Steven Arcidiacono

EVOLUTION OF REPETITIVE PROTEINS: SPIDER SILKS FROM NEPHILA CLAVIPES (TETRAGNATHIDAE) AND ARANEUS BICENTENARIUS (ARANEIDAE)

Spider silks are highly repetitive proteins, characterized by regions of polyalanine and glycine-rich repeating units. We have obtained two variants of the Spidroin 1 (NCF-1) silk gene sequence from Nephila clavipes. One sequence (1726 bp) was from a cloned cDNA, and the other (1951 bp) was from PCR of genomic DNA. When these sequences are compared with each other and the previously published Spidroin 1 sequence, there are differences due to sequence rearrangements, as well as single base substitutions. These variations are similar to those that have been reported from other highly repetitive genes, and probably represent the results of unequal cross-overs. We have also obtained 708 bp of sequence from pCR of genomic DNA from Araneus biocentenarius. This sequence shows considerable similarity to a dragline sequence (ADF-3) from A. diadematus, as well as Spidroin 2 (NCF-2) from N. clavipes. Minor but consistent differences in the repeating unit sequence between A. bicentenarius and A. diadematus suggest that concerted evolution or gene conversion processes are acting to maintain similarity among repeat units within a single gene.

Cy5 labeled antimicrobial peptides for enhanced detection of Escherichia coli O157:H7

Fluorescently labeled antimicrobial peptides were evaluated as a potential replacement of labeled antibodies in a sandwich assay for the detection of Escherichia coli O157:H7. Antimicrobial peptides naturally bind to the lipopolysaccharide component of bacterial cell walls as part of their mode of action. Because of their small size relative to antibodies peptides can bind to cell surfaces with greater density, thereby increasing the optical signal and improving sensitivity. This method combines the specificity of a capture antibody with the increased sensitivity provided by using a labeled peptide as a detection molecule. The antimicrobial peptides cecropin P1, SMAP29, and PGQ were labeled with the fluorescent dye Cy5 via maleimide linker chemistry. Preliminary screening using a whole-cell solution binding assay revealed that Cy5 cecropin P1 enhanced the detection of E. coli O157:H7 relative to a Cy5 labeled anti-E. coli O157:H7 antibody 10-fold. Detection sensitivity of antibody and peptide were also compared with a prototype immuno-magnetic bead biosensor. Detection using Cy5 cecropin P1 resulted in a 10-fold improvement in sensitivity. Correlation of peptide antimicrobial activity with detection of E. coli O157:H7 indicated that activity was not predictive of the sensitivity of the fluorescent assay.

Biosynthesis and Processing of Silk Proteins

Silks produced by silkworms (e.g., Bombyx mori ) and orb-web weaving spiders (e.g., Nephila clavipes ) are essentially pure protein, that is, complexes of amino acid polymers. They are the most common fibers spun by biological systems. There has been a long-standing interest in the use of these and similar fibers in textiles, cables, fiber reinforcement in composites, in addition, for example, to cross hairs in optical instruments, and fishing nets. Both nylon, a homo-polymer of the amino acid glycine, and Kevlar, a polymer of a nonnatural aromatic amino acid, can be considered modified, synthetic versions of silk and are used for some of the applications mentioned above. The potential for genetic manipulation, through recombinant DNA technology, of the natural biosynthetic process for these natural proteins (see the article by Cappello in this issue) has renewed interest in the production of new silklike proteins. The natural silks are characterized by a β -sheet secondary structure which is stabilized by interchain hydrogen bonds and intersheet hydrophobic interactions (Figure 1). Silks can be considered block copolymers, with crystalline domains consisting of short side chain monomers (the amino acids glycine, alanine, and serine) interspersed in amorphous domains consisting of bulkier side chain amino acids. This family of fibers is naturally tailored to perform functions such as catching prey (orb web) or serving as a barrier against environmental challenges (cocoon). The domestic silkworm ( B. mori ) produces only one type of silk, cocoon silk, at only one stage in its lifecyle, during the fifth larval instar just before molt to the pupa. The silk is produced in modified salivary glands and spun from the mouth.

Membrane permeability and antimicrobial kinetics of cecropin P1 against Escherichia coli.

The interaction of cecropin P1 (CP1) with Escherichiacoli was investigated to gain insight into the time‐dependent antimicrobial action. Biophysical characterizations of CP1 with whole bacterial cells were performed using both fluorescent and colorimetric assays to investigate the role of membrane permeability and lipopolysaccharide (LPS) binding in lytic behavior. The kinetics of CP1 growth inhibition assays indicated a minimal inhibitory concentration (MIC) of 3 µM. Bactericidal kinetics at the MIC indicated rapid killing of E.coli (<30 min). Membrane permeability studies illustrated permeation as a time‐dependent event. Maximum permeability at the MIC occurred within 30 min, which correlates to the bactericidal action. Further investigation showed that the immediate permeabilizing action of CP1 is concentration‐dependent, which correlates to the concentration‐dependent nature of the inhibition assays. At the MIC and above, the immediate permeability was significant enough that the cells could not recover and exhibit growth. Below the MIC, immediate permeability was evident, but the level was insufficient to inhibit growth. Dansyl polymyxin B displacement studies showed LPS binding is essentially the same at all concentrations investigated. However, it does appear that only the immediate interaction is important, because binding continued to increase over time beyond cell viability. Our studies correlated CP1 bactericidal kinetics to membrane permeability suggesting CP1 concentration‐dependent killing is driven by the extent of the immediate permeabilizing action of the peptide. Copyright

Aqueous-Based Spinning of Fibers from Self-Assembling Structural Proteins

There has been long-standing interest in generating fibers from structural proteins and a great deal of work has been done in attempting to mimic dragline spider silk. Dragline silk balances stiffness, strength, extensibility, and high energy to break. Mimicking these properties through aqueous-based spinning of recombinant silk protein is a significant challenge; however, an approach has been developed that facilitates the formation of fibers approaching the mechanical properties seen with natural dragline silk. Due to the multitude of solution, spinning and post-spinning variables one has to consider, the method entails a multivariate approach to protein solution processing and fiber spinning. Optimization to maximize mechanical integrity of the fibers is performed by correlating the solution and spinning variables to mechanical properties and using this information for subsequent fiber spinning studies. Here, the method is described in detail and emphasizes the lessons learned during the iterative variable analysis process, which can be used as a basis for aqueous-based fiber spinning of other structural proteins.

The current state and future direction of DoD gut microbiome research: a summary of the first DoD gut microbiome informational meeting

The gut microbiome is increasingly recognized as integral to human health, and is emerging as a mediator of human physical and cognitive performance. This has led to the recognition that US Department of Defense (DoD) research supporting a healthy and resilient gut microbiome will be critical to optimizing the health and performance of future Warfighters. To facilitate knowledge dissemination and collaboration, identify resource capabilities and gaps, and maximize the positive impact of gut microbiome research on the Warfighter, DoD partners in microbiome research participated in a 2-day informational meeting co-hosted by the Natick Soldier Research, Engineering and Development Center (NSRDEC) and the US Army Research Institute of Environmental Medicine (USARIEM) on 16–17 November 2015. Attendee presentations and discussions demonstrated that multiple DoD organizations are actively advancing gut microbiome research. Common areas of research included the influence of military-relevant stressors on interactions between the microbiome and Warfighter biology, manipulation of the microbiome to influence Warfighter health, and use of the microbiome as a biomarker of Warfighter health status. Although resources and capabilities are available, they vary across laboratories and it was determined that centralizing certain DoD capabilities could accelerate progress. More significantly, the meeting created a foundation for a coordinated gut microbiome and nutrition research program aligning key DoD partners in the area of microbiome research. This report details the presentations and discussions presented during the 1st DoD Gut Microbiome Informational Meeting.

Kinetic microplate assay for determining immobilized antimicrobial peptide activity

Antimicrobial peptide immobilization onto surfaces is of great interest, although characterization of activity can be problematic. The kinetic microplate method described here determines the minimum bactericidal concentration (MBC) of immobilized antimicrobial peptides through a combination and modification of traditional solution assays, overcoming the difficulties of working with a solid substrate. The technique enables rapid, accurate evaluation of immobilized peptide lytic behavior, elucidating both dose- and time-dependent activity at multiple concentrations. Furthermore, the method yields information regarding sublethal concentrations not realized in the traditional assays.

Immobilization and orientation-dependent activity of a naturally occurring antimicrobial peptide.

A naturally occurring antimicrobial peptide, SMAP‐29, was synthesized with an n‐terminal or c‐terminal cysteine, termed c_SMAP and SMAP_c, respectively, for site‐directed immobilization to superparamagnetic beads. Immobilized SMAP orientation‐dependent activity was probed against multiple bacteria of clinical interest including Acinetobacter baumannii, Pseudomonas aeruginosa, Bacillus anthracis sterne and Staphylococcus aureus. A kinetic microplate assay was employed to reveal both concentration and time‐dependent activity for elucidation of minimum bactericidal concentration (MBC) and sub‐lethal effects. Immobilized SMAP activity was equivalent or reduced compared with soluble SMAP_c and c_SMAP regardless of immobilization orientation, with only one exception. A comparison of immobilized SMAP_c and c_SMAP activity revealed a bacteria‐specific potency dependent on immobilization orientation, which was contrary to that seen in solution, wherein SMAP_c was more potent against all bacteria than c_SMAP. Sub‐MBC kinetic studies displayed the influence of peptide exposure to the cells with multiple bacteria exhibiting increased susceptibility and efficacy at lower concentrations upon extended exposure (i.e. MBC enhancement). For instances in which complete killing was not achieved, two predominant effects were evident: retardation of growth rate and an increased lag phase. Both effects, seen independently and concomitantly, indicate some degree of induced cellular damage that can serve as a predictor toward eventual cell death. SMAP_c immobilized on glass through standard silanization chemistry was also investigated to ascertain the influence of substrate on activity against select bacteria. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Nephila Clavipes Dragline Silk: Approaches to a Recombinantly Produced Silk Protein

Spider silks exhibit an unusual combination of strength and toughness that distinguishes them from other natural and synthetic fibers. Silk proteins perform a key natural function as structural fibers, to absorb impact energy from flying insects without breaking. They dissipate energy over a broad area and balance stiffness, strength and extensibility (1,2). In addition to their unusual mechanical properties and visual lustre, silks also exhibit interesting interference patterns within the electromagnetic spectrum (3), unusual viscometric patterns related to processing (4), and piezoelectric properties (3,5,6). These properties suggest they would be good candidates for high performance fiber and composite applications. However, the spider is not capable of producing sufficient quantities of proteins to enable thorough evaluation of their potential. Consequently, we are pursuing recombinant DNA techniques to clone and express adequate quantities of recombinant spider silk for these studies.

Antimicrobial and Antifungal Treatments for US Army Applications

The US Army has been battling against microbial and fungal degradation since its founding in 1775. Advances in small molecule treatments provided some level of protection for Army materiel since WWII, but issues with supply, long-term Soldier health and environmental concerns led to research on alternatives. Synthetic polymers emerged as the primary replacement to treated cotton canvas, successfully supplanting it in most of the shelter components. However, recent evidence suggests that fungal growth on synthetics could still be a problem, and with a greater emphasis on personalized Soldier health, research, and development on antimicrobial and antifungal treatments for military textiles is experiencing a resurgence. Novel technologies in shelters, uniforms, and Soldier health, as well as new testing protocols, will be necessary to protect Soldiers and continue to field a strong and effective fighting force.