Effects of polymer molecular weight on the size, activity, and stability of PEG-functionalized trypsin

Polymer conjugation increases an enzyme's circulation time and stability for use as a therapeutic agent, but this attachment indubitably affects its properties. Covalent attachment of multiple polyethylene glycol chains with sizes of either 2, 5, 10, or 20 kDa increases the molecular weight and hydrodynamic radius of the model enzyme trypsin. The sizes of these polymer-enzyme conjugates are increased to be within the recommended limits for PDEPT applications. The T(d) increases from 49 to 60 °C to expand the enzyme's workable range of conditions. This functionalization with PEG polymers of varying lengths maintains trypsin's enzymatic activity. Conjugate activities are 79-120% that of native trypsin at room temperature and 221-432% that of trypsin at 37 °C.     

Evolution of an interloop disulfide bond in high-affinity antibody mimics based on fibronectin type III domain and selected by yeast surface display: molecular convergence with single-domain camelid and shark antibodies

The 10th human fibronectin type III domain ((10)Fn3) is one of several protein scaffolds used to design and select families of proteins that bind with high affinity and specificity to macromolecular targets. To date, the highest affinity (10)Fn3 variants have been selected by mRNA display of libraries generated by randomizing all three complementarity-determining region -like loops of the (10)Fn3 scaffold. The sub-nanomolar affinities of such antibody mimics have been attributed to the extremely large size of the library accessible by mRNA display (10(12) unique sequences). Here we describe the selection and affinity maturation of (10)Fn3-based antibody mimics with dissociation constants as low as 350 pM selected from significantly smaller libraries (10(7)-10(9) different sequences), which were constructed by randomizing only 14 (10)Fn3 residues. The finding that two adjacent loops in human (10)Fn3 provide a large enough variable surface area to select high-affinity antibody mimics is significant because a smaller deviation from wild-type (10)Fn3 sequence is associated with a higher stability of selected antibody mimics. Our results also demonstrate the utility of an affinity-maturation strategy that led to a 340-fold improvement in affinity by maximizing sampling of sequence space close to the original selected antibody mimic. A striking feature of the highest affinity antibody mimics selected against lysozyme is a pair of cysteines on adjacent loops, in positions 28 and 77, which are critical for the affinity of the (10)Fn3 variant for its target and are close enough to form a disulfide bond. The selection of this cysteine pair is structurally analogous to the natural evolution of disulfide bonds found in new antigen receptors of cartilaginous fish and in camelid heavy-chain variable domains. We propose that future library designs incorporating such an interloop disulfide will further facilitate the selection of high-affinity, highly stable antibody mimics from libraries accessible to phage and yeast surface display methods.     

Ultrastructure of endothelium in ovules of Penstemon gentianoides Poir. (Scrophulariaceae) at mature embryo sac phase

In this study ultrastructural differences between endothelial cells of different location in Penstemon gentianoides have been examined with electron microscope at mature embryo sac phase. Embryo sac is of the Polygonum type and surrounded by endothelium except the micropylar region. The cuticle is located primarily around the chalazal three-fourths of the embryo sac. Endothelium cells around the chalaza and toward the micropylar region are rich in cytoplasmic organelles. The cytoplasm of endothelial cells near the central cell has large vacuoles and few organelles. There are also plasmodesmas on the anticlinal walls of endothelial cells. The endothelium and the micropylar integumentary cells play a role in transport of metabolites into the embryo sac.     

Computational design of antibody-affinity improvement beyond in vivo maturation

Antibodies are used extensively in diagnostics and as therapeutic agents. Achieving high-affinity binding is important for expanding detection limits, extending dissociation half-times, decreasing drug dosages and increasing drug efficacy. However, antibody-affinity maturation in vivo often fails to produce antibody drugs of the targeted potency, making further affinity maturation in vitro by directed evolution or computational design necessary. Here we present an iterative computational design procedure that focuses on electrostatic binding contributions and single mutants. By combining multiple designed mutations, a tenfold affinity improvement to 52 pM was engineered into the anti-epidermal growth factor receptor drug cetuximab (Erbitux), and a 140-fold improvement in affinity to 30 pM was obtained for the anti-lysozyme model antibody D44.1. The generality of the methods was further demonstrated through identification of known affinity-enhancing mutations in the therapeutic antibody bevacizumab (Avastin) and the model anti-fluorescein antibody 4-4-20. These results demonstrate computational capabilities for enhancing and accelerating the development of protein reagents and therapeutics.     

Temporal dynamics of oocyte development, plasma sex steroids and somatic energy reserves during seasonal ovarian maturation in captive Murray cod Maccullochella peelii peelii

The temporal dynamics of oocyte growth, plasma sex steroids and somatic energy stores were examined during a 12 month ovarian maturation cycle in captive Murray cod Maccullochella peelii peelii under simulated natural photothermal conditions. Ovarian function was found to be relatively uninhibited in captivity, with the exception that post-vitellogenic follicles failed to undergo final maturation, resulting in widespread pre-ovulatory atresia. Seasonal patterns of oocyte growth were characterised by cortical alveoli accumulation in March, deposition of lipids in April, and vitellogenesis between May and September. Two distinct batches of vitellogenic oocytes were found in Murray cod ovaries, indicating a capacity for multiple spawns. Plasma profiles of 17β-oestradiol and testosterone were both highly variable during the maturation period suggesting that multiple roles exist for these steroids during different stages of oocyte growth. Condition factor, liver size and visceral fat stores were all found to increase prior to, or during the peak phase of vitellogenic growth. Murray cod appear to strategically utilise episodes of high feeding activity to accrue energy reserves early in the reproductive cycle prior to its deployment during periods of rapid ovarian growth.     

Incorporation of CpG oligonucleotide ligand into protein-loaded particle vaccines promotes antigen-specific CD8 T-cell immunity

The development of multicomponent biotherapeutic carriers is an important challenge in the field of drug delivery, particularly in the area of protein-based vaccines. While the delivery of protein antigens to antigen presenting cells (APCs) is crucial for this type of vaccination, the incorporation of additional adjuvants may be just as important in order to generate more potent immune responses. This article presents the synthesis and biological evaluation of carrier particles that both deliver a protein payload to APCs and display receptor ligands for the enhancement of APC immunostimulation. Particles displaying CpG oligonucleotide ligands for Toll-like receptor 9 were synthesized. The addition of CpG DNA to the particles led to a 45-fold increase in the secretion of interleukin-12, a cytokine that aids in T-cell activation, and a significant increase in the expression of costimulatory molecules by APCs. Moreover, vaccination with particles containing both ovalbumin (OVA) and CpG DNA induced a superior OVA-specific CD8 T-cell response in vivo, as measured by increased OVA-specific CD8 T-cell proliferation, secretion of the proinflammatory cytokine IFN-gamma, and the induction of OVA-specific cytotoxicity.     

Analysis of intergenic spacer region length polymorphisms to investigate the halophilic archaeal diversity of stromatolites and microbial mats

Hamelin Pool in Western Australia is one of the two major sites in the world with active marine stromatolite formation. Surrounded by living smooth and pustular mats, these ancient laminated structures are associated with cyanobacterial communities. Recent studies have identified a wide diversity of bacteria and archaea in this habitat. By understanding and evaluating the microbial diversity of this environment we can obtain insights into the formation of early life on Earth, as stromatolites have been dated in the geological record as far back as 3.5 billion years. Automated ribosomal intergenic spacer analysis (ARISA) patterns were shown to be a useful method to genetically discriminate halophilic archaea within this environment. Patterns of known halophilic archaea are consistent, by replicate analysis, and the halophilic strains isolated from stromatolites have novel intergenic spacer profiles. ARISA–PCR, performed directly on extracted DNA from different sample sites, provided significant insights into the extent of previous unknown diversity of halophilic archaea within this environment. Cloning and sequence analysis of the spacer regions obtained from stromatolites confirmed the novel and broad diversity of halophilic archaea in this environment.