Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.     

Understory Plant Community Composition Is Associated with Fine-Scale Above- and Below-Ground Resource Heterogeneity in Mature Lodgepole Pine (Pinus contorta) Forests

Understory plant communities play critical ecological roles in forest ecosystems. Both above- and below-ground ecosystem properties and processes influence these communities but relatively little is known about such effects at fine (i.e., one to several meters within-stand) scales, particularly for forests in which the canopy is dominated by a single species. An improved understanding of these effects is critical for understanding how understory biodiversity is regulated in such forests and for anticipating impacts of changing disturbance regimes. Our primary objective was to examine the patterns of fine-scale variation in understory plant communities and their relationships to above- and below-ground resource and environmental heterogeneity within mature lodgepole pine forests. We assessed composition and diversity of understory vegetation in relation to heterogeneity of both the above-ground (canopy tree density, canopy and tall shrub basal area and cover, downed wood biomass, litter cover) and below-ground (soil nutrient availability, decomposition, forest floor thickness, pH, and phospholipid fatty acids (PLFAs) and multiple carbon-source substrate-induced respiration (MSIR) of the forest floor microbial community) environment. There was notable variation in fine-scale plant community composition; cluster and indicator species analyses of the 24 most commonly occurring understory species distinguished four assemblages, one for which a pioneer forb species had the highest cover levels, and three others that were characterized by different bryophyte species having the highest cover. Constrained ordination (distance-based redundancy analysis) showed that two above-ground (mean tree diameter, litter cover) and eight below-ground (forest floor pH, plant available boron, microbial community composition and function as indicated by MSIR and PLFAs) properties were associated with variation in understory plant community composition. These results provide novel insights into the important ecological associations between understory plant community composition and heterogeneity in ecosystem properties and processes within forests dominated by a single canopy species.     

Next-Generation Sequencing of a Single Domain Antibody Repertoire Reveals Quality of Phage Display Selected Candidates

Next-Generation Sequencing and bioinformatics are powerful tools for analyzing the large number of DNA sequences present in an immune library. In this work, we constructed a cDNA library of single domain antibodies from a llama immunized with staphylococcal enterotoxin B. The resulting library was sequenced, resulting in approximately 8.5 million sequences with 5.4 million representing intact, useful sequences. The sequenced library was interrogated using sequences of known SEB-binding single domain antibodies from the library obtained through phage display panning methods in a previous study. New antibodies were identified, produced, and characterized, and were shown to have affinities and melting temperatures comparable to those obtained by traditional panning methods. This demonstrates the utility of using NGS as a complementary tool to phage-displayed biopanning as a means for rapidly obtaining additional antibodies from an immune library. It also shows that phage display, using a library of high diversity, is able to select high quality antibodies even when they are low in frequency.     

Consequences of elevated temperature and pCO2 on insect folivory at the ecosystem level: perspectives from the fossil record

Paleoecological studies document the net effects of atmospheric and climate change in a natural laboratory over timescales not accessible to laboratory or ecological studies. Insect feeding damage is visible on well‐preserved fossil leaves, and changes in leaf damage through time can be compared to environmental changes. We measured percent leaf area damaged on four fossil leaf assemblages from the Bighorn Basin, Wyoming, that range in age from 56.1 to 52.65 million years (Ma). We also include similar published data from three US sites 49.4 to ~45 Ma in our analyses. Regional climate was subtropical or warmer throughout this period, and the second oldest assemblage (56 Ma) was deposited during the Paleocene–Eocene Thermal Maximum (PETM), a geologically abrupt global warming event caused by massive release of carbon into the atmosphere. Total and leaf‐chewing damage are highest during the PETM, whether considering percent area damaged on the bulk flora, the average of individual host plants, or a single plant host that occurs at multiple sites. Another fossil assemblage in our study, the 52.65 Ma Fifteenmile Creek paleoflora, also lived during a period of globally high temperature and pCO₂, but does not have elevated herbivory. Comparison of these two sites, as well as regression analyses conducted on the entire dataset, demonstrates that, over long timescales, temperature and pCO₂ are uncorrelated with total insect consumption at the ecosystem level. Rather, the most important factor affecting herbivory is the relative abundance of plants with nitrogen‐fixing symbionts. Legumes dominate the PETM site; their prevalence would have decreased nitrogen limitation across the ecosystem, buffering generalist herbivore populations against decreased leaf nutritional quality that commonly occurs at high pCO₂. We hypothesize that nitrogen concentration regulates the opposing effects of elevated temperature and CO₂ on insect abundance and thereby total insect consumption, which has important implications for agricultural practices in today's world of steadily increasing pCO₂.     

Body size and proportions in chimpanzees,with special reference toPan troglodytes schweinfurthii from Gombe National Park,Tanzania

Body weight, cranial capacity, linear and joint area data from ten free-ranging adult chimpanzees from Gombe National Park, Tanzania with known life histories allow study of variation in a local population and comparison to other populations ofPan troglodytes and toPan paniscus. Because individuals in the Gombe population are small compared to other common chimpanzees, they provide a useful comparison toPan paniscus. Body weight and some linear dimensions overlap withPan paniscus. However, cranial capacity, tooth size, and body proportions of Gombe individuals lie within the range of otherPan troglodytes and are distinct fromPan paniscus.     

The role of phospholamban in the regulation of calcium transport by cardiac sarcoplasmic reticulum

The calcium transport mechanism of cardiac sarcoplasmic reticulum (SR) is regulated by a phosphoregulatory mechanism involving the phosphorylation-dephosphorylation of an integral membrane component, termed phospholamban. Phospholamban, a 27,000 Da proteolipid, contains phosphorylation sites for three independent protein kinases: 1) cAMP-dependent, 2) Ca²⁺-calmodulin-dependent, and 3) Ca²⁺-phospholipid-dependent. Phosphorylation of phospholamban by any one of these kinases is associated with stimulation of the calcium transport rates in isolated SR vesicles. Dephosphorylation of phosphorylated phospholamban results in the reversal of the stimulatory effects produced by the protein kinases. Studies conducted on perfused hearts have shown that during exposure to beta-adrenergic agents, a good correlation exists between the in situ phosphorylation of phospholamban and the relaxation of the left ventricle. Phosphorylation of phospholamban in situ is also associated with stimulation of calcium transport rates by cardiac SR, similar to in vitro findings. Removal of beta-adrenergic agents results in the reversal of the inotropic response and this is associated with dephosphorylation of phospholamban. These findings indicate that a phospho-regulatory mechanism involving phospholamban may provide at least one of the controls for regulation of the contractile properties of the myocardium.     

Ten generations of Drosophila melanogaster reared axenically on a fatty acid-free holidic diet

Wild-type Drosophila melanogaster were axenically raised on a completely synthetic fatty acid-free diet for at least ten consecutive generations, confirming that these insects do not require dietary polyunsaturated fatty acids. Capillary column gas-chromatographic analysis of lipids extracted from adults reared on yeast medium showed a peak which cochromatographed with linoleic acid, representing about 1.2% and 0.15% of all fatty acids in phospholipids and triacylglycerols, respectively. In flies reared on the synthetic diet for one generation or for five or more generations, the linoleic acid peak was still present but in about tenfold lower proportions of total fatty acids. This was true of both phospholipid and triglyceride fractions.     

Predator-prey interactions in natural communities.

The familiar concepts of harvest and yield are developed for the purpose of describing predator-prey interactions in a community context. In this regard the functional response (appropriate for one predator-one prey systems) is replaced by a community harvest function. Conditions for the stability of an ecological community are obtained. Exploring the dynamics of predator-prey interactions within this framework leads to new interpretations of other dynamical models such as the Lotka-Volterra model. The concept of a community moving attractor point is introduced in order to describe the changes in all populations over time.     

Partial characterization of Ia antigens from murine lymphoid cells

Congenic anti-Ia antisera were used to bind radiolabelled Ia antigens from cells of various strains of mice of knownH-2 haplotype. The results indicate that Ia antigens are proteins of molecular weight 30,000 to 35,000 daltons. The Ia antigens are distinct from known H-2 antigens as judged by independent immunoprecipitation as well as by molecular weight. Ia antigens are synthesized by, and are present on the surface of lymphoid cells as evidenced by incorporation studies using³H-leucine and enzymatic radioiodination of cells, respectively. Tissue distribution of cell surface Ia suggests that Ia antigens are on B cells. Ia antigens were detected in the incubation media of³H-leucine labeled splenocytes suggesting that antigens may be secreted.