Thermal stress responses in the bacterial biosphere of the Great Barrier Reef sponge,Rhopaloeides odorabile

Marine sponges are diverse, abundant and provide a crucial coupling point between benthic and pelagic habitats due to their high filtration rates. They also harbour extensive microbial communities, with many microbial phylotypes found exclusively in sponge hosts and not in the seawater or surrounding environment, i.e. so‐called sponge‐specific clusters (SCs) or sponge‐ and coral‐specific clusters (SCCs). We employed DNA (16S rRNA gene) and RNA (16S rRNA)‐based amplicon pyrosequencing to investigate the effects of sublethal thermal stress on the bacterial biosphere of the Great Barrier Reef sponge Rhopaloeides odorabile. A total of 8381 operational taxonomic units (OTUs) (97% sequence similarity) were identified, affiliated with 32 bacterial phyla from seawater samples, 23 bacterial phyla from sponge DNA extracts and 18 bacterial phyla from sponge RNA extracts. Sublethal thermal stress (31°C) had no effect on the present and/or active portions of the R. odorabile bacterial community but a shift in the bacterial assemblage was observed in necrotic sponges. Over two‐thirds of DNA and RNA sequences could be assigned to previously defined SCs/SCCs in healthy sponges whereas only 12% of reads from necrotic sponges could be assigned to SCs/SCCs. A rapid decline in host health over a 1°C temperature increment suggests that sponges such as R. odorabile may be highly vulnerable to the effects of global climate change.     

Larval host plant affects fitness consequences of egg size variation in the seed beetle Stator limbatus

Egg size variation often has large effects on the fitness of progeny in insects. However, many studies have been unable to detect an advantage of developing from large eggs, suggesting that egg size variation has implications for offspring performance only under adverse conditions, such as during larval competition, periods of starvation, desiccation, or when larvae feed on low-quality resources. We test this hypothesis by examining the consequences of egg size variation for survivorship and development of a seed-feeding insect, Stator limbatus, on both a low-quality (Cercidium floridum) and a high-quality (Acacia greggii) host plant. Our results are consistent with the hypothesis. S. limbatus larval performance was affected by egg size only when developing on the poor-quality host (C. floridum); larvae from large eggs survived better on C. floridum than those from small eggs, while there was no evidence of an effect of egg size on progeny development time, body weight, or survivorship when larvae developed on A. greggii. These results indicate intense selection for large eggs within C. floridum-associated populations, but not in A. greggii-associated populations, so that egg size is predicted to vary among populations associated with different hosts. Our results also support this hypothesis; females from a C. floridum-associated population (Scottsdale) laid larger eggs than females from an A. greggii-associated population (Black Canyon City).     

Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab

The aggregation of biotherapeutics is a major hindrance to the development of successful drug candidates; however, the propensity to aggregate is often identified too late in the development phase to permit modification to the protein's sequence. Incorporating rational design for the stability of proteins in early discovery has numerous benefits. We engineered out aggregation-prone regions on the Fab domain of a therapeutic monoclonal antibody, bevacizumab, to rationally design a biobetter drug candidate. With the purpose of stabilizing bevacizumab with respect to aggregation, 2 strategies were undertaken: single point mutations of aggregation-prone residues and engineering a glycosylation site near aggregation-prone residues to mask these residues with a carbohydrate moiety. Both of these approaches lead to comparable decreases in aggregation, with an up to 4-fold reduction in monomer loss. These single mutations and the new glycosylation pattern of the Fab domain do not modify binding to the target. Biobetters with increased stability against aggregation can therefore be generated in a rational manner, by either removing or masking the aggregation-prone region or crowding out protein-protein interactions.     

A Link between ORC-Origin Binding Mechanisms and Origin Activation Time Revealed in Budding Yeast

Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Finally, the ‘chromatin-dependent’ class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time.     

1,25-Dihydroxyvitamin D3 receptor in bovine thymus gland

1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃] receptor was characterized after partial purification of thymus cytosol by ammonium sulfate fractionation. The 1,25-(OH)₂D₃ receptor sediments at 3.7S in 5–20% sucrose gradients. The binding of 1,25-(OH)₂D₃ in thymic cytosol was a saturable process with high affinity (Kd = 0.12?0.48 nM) at 4°C. Competition for 1,25-(OH)₂[³H]D₃ receptor by nonradioactive analogs demonstrated the affinities of these analogs to be in order; 1,25-(OH)₂D₃ = 1,24R,25-(OH)₃D₃ = 1,25S,26-(OH)₃D₃ = 1,25R,26-(OH)₃D₃ > 1,25-(OH)₂D₃-26,23 lactone > 25-OHD₃ > 23R,25-(OH)₂D₃ > 24R,25-(OH)₂D₃ > 23S,25-(OH)₂D₃ ? 25-OHD₃-26,23 lactone. The receptor bound to DNA cellulose columns in low salt buffer and eluted as a single peak at 0.21 M KCl. These findings provide evidence that the thymus possesses a 1,25-(OH)₂D₃ receptor with properties indistinguishable from 1,25-(OH)₂D₃ receptors in other tissues.     

Stress-induced phosphorylation and proteasomal degradation of mitofusin 2 facilitates mitochondrial fragmentation and apoptosis

Mitochondria play central roles in integrating pro- and antiapoptotic stimuli, and JNK is well known to have roles in activating apoptotic pathways. We establish a critical link between stress-induced JNK activation, mitofusin 2, which is an essential component of the mitochondrial outer membrane fusion apparatus, and the ubiquitin-proteasome system (UPS). JNK phosphorylation of mitofusin 2 in response to cellular stress leads to recruitment of the ubiquitin ligase (E3) Huwe1/Mule/ARF-BP1/HectH9/E3Histone/Lasu1 to mitofusin 2, with the BH3 domain of Huwe1 implicated in this interaction. This results in ubiquitin-mediated proteasomal degradation of mitofusin 2, leading to mitochondrial fragmentation and enhanced apoptotic cell death. The stability of a nonphosphorylatable mitofusin 2 mutant is unaffected by stress and protective against apoptosis. Conversely, a mitofusin 2 phosphomimic is more rapidly degraded without cellular stress. These findings demonstrate how proximal signaling events can influence both mitochondrial dynamics and apoptosis through phosphorylation-stimulated degradation of the mitochondrial fusion machinery.     

Clumping factor A of Staphylococcus aureus inhibits phagocytosis by human polymorphonuclear leucocytes

Staphylococcus aureus is a major cause of nosocomial and community-acquired infection. It expresses several factors that promote avoidance of phagocytosis by polymorphonuclear leucocytes. Clumping factor A (ClfA) is a fibrinogen-binding surface protein of S. aureus that is an important virulence factor in several infection models. This study investigated whether ClfA is an antiphagocytic factor, and whether its antiphagocytic properties were based on its ability to bind fibrinogen. In S. aureus, ClfA was shown to be of equal importance to protein A, the antiphagocytic properties of which are well established. ClfA expressed in a surrogate Gram-positive host was also found to be antiphagocytic. A ClfA mutant that was unable to bind fibrinogen had a similar antiphagocytic effect to native ClfA in the absence of fibrinogen. ClfA inhibited phagocytosis in the absence of fibrinogen, and showed enhanced inhibition in the presence of fibrinogen.