Crystal structures of the two major aggrecan degrading enzymes, ADAMTS4 and ADAMTS5

Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor-bound enzymes exhibit two essentially different catalytic-site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.     

RAM, a gene of yeast required for a functional modification of RAS proteins and for production of mating pheromone a-factor

We have identified a gene (SUPH) of S. cerevisiae that is required for both RAS function and mating by cells of a mating type. supH is allelic to ste16, a gene required for the production of the mating pheromone a-factor. Both RAS and a-factor coding sequences terminate with the potential acyltransferase recognition sequence Cys-A-A-X, where A is an aliphatic amino acid. Mutations in SUPH-STE16 prevent the membrane localization and maturation of RAS protein, as well as the fatty acid acylation of it and other membrane proteins. We propose the designation RAM (RAS protein and a-factor maturation function) for SUPH and STE16. RAM may encode an enzyme responsible for the modification and membrane localization of proteins with this C-terminal sequence.     

Differential resistance of insect sodium channels with kdr mutations to deltamethrin, permethrin and DDT

Knockdown resistance (kdr) in insects, caused by inherited nucleotide polymorphisms in the voltage-gated sodium channel (VGSC) gene, is a major threat to the efficacy of pyrethroid insecticides. Classic kdr, resulting from an L1014F substitution in the VGSC is now present in numerous pest species. Two other substitutions at the L1014 locus have also been reported, L1014S and L1014H. Here we have used expression of L1014 modified Drosophila para VGSCs in Xenopus oocytes with two-electrode voltage clamp to characterise all three mutations. The mutations L1014F and L1014H caused significant depolarizing shifts in the half activation voltage (V_50,act) from −17.3 mV (wild-type) to −13.1 and −13.5 mV respectively, whereas L1014S caused no shift in V_50,act but its currents decayed significantly faster than wild-type channels. Treatment of the wild-type channel with deltamethrin (≥1 nM), permethrin (≥30 nM) or DDT (≥1 ??M) resulted in hyperpolarizing shifts in V_50,act. Deltamethrin, permethrin and DDT also produced “tail currents” with EC₅₀s of 0.043, 0.40 and 65 ??M and maximum modifications of 837, 325 and 7% respectively. L1014F provided a high level of resistance against all insecticides for both measured parameters. L1014H most effectively combated deltamethrin induced tail currents while L1014S strongly resisted the large DDT induced shifts in V_50,act. We conclude that L1014H and L1014S may have arisen through heavy exposure to specific pyrethroids and DDT respectively.     

Genomic imbalances are confined to non-proliferating cells in paediatric patients with acute myeloid leukaemia and a normal or incomplete karyotype

Leukaemia is often associated with genetic alterations such as translocations, amplifications and deletions, and recurrent chromosome abnormalities are used as markers of diagnostic and prognostic relevance. However, a proportion of acute myeloid leukaemia (AML) cases have an apparently normal karyotype despite comprehensive cytogenetic analysis. Based on conventional cytogenetic analysis of banded chromosomes, we selected a series of 23 paediatric patients with acute myeloid leukaemia and performed whole genome array comparative genome hybridization (aCGH) using DNA samples derived from the same patients. Imbalances involving large chromosomal regions or entire chromosomes were detected by aCGH in seven of the patients studied. Results were validated by fluorescence in situ hybridization (FISH) to both interphase nuclei and metaphase chromosomes using appropriate bacterial artificial chromosome (BAC) probes. The majority of these copy number alterations (CNAs) were confirmed by FISH and found to localize to the interphase rather than metaphase nuclei. Furthermore, the proliferative states of the cells analyzed by FISH were tested by immunofluorescence using an antibody against the proliferation marker pKi67. Interestingly, these experiments showed that, in the vast majority of cases, the changes appeared to be confined to interphase nuclei in a non-proliferative status.     

The future of bioenergy

Energy from biomass plays a large and growing role in the global energy system. Energy from biomass can make significant contributions to reducing carbon emissions, especially from difficult‐to‐decarbonize sectors like aviation, heavy transport, and manufacturing. But land‐intensive bioenergy often entails substantial carbon emissions from land‐use change as well as production, harvesting, and transportation. In addition, land‐intensive bioenergy scales only with the utilization of vast amounts of land, a resource that is fundamentally limited in supply. Because of the land constraint, the intrinsically low yields of energy per unit of land area, and rapid technological progress in competing technologies, land intensive bioenergy makes the most sense as a transitional element of the global energy mix, playing an important role over the next few decades and then fading, probably after mid‐century. Managing an effective trajectory for land‐intensive bioenergy will require an unusual mix of policies and incentives that encourage appropriate utilization in the short term but minimize lock‐in in the longer term.     

First evidence of fish larvae producing sounds

The acoustic ecology of marine fishes has traditionally focused on adults, while overlooking the early life-history stages. Here, we document the first acoustic recordings of pre-settlement stage grey snapper larvae (Lutjanus griseus). Through a combination of in situ and unprovoked laboratory recordings, we found that L. griseus larvae are acoustically active during the night, producing ‘knock’ and ‘growl’ sounds that are spectrally and temporally similar to those of adults. While the exact function and physiological mechanisms of sound production in fish larvae are unknown, we suggest that these sounds may enable snapper larvae to maintain group cohesion at night when visual cues are reduced.     

Interleukin-7 deficiency in rheumatoid arthritis: consequences for therapy-induced lymphopenia

We previously demonstrated prolonged, profound CD4⁺ T-lymphopenia in rheumatoid arthritis (RA) patients following lymphocyte-depleting therapy. Poor reconstitution could result either from reduced de novo T-cell production through the thymus or from poor peripheral expansion of residual T-cells. Interleukin-7 (IL-7) is known to stimulate the thymus to produce new T-cells and to allow circulating mature T-cells to expand, thereby playing a critical role in T-cell homeostasis. In the present study we demonstrated reduced levels of circulating IL-7 in a cross-section of RA patients. IL-7 production by bone marrow stromal cell cultures was also compromised in RA. To investigate whether such an IL-7 deficiency could account for the prolonged lymphopenia observed in RA following therapeutic lymphodepletion, we compared RA patients and patients with solid cancers treated with high-dose chemotherapy and autologous progenitor cell rescue. Chemotherapy rendered all patients similarly lymphopenic, but this was sustained in RA patients at 12 months, as compared with the reconstitution that occurred in cancer patients by 3–4 months. Both cohorts produced naïve T-cells containing T-cell receptor excision circles. The main distinguishing feature between the groups was a failure to expand peripheral T-cells in RA, particularly memory cells during the first 3 months after treatment. Most importantly, there was no increase in serum IL-7 levels in RA, as compared with a fourfold rise in non-RA control individuals at the time of lymphopenia. Our data therefore suggest that RA patients are relatively IL-7 deficient and that this deficiency is likely to be an important contributing factor to poor early T-cell reconstitution in RA following therapeutic lymphodepletion. Furthermore, in RA patients with stable, well controlled disease, IL-7 levels were positively correlated with the T-cell receptor excision circle content of CD4⁺ T-cells, demonstrating a direct effect of IL-7 on thymic activity in this cohort.     

Unlocking the potential of metagenomics through replicated experimental design

Metagenomics holds enormous promise for discovering novel enzymes and organisms that are biomarkers or drivers of processes relevant to disease, industry and the environment. In the past two years, we have seen a paradigm shift in metagenomics to the application of cross-sectional and longitudinal studies enabled by advances in DNA sequencing and high-performance computing. These technologies now make it possible to broadly assess microbial diversity and function, allowing systematic investigation of the largely unexplored frontier of microbial life. To achieve this aim, the global scientific community must collaborate and agree upon common objectives and data standards to enable comparative research across the Earth's microbiome. Improvements in comparability of data will facilitate the study of biotechnologically relevant processes, such as bioprospecting for new glycoside hydrolases or identifying novel energy sources.     

Platelets present antigen in the context of MHC class I

Platelets are most recognized for their vital role as the cellular mediator of thrombosis, but platelets also have important immune functions. Platelets initiate and sustain vascular inflammation in many disease conditions, including arthritis, atherosclerosis, transplant rejection, and severe malaria. We now demonstrate that platelets express T cell costimulatory molecules, process and present Ag in MHC class I, and directly activate naive T cells in a platelet MHC class I-dependent manner. Using an experimental cerebral malaria mouse model, we also demonstrate that platelets present pathogen-derived Ag to promote T cell responses in vivo, and that platelets can be used in a cell-based vaccine model to induce protective immune responses. Our study demonstrates a novel Ag presentation role for platelets.     

Genetic and ecological consequences of recent habitat fragmentation in a narrow endemic plant species within an urban context

Understanding the timescales that shape spatial genetic structure is pivotal to ascertain the impact of habitat fragmentation on the genetic diversity and reproductive viability of long-lived plant populations. Combining genetic and ecological information with current and past fragmentation conditions allows the identification of the main drivers important in shaping population structure and declines in reproduction, which is crucial for informing conservation strategies. Using historic aerial photographs, we defined the past fragmentation conditions for the shrub Conospermum undulatum, a species now completely embedded in an urban area. We explored the impact of current and past conditions on its genetic layout and assessed the effects of genetic and environmental factors on its reproduction. The historically high structural connectivity was evident in the genetics of the species. Despite the current intense fragmentation, we found similar levels of genetic diversity across populations and a weak spatial genetic structure. Historical connectivity was negatively associated with genetic differentiation among populations and positively related to within-population genetic diversity. Variation partitioning of reproductive performance explained?~?66% of the variance, showing significant influences for genetic (9%), environmental (15%), and combined (42%) fractions. Our study highlights the importance of considering the historical habitat dynamics when investigating fragmentation consequences in long-lived plants. A detailed characterization of fragmentation from 1953 has shown how low levels of genetic fixation are due to extensive gene flow through the non-fragmented landscape. Moreover, knowledge of the relationships between genetic and environmental variation and reproduction can help to implement effective conservation strategies, particularly in highly dynamic landscapes.