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AATCC TM100 is one of a number of standard test methods commonly used for quantitative assessment of antimicrobial textiles performance. However, as currently written, the method allows for a number of steps to be conducted with several options, with reproducibility between testing laboratories being problematic. Options for the number of swatches and inoculum carrier (nutritive vs non-nutritive) were hypothesized to be primary drivers for variability. The present study attempted to identify sources of inter-laboratory variability. It was shown that specifying the number of swatches and inoculum carrier did not eliminate variability, as these parameters were not alone responsible for the outcome. Many other parameters were assumed to not affect the outcome. Additional systematic examination of these parameters was needed to determine their role in causing inter-laboratory variability.

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.

In vitro fermentation systems offer significant opportunity for deconvoluting complex metabolic dynamics within polymicrobial communities, particularly those associated with the human gut microbiome. In vitro gut models have broad experimental capacity allowing rapid evaluation of multiple parameters, generating knowledge to inform design of subsequent in vivo studies. Here, our method describes an in vitro fermentation test bed to provide a physiologically-relevant assessment of engineered probiotics circuit design functions. Typically, engineered probiotics are evaluated under pristine, monoor co-culture conditions and transitioned directly into animal or human studies, commonly resulting in a loss of desired function when introduced to complex gut communities. Our method encompasses a systematic workflow entailing fermentation, molecular and functional characterization, and statistical analyses to validate an engineered probiotic’s persistence, plasmid stability and reporter response. To demonstrate the workflow, simplified polymicrobial communities of human gut microbial commensals were utilized to investigate the probiotic Escherichia coli Nissle 1917 engineered to produce a fluorescent reporter protein. Commensals were assembled with increasing complexity to produce a mock community based on nutrient utilization. The method assesses engineered probiotic persistence in a competitive growth environment, reporter production and function, effect of engineering on organism growth and influence on commensal composition. The in vitro test bed represents a new element within the Design-Build-Test-Learn paradigm, providing physiologically-relevant feedback for circuit re-design and experimental validation for transition of engineered probiotics to higher fidelity animal or human studies.

OBJECTIVES: The human small intestine is a complex and dynamic organ tasked with enzymatic digestion and absorption of nutrients. Design of a small intestine model can provide detailed systematic knowledge of these processes; model design challenges include differential pH and oxygen availability along the length of the small intestine, food-dependent host secretion of digestive compounds, complex nutrient absorption processes, and microbiome interactions with both food and host. Numerous in vitro models have been developed to simulate the small intestine, but physiological relevance is limited. Here, we present an in vitro fermentation model of the small intestine to include microbiota and enhance physiological relevance. METHODS: A stepwise biofidelic model design approach was implemented with initial stages consisting of simulating ileum conditions, including pH and residence time, utilizing an automated bioreactor system for real-time monitoring and control of fermentation parameters, with incorporation of digestive enzymes and bile acids for breakdown of food inputs. Nutrient absorption, simulated using hollow-fiber columns to emulate passive diffusion, was initially optimized using small molecules to mimic dietary digestion byproducts; validation with food components, such as starch or whey powder, is planned. A mock microbial community, with organisms selected to represent major phyla and functions of the small intestine microbiota, was designed, implemented, and characterized in fermentations representing “fed-state” ileum conditions. RESULTS: Design and validation of the model with mock food components will be presented, along with steps taken to integrate in situ nutrient absorption and mock microbial community. Initial characterization of the microbial community indicates synergistic growth dynamics and nutrient utilization under “fed-state” conditions. CONCLUSIONS: These efforts will be the foundation for our long-term goal of simulating the small intestine to complement our large intestine fermentation model, jA(2)COB, creating a complete in vitro fermentation model of the lower GI tract. Insight gleaned from this model, alone or in concert with in vivo studies, can inform nutritional strategies to restore and maintain host gut homeostasis. FUNDING SOURCES: Funded by U.S. Army NSRDEC core applied research funds.

OBJECTIVES: From a military perspective, the gut microbiome serves as an ideal tool to enhance Soldier gut and immune health and to improve survivability and performance. Our work employs in vitro tools as a means to elucidate systematic processes of colonic bacterial metabolism of dietary inputs under native and stressed conditions. This talk will focus on the use of in vitro fermentation to investigate both the prebiotic potential of cranberry proanthocyanidins (PAC) within a healthy microbiome and stress-induced alterations in microbial inter-species competition for fermentable fiber. METHODS: Fermentations were performed in triplicate, utilizing fecal inocula derived from at least three individuals, in a nutrient-rich anaerobic medium with sampling at 0 and 24 hrs. Within PAC supplementation studies, samples were analyzed for bacterial identification (16 s rRNA sequencing) and metabolite content (GC-FID and GC/MS). Stressed metabolism studies, which utilized fecal samples before and after a 21-day change in diet challenge (habitual vs. Meal Ready-to-Eat), employed media supplemented with resistant starch under ascending colon domain-specific conditions; samples taken were analyzed for bacterial identification (16 s rRNA) and enumeration of select organisms (qPCR). RESULTS: Bacterial population dynamics within PAC supplementation studies indicated a dose-dependent increase in several beneficial taxa, including Ruminococcus spp (P

Stress, a ubiquitous part of daily human life, has varied biological effects which are increasingly recognized as including modulation of commensal microorganisms residing in the gastrointestinal tract, the gut microbiota. In turn, the gut microbiota influences the host stress response and associated sequelae, thereby implicating the gut microbiota as an important mediator of host health. This narrative review aims to summarize evidence concerning the impact of psychological, environmental, and physical stressors on gut microbiota composition and function. The stressors reviewed include psychological stress, circadian disruption, sleep deprivation, environmental extremes (high altitude, heat, and cold), environmental pathogens, toxicants, pollutants, and noise, physical activity, and diet (nutrient composition and food restriction). Stressors were selected for their direct relevance to military personnel, a population that is commonly exposed to these stressors, often at extremes, and in combination. However, the selected stressors are also common, alone or in combination, in some civilian populations. Evidence from preclinical studies collectively indicates that the reviewed stressors alter the composition, function and metabolic activity of the gut microbiota, but that effects vary across stressors, and can include effects that may be beneficial or detrimental to host health. Translation of these findings to humans is largely lacking at present. This gap precludes concluding with certainty that transient or cumulative exposures to psychological, environmental, and physical stressors have any consistent, meaningful impact on the human gut microbiota. However, provocative preclinical evidence highlights a need for translational research aiming to elucidate the impact of stressors on the human gut microbiota, and how the gut microbiota can be manipulated, for example by using nutrition, to mitigate adverse stress responses.

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.

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.

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 microM. Bactericidal kinetics at the MIC indicated rapid killing of E.coli (

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.

Despite its remarkable materials properties, the structure of spider dragline silk has remained unsolved. Results from two probe microscopy techniques provide new insights into the structure of spider dragline silk. A soluble synthetic protein from dragline silk spontaneously forms nanofibers, as observed by atomic force microscopy. These nanofibers have a segmented substructure. The segment length and amino acid sequence are consistent with a slab-like shape for individual silk protein molecules. The height and width of nanofiber segments suggest a stacking pattern of slab-like molecules in each nanofiber segment. This stacking pattern produces nano-crystals in an amorphous matrix, as observed previously by NMR and x-ray diffraction of spider dragline silk. The possible importance of nanofiber formation to native silk production is discussed. Force spectra for single molecules of the silk protein demonstrate that this protein unfolds through a number of rupture events, indicating a modular substructure within single silk protein molecules. A minimal unfolding module size is estimated to be around 14 nm, which corresponds to the extended length of a single repeated module, 38 amino acids long. The structure of this spider silk protein is distinctly different from the structures of other proteins that have been analyzed by single-molecule force spectroscopy, and the force spectra show correspondingly novel features.

Spider silks are protein-based “biopolymer” filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to “biomimic” the process of spider silk production by expressing in mammalian cells the dragline silk genes ( ADF-3 / MaSpII and MaSpI ) of two spider species. We produced soluble recombinant (rc)–dragline silk proteins with molecular masses of 60 to 140 kilodaltons. We demonstrated the wet spinning of silk monofilaments spun from a concentrated aqueous solution of soluble rc–spider silk protein (ADF-3; 60 kilodaltons) under modest shear and coagulation conditions. The spun fibers were water insoluble with a fine diameter (10 to 40 micrometers) and exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. Dope solutions with rc–silk protein concentrations >20% and postspinning draw were necessary to achieve improved mechanical properties of the spun fibers. Fiber properties correlated with finer fiber diameter and increased birefringence.

Spiders have captured the interest of scientists for many years because spider silks are among the toughest materials, having properties that surpass some man-made synthetic materials. Spinning recombinant silk to duplicate those properties has proved to be extremely difficult. This is the first known report of spinning recombinant silk fibers in an aqueous environment. The method seeks to keep the protein soluble throughout the process, not unlike the way the spider stores and spins silk. Recombinant silk proteins were produced by bacterial fermentation in which the cell pellets were lyophilized and lysed with organic acid. Silk protein was purified from the lysate by chromatography and processed in dilute denaturing buffer into a fiber spinning solution. Circular dichroism measurements of the silk solutions revealed an increase in β-sheet content as a function of time. Time-dependent self-association of silk protein was monitored in solution by dynamic light scattering. Furthermore, the observed increas...

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.

A partial cDNA clone, from the 3' end of the dragline silk gene was isolated from Nephila clavipes major ampullate glands. This clone contains a 1.7-kb insert, consisting of a repetitive coding region of 1.4-kb and a 0.3-kb nonrepetitive coding region; 1.5-kb of the 1.7-kb fragment was cloned into Escherichia coli and a 43-kDa recombinant silk protein was expressed. Characterization of the purified protein by Western blot, amino acid composition analysis, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry confirms it to be spider dragline silk.

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.

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.

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.

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 aβ-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.

Antimicrobial peptides are part of the innate immune system of vertebrates and invertebrates. They are active against gram-negative and gram-positive bacteria, fungi, and protozoa. Currently, most antimicrobial peptides are extracted from host organisms or produced by solid- phase peptide synthesis. Recombinant protein expression in Escherichia coli is a tool for greater production yields at a decreased cost and reduces the use of hazardous ma- terials. We have constructed a concatamer of indolicidin and successfully expressed a fusion product with thiore- doxin in E. coli BL21DE3. Codons for methionine residues flanking individual indolicidin genes were incorporated for cyanogen bromide cleavage of the fusion protein and liberation of active monomeric indolicidin. Peptide yields of 150 μg/l monomeric indolicidin were achieved in this first report of recombinant production of indolicidin with demonstrated antimicrobial activity.

A procedure has been developed for the isolation of recombinant spider silk proteins based upon their unique stability and solubilization characteristics. Three recombinant silk proteins, (SpI)7, NcDS, and [(SpI)4/(SpII)1]4, were purified by extraction with organic acids followed by affinity or ion exchange chromatography resulting in 90-95% pure silk solutions. The protein yield of NcDS (15 mg/L culture) and (SpI)7 (35 mg/L) increased 4- and 5-fold, respectively, from previously reported values presumably due to a more complete solubilization of the expressed recombinant protein. [(SpI)4/(SpII)1]4, a hybrid protein based on the repeat sequences of spidroin I and spidroin II, had a yield of 12.4 mg/L. This method is an effective, reproducible technique that has broad applicability for a variety of silk proteins as well as other acid stable biopolymers.

The polymerase chain reaction (PCR) has been used to amplify the portion of the Spidroin 1 gene that codes for the C-terminal part of the silk protein of the spider Nephila clavipes. Along with some substitution mutations of minor consequence, the PCR-derived sequence reveals an additional base missing from the previously published Nephila Spidroin 1 sequence. Comparison of the PCR-derived sequence with the equivalent region of Spidroin 2 indicates that the insertion of this single base results in greatly increased similarity in the resulting amino acid sequences of Spidroin 1 and Spidroin 2 (75% over 97 amino acids). The same PCR primers also amplified a fragment of the same length from Araneus bicentenarius. This sequence is also very similar to Spidroin 1 of Nephila (71% over 238 bases excluding the PCR primers, which translates into 76% over 79 amino acids).

Porins are essential pore-forming proteins found in the outer membrane of several gram-negative bacteria. Investigating the relationships between molecular structure and function involves an extremely time-consuming and labor-intensive purification procedure. We report a method for rapid extraction of the outer membrane protein, OmpF, from freeze-dried Escherichia coli cells using valeric acid, alleviating the effort and time in sample preparation. Extraction results in a highly enriched fraction containing OmpF as 76% of the total protein content. The apparent molecular mass determined by SDS-PAGE mobility was 38,900, similar to that of the monomeric form of OmpF. N-terminal sequencing yielded 23 amino acids with 100% identity to the published OmpF sequence. The trimeric form of OmpF was observed in unheated samples run on SDS-PAGE and analysis of these samples by periodic acid/silver staining revealed the presence of unbound lipopolysaccharides. Furthermore, this method should prove useful for isolating other outer membrane proteins.

Despite the extensive interest in its remarkable materials properties, the structure of spider dragline silk is not yet known. Indirect structural information is thus of particular interest. New data are presented here from two probe microscopy techniques – atomic force microscopy (AFM) and single-molecule force spectroscopy, or ‘molecular pulling.’ Using a soluble synthetic protein from dragline silk, segmented silk nanofibers have been observed by AFM. Molecular pulling (Force Spectroscopy) has revealed sawtooth-like rupture peaks, indicative of the sacrificial bonds and hidden lengths seen in molecules of other self-healing biomaterials. A model is presented for the folding of single dragline-silk molecules and the arrangement of these molecules into nanofibers. The relationship is discussed between these molecular / nanoscale results and the materials properties of natural and artificial dragline silks.