No abstract

The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombinant protein purification and fiber formation. With an improved gene construction and cloning scheme, this technique is adaptable for the production of any repetitive fibrous proteins, and ensures the exact reproduction of native repeat sequences, analogs or chimeric versions. The proteins are solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 25–30% (wt/vol) for extrusion into fibers. This protocol, routinely used to spin single micrometer-size fibers from several recombinant silk-like proteins from different spider species, is a powerful tool to generate protein libraries with corresponding fibers for structure–function relationship investigations in protein-based biomaterials. This protocol may be completed in 40 d.

Antibiotic resistance, prompted by the overuse of antimicrobial agents, may arise from a variety of mechanisms, particularly horizontal gene transfer of virulence and antibiotic resistance genes, which is often facilitated by biofilm formation. The importance of phenotypic changes seen in a biofilm, which lead to genotypic alterations, cannot be overstated. Irrespective of if the biofilm is single microbe or polymicrobial, bacteria, protected within a biofilm from the external environment, communicate through signal transduction pathways (e.g., quorum sensing or two-component systems), leading to global changes in gene expression, enhancing virulence, and expediting the acquisition of antibiotic resistance. Thus, one must examine a genetic change in virulence and resistance not only in the context of the biofilm but also as inextricably linked pathologies. Observationally, it is clear that increased virulence and the advent of antibiotic resistance often arise almost simultaneously; however, their genetic connection has been relatively ignored. Although the complexities of genetic regulation in a multispecies community may obscure a causative relationship, uncovering key genetic interactions between virulence and resistance in biofilm bacteria is essential to identifying new druggable targets, ultimately providing a drug discovery and development pathway to improve treatment options for chronic and recurring infection.

Poly(amidoamine) (PAMAM) dendrimers are increasingly studied as model nanoparticles for a variety of biomedical applications, notably in systemic administrations. However, with respect to blood contacting applications, amine-terminated dendrimers have recently been shown to activate platelets and cause a fatal, disseminated intravascular coagulation (DIC)-like condition in mice and rats. We here demonstrate that, upon addition to blood, cationic G7 PAMAM dendrimers induce fibrinogen aggregation, which may contribute to the in vivo DIC-like phenomenon. We demonstrate that amine-terminated dendrimers act directly on fibrinogen in a thrombin-independent manner to generate dense, high-molecular-weight fibrinogen aggregates with minimal fibrin fibril formation. In addition, we hypothesize this clot-like behavior is likely mediated through electrostatic interactions between the densely charged cationic dendrimer surface and negatively charged fibrinogen domains. Interestingly, cationic dendrimers also induced aggregation of albumin, suggesting that many negatively charged blood proteins may be affected by cationic dendrimers. To investigate this further, zebrafish embryos (ZFE) were employed to more specifically determine the speed of this phenomenon and the pathway- and dose-dependency of the resulting vascular occlusion phenotype. These novel findings show that G7 PAMAM dendrimers significantly and adversely impact many blood components to produce rapid coagulation and strongly suggest that these effects are independent of classic coagulation mechanisms. These results also strongly suggest the need to fully characterize amine-terminated PAMAM dendrimers in regards to their adverse effects on both coagulation and platelets, which may contribute to blood toxicity.

Spiders have evolved a complex system of silk producing glands. Each of the glands produces silk with strength and elasticity tailored to its biological purpose. Sequence analysis of the major ampullate silk reveals four highly conserved concatenated blocks of amino acids: (GA) n , A n , GPGXX, and GGX. While the GPGXX motif, which has been hypothesized to be responsible for the extensibility of the fiber, displays natural variation in its precise sequence arrangement and content, correlating these differences with particular fiber properties has been difficult. Three genetic constructs based on the Argiope aurantia sequence were engineered to progressively increase the number of GPGXX repeats in a head-to-tail assembly prior to interruption by another motif. Circular dichroism and Fourier transform infrared spectroscopy of synthetic spider silk spin dopes show secondary structures that correspond to an increase in the repeat number of GPGXX regions and an increase in the extensibility of synthetically spun recombinant fibers.

Background: There is a steady increase in the prevalence of chronic kidney disease (CKD) in the United States. Primary care physicians (PCPs) can engage in strategies that are proven to be effective in reducing the progression rate of kidney disease. The National Kidney Foundation has released evidencebased guidelines called the Kidney Disease Outcome Quality Improvement Initiative (K/DOQI) that detail these strategies. No information exists regarding adoption of these guidelines in primary care.Methods: A qualitative study in a practice-based research network (PBRN) was undertaken to explore common PCP practices and knowledge regarding CKD. A typical case sampling strategy was followed. Semi-structured interviews and exit surveys were conducted with 10 PCPs from randomly selected PBRN practices. Three reviewers conducted content analysis using the immersion-crystallization approach.Results

The major ampullate fiber of both Nephila clavipes and Argiope aurantia is composed of two different proteins, MaSp1 and MaSp2. Each of these proteins has a highly conserved pattern of silk-associated amino acid motifs. The GPGXX motif is the only source of proline and is unique to MaSp2. On the basis of the percent of proline, Nephila clavipes major ampullate silk was calculated to consist of 19% MaSp2 and 81% MaSp1, while Argiope aurantia was calculated to have a significantly higher MaSp2 content of 59% with MaSp1 comprising the remaining 41%. To investigate the functional implications of the difference in protein composition, major ampullate silk fibers from Nephila clavipes and Argiope aurantia were mechanically tested and compared. Stress-strain curves produced from polynomial regression show that the two significant differences between major ampullate silk fibers from Nephila clavipes and Argiope aurantia are the average peak load stress and Young's modulus, with Argiope higher for both.

Major ampullate (dragline) spider silk is a coveted biopolymer due to its combination of strength and extensibility. The dragline silk of different spiders have distinct mechanical properties that can be qualitatively correlated to the protein sequence. This study uses amino acid analysis and carbon-13 solid-state NMR to compare the molecular composition, structure and dynamics of major ampullate dragline silk of four orb-web spider species (Nephila clavipes, Araneus gemmoides, Argiope aurantia and Argiope argentata) and one cobweb species (Latrodectus hesperus). The mobility of the protein backbone and amino acid side chains in water exposed silk fibers is shown to correlate to the proline content. This implies that regions of major ampullate spidroin 2 protein, which is the only dragline silk protein with any significant proline content, become significantly hydrated in dragline spider silk.