolation by distance in Mesoamerican mahogany. The results of this study demonstrate greater phylogeographic structure than has been found across Amazon basin S. macrophylla. Our findings suggest a relatively complex Mesoamerican biogeographic history and lead to the prediction that other Central American trees will show similar patterns of regional differentiation.