In vivo bone strain and ontogenetic growth patterns in relation to life-history strategies and performance in two vertebrate taxa: goats and emu

This study examined ontogenetic patterns of limb loading, bone strains, and relative changes in bone geometry to explore the relationship between in vivo mechanics and size-related changes in the limb skeleton of two vertebrate taxa. Despite maintaining similar relative limb loads during ontogeny, bone strain magnitudes in the goat radius and emu tibiotarsus generally increased. However, while the strain increases in the emu tibiotarsus were mostly insignificant, strains within the radii of adult goats were two to four times greater than in young goats. The disparity between ontogenetic strain increases in these taxa resulted from differences in ontogenetic scaling patterns of the cross-sectional bone geometry. While the cross-sectional and second moments of area scaled with negative allometry in the goat radius, these measures were not significantly different from isometry in the emu tibiotarsus. Although the juveniles of both taxa exhibited lower strains and higher safety factors than the adults, the radii of the young goats were more robust relative to the adult goats than were the tibiotarsi of the young compared with adult emu. Differences in ontogenetic growth and strain patterns in the limb bones examined likely result from different threat avoidance strategies and selection pressures in the juveniles of these two taxa.     

The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS

It is well established that the humoral immune response can generate antibodies to many different antigens. The antibody diversity required to achieve this is believed to be substantial. However, the extent to which the immune repertoire can generate structural diversity against a single target antigen has never been addressed. Here, we have used phage display to demonstrate the extraordinary capacity of the human antibody repertoire. Over 1000 antibodies, all different in amino acid sequence, were generated to a single protein, B-lymphocyte stimulator (BLyS™ protein). This is a highly diverse panel of antibodies as exemplified by the extensive heavy and light chain germline usage: 42/49 functional heavy chain germlines and 19/33 V_λ and 13/35 V_κ light chain germlines were all represented in the panel of antibodies. Moreover, a high level of sequence diversity was observed in the V_H CDR3 domains of these antibodies, with 568 different amino acid sequences identified. Thus we have demonstrated that specific recognition of a single antigen can be achieved from many different VDJ combinations, illustrating the remarkable problem-solving ability of the human immune repertoire. When studied in a biochemical assay, around 500 (40%) of these antibodies inhibited the binding of BLyS to its receptors on B-cell lines. The most potent antibodies inhibited BLyS binding with sub-nanomolar IC₅₀ values and with sub-nanomolar affinities. Such antibodies provide excellent choices as candidates for the treatment of BLyS-associated autoimmune diseases.     

Importance of mangrove plantations for climate change mitigation in Bangladesh

Mangroves have been identified as blue carbon ecosystems that are natural carbon sinks. In Bangladesh, the establishment of mangrove plantations for coastal protection has occurred since the 1960s, but the plantations may also be a sustainable pathway to enhance carbon sequestration, which can help Bangladesh meet its greenhouse gas (GHG) emission reduction targets, contributing to climate change mitigation. As a part of its Nationally Determined Contribution (NDC) under the Paris Agreement 2016, Bangladesh is committed to limiting the GHG emissions through the expansion of mangrove plantations, but the level of carbon removal that could be achieved through the establishment of plantations has not yet been estimated. The mean ecosystem carbon stock of 5–42 years aged (average age: 25.5 years) mangrove plantations was 190.1 (±30.3) Mg C ha⁻¹, with ecosystem carbon stocks varying regionally. The biomass carbon stock was 60.3 (±5.6) Mg C ha⁻¹ and the soil carbon stock was 129.8 (±24.8) Mg C ha⁻¹ in the top 1 m of which 43.9 Mg C ha⁻¹ was added to the soil after plantation establishment. Plantations at age 5 to 42 years achieved 52% of the mean ecosystem carbon stock calculated for the reference site (Sundarbans natural mangroves). Since 1966, the 28,000 ha of established plantations to the east of the Sundarbans have accumulated approximately 76,607 Mg C year⁻¹ sequestration in biomass and 37,542 Mg C year⁻¹ sequestration in soils, totaling 114,149 Mg C year⁻¹. Continuation of the current plantation success rate would sequester an additional 664,850 Mg C by 2030, which is 4.4% of Bangladesh's 2030 GHG reduction target from all sectors described in its NDC, however, plantations for climate change mitigation would be most effective 20 years after establishment. Higher levels of investment in mangrove plantations and higher plantation establishment success could contribute up to 2,098,093 Mg C to blue carbon sequestration and climate change mitigation in Bangladesh by 2030.     

Multiplication of the agent of X-disease in a non-vector leafhopper Macrostelesfascifrons

The relative titre of the causal agent of X-disease of stone fruits in the non-vector leafhopper Macrosteles fascifrons was tested by injecting dilutions of M. fascifrons extracts into non-infective Colladonus montanus leafhopper vectors. The recipient C. montanus were fed on celery test plants which were then observed for X-disease symptoms. M. fascifrons were assayed at various intervals for up to 37 days after they were fed on X-diseased celery or injected with infectious extracts of the X-agent. Infectivity was detected in M. fascifrons only after 25 or 37 days in separate trials. Whole body extracts but not extracts from detached heads of M. fascifrons that had fed on X-diseased celery were infectious, whereas extracts prepared from the heads of M. fascifrons previously injected with X-agent extracts were infectious. This infectivity was retained for up to four serial passages in M. fascifrons. Electron microscopy of M. fascifrons that had been injected with extracts of the X-disease agent revealed mycoplasma-like organisms (MLO) only intercellularly and appressed to various organs in the haemocoele. No MLO were observed in uninjected M. fascifrons or those injected with extracts from non-infectious C. montanus. These results suggest that, despite multiplication of the X-agent in vivo. barriers in the gut and salivary glands prevent its transmission to plants by M. fascifrons.     

The effect of introducing aphid-pathogenic Entomophthoraceae into field populations of Aphis fabae

During two warm, dry seasons (1975 and 1976) aphid-pathogenic species of Entomophthoraceae introduced into Aphis fabae populations on field beans established briefly but failed to spread. In cool moist seasons (1977 and 1978), the fungi, principally Erynia neoaphidis and Neozygites fresenii, spread more rapidly in treated than untreated plots. In 1977, the maximum aphid population in plots treated with fungi reached a mean of 9000/plant on 19 July compared with 16 000/plant 1 wk later in untreated plots. The corresponding yield of beans in treated plots was twice that in untreated plots though only half that in plots kept free of aphids with pirimicarb. In two experiments in 1978, the maximum aphid population in treated areas was 700/plant, half that in untreated areas and 1 to 2 wk earlier. Yields from treated plots, however, were not significantly different from those from untreated plots. In nature, the fungi attack A. fabae populations in England only after they have become damaging, partly because fungal inoculum is sparse when the aphid population is small.     

Mapping the interactions present in the transition state for unfolding/folding of FKBP12.

The structure of the transition state for folding/unfolding of the immunophilin FKBP12 has been characterised using a combination of protein engineering techniques, unfolding kinetics, and molecular dynamics simulations. A total of 34 mutations were made at sites throughout the protein to probe the extent of secondary and tertiary structure in the transition state. The transition state for folding is compact compared with the unfolded state, with an approximately 30 % increase in the native solvent-accessible surface area. All of the interactions are substantially weaker in the transition state, as probed by both experiment and molecular dynamics simulations. In contrast to some other proteins of this size, no element of structure is fully formed in the transition state; instead, the transition state is similar to that found for smaller, single-domain proteins, such as chymotrypsin inhibitor 2 and the SH3 domain from alpha-spectrin. For FKBP12, the central three strands of the beta-sheet, beta-strand 2, beta-strand 4 and beta-strand 5, comprise the most structured region of the transition state. In particular Val101, which is one of the most highly buried residues and located in the middle of the central beta-strand, makes approximately 60 % of its native interactions. The outer beta-strands and the ends of the central beta-strands are formed to a lesser degree. The short alpha-helix is largely unstructured in the transition state, as are the loops. The data are consistent with a nucleation-condensation model of folding, the nucleus of which is formed by side-chains within beta-strands 2, 4 and 5, and the C terminus of the alpha-helix. The precise residues involved in the nucleus differ in the two simulated transition state ensembles, but the interacting regions of the protein are conserved. These residues are distant in the primary sequence, demonstrating the importance of tertiary interactions in the transition state. The two independently derived transition state ensembles are structurally similar, which is consistent with a Bronsted analysis confirming that the transition state is an ensemble of states close in structure.     

Diet of the Antarctic starry skate Amblyraja georgiana (Rajidae, Chondrichthyes) at South Georgia (Southern Ocean)

Stomach contents were identified from 206 Antarctic starry skate (Amblyraja georgiana) that were collected during three groundfish surveys (September 2007, April 2008 and January 2009) at South Georgia, Southern Ocean. The diet of A. georgiana varied with skate size and between years. Preferred prey included fish (particularly for larger individuals) and Antarctic krill, Euphausia superba, as well as amphipods, polychaetes and other benthic fauna. The skate A. georgiana appears to be an opportunistic predator, and the clear presence of Antarctic krill in this demersal predator’s diet may indicate a benthic habit of this euphausiid species, which has hitherto mainly been considered as occupying a purely pelagic niche.     

Sensitivity enhancement of surface plasmon resonance biosensing of small molecules

Surface plasmon resonance (SPR) biosensor formats using gold nanoparticle or protein signal amplification for the sensitive assay of small molecules were developed using progesterone as a model compound. Progesterone was immobilized to a dextran surface in the Biacore biosensor through in situ covalent immobilization using an oligoethylene glycol linker attached to the 4 position of the steroid. This surface produced stable antibody binding for in excess of 1100 assay cycles. Using this surface, assays were developed for progesterone using 10- and 20-nm gold-streptavidin labels attached to biotinylated monoclonal antibody in both label prebinding and sequential binding formats. Prelabeling formats gave no signal enhancement but produced assays with limits of detection of 143 pg/ml, compared with approximately 1 ng/ml in previous studies. Sequential binding formats gave signal enhancements of 2.2-fold over the monoclonal antibody and a limit of detection of 23.1 pg/ml. It was found that secondary antibody labeling gave 8.1-fold signal enhancements and a limit of detection of 20.1 pg/ml, whereas use of secondary antibody-25 nm gold complexes provided more signal enhancement (13-fold) and a further improvement in limit of detection of 8.6 pg/ml.     

A recurring theme in protein engineering: the design, stability and folding of repeat proteins

Repeat proteins are ubiquitous and are involved in a myriad of essential processes. They are typically non-globular structures that act as diverse scaffolds for the mediation of protein-protein interactions. These excitingly different structures, which arise from tandem arrays of a repeated structural motif, have generated significant interest with respect to protein engineering and design. Recent advances have been made in the design and characterisation of repeat proteins. The highlights include re-engineering of binding specificity, quantitative models of repeat protein stability and kinetic studies of repeat protein folding.     

Determinants of diastolic myocardial tissue Doppler velocities: influences of relaxation and preload

Myocardial tissueDoppler echocardiography (TDE) has been proposed as a tool for theassessment of diastolic function. Controversy exists regarding whetherTDE measurements are influenced by preload. In this study, leftventricular volume and high-fidelity pressures were obtained ineight closed-chest dogs during intermittent caval occlusion. The timeconstant of isovolumic ventricular relaxation () was alteredwith varying doses of dobutamine and esmolol. Peak early diastolicmyocardial (E_m) and transmitral (E)velocities were measured before and after preload reduction. Therelative effects of changes in preload and relaxation were determinedfor E_m and compared with their effects onE. The following results were observed: caval occlusionsignificantly decreased E (E = 16.4 ± 3.3 cm/s, 36.6 ± 13.7%, P < 0.01) andE_m (E_m = 1.3 ± 0.4 cm/s, 32.5 ± 26.1%, P < 0.01) underbaseline conditions. However, preload reduction was similar forE under all lusitropic conditions (P = notsignificant), but these effects on E_m decreasedwith worsening relaxation. At  < 50 ms, changes inE_m with preload reduction were significantlygreater (E_m = 2.8 ± 0.6 cm/s) than at  = 50-65 ms (E_m = 1.2 ± 0.2 cm/s) and at  >65 ms(E_m = 0.5 ± 0.1 cm/s,P < 0.05). We concluded that TDEE_m is preload dependent. However, this effectdecreases with worsening relaxation.