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Flooding of sweetpotatoes in the field leads to development of soft rot on the storage roots while they remain submerged or on subsequent harvest and storage. Incidences of flooding after periods of intense rainy weather are on the rise in the southeastern United States, which is home to the majority of sweetpotato production in the nation. In an effort to characterize the causative agent(s) of this devastating disease, here we describe two distinct bacterial strains isolated from soft-rotted sweetpotato storage roots retrieved from an intentionally flooded field. Both of these anaerobic spore-forming isolates were identified as members of the genus Clostridium based on sequence similarity of multiple housekeeping genes, and both were confirmed to cause soft rot disease on sweetpotato and other vegetable crops. Despite these common features, the isolates were distinguishable by several phenotypic and biochemical properties, and phylogenetic analysis placed them in separate well-supported clades within the genus. Overall, our results demonstrate that multiple plant-pathogenic Clostridium species can cause soft rot disease on sweetpotato and suggest that a variety of other plant hosts may also be susceptible.

Flooding of sweetpotatoes in the field leads to development of soft rot on the storage roots while they remain submerged or on subsequent harvest and storage. Incidences of flooding after periods of intense rainy weather are on the rise in the southeastern United States, which is home to the majority of sweetpotato production in the nation. In an effort to characterize the causative agent(s) of this devastating disease, here we describe two distinct bacterial strains isolated from soft-rotted sweetpotato storage roots retrieved from an intentionally flooded field. Both of these anaerobic spore-forming isolates were identified as members of the genus

CJ Boyer,1 J Ambrose Jr,2 S Das,1 A Humayun,1 D Chappidi,1 R Giorno,3 DK Mills2,3 1Molecular Science and Nanotechnology, College of Engineering & Science, Louisiana Tech University, Ruston, LA, USA; 2Center for Biomedical Engineering and Rehabilitation Science, Louisiana Tech University, Ruston, LA, USA; 3School of Biological Sciences, Louisiana Tech University, Ruston, LA, USA Introduction: Invasive medical devices are used in treating millions of patients each day. Bacterial adherence to their surface is an early step in biofilm formation that may lead to infection, health complications, longer hospital stays, and death. Prevention of bacterial adherence and biofilm development continues to be a major healthcare challenge. Accordingly, there is a pressing need to improve the anti-microbial properties of medical devices. Materials and Methods: Polydimethylsiloxane (PDMS) was doped with halloysite nanotubes (HNTs), and the PDMS-HNT composite surfaces were coated with PDMS-b-polyethylene oxide (PEO) and antibacterials. The composite material properties were examined using SEM, energy dispersive spectroscopy, water contact angle measurements, tensile testing, UV-Vis spectroscopy, and thermal gravimetric analysis. The antibacterial potential of the PDMS-HNT composites was compared to commercial urinary catheters using cultures of E. coli and S. aureus. Fibrinogen adsorption studies were also performed on the PDMS-HNT-PEO composites. Results: HNT addition increased drug load during solvent swelling without reducing material strength. The hydrophilic properties provided by PEO were maintained after HNT addition, and the composites displayed protein-repelling properties. Additionally, composites showed superiority over commercial catheters at inhibiting bacterial growth. Conclusion: PDMS-HNT composites showed superiority regarding their efficacy at inhibiting bacterial growth, in comparison to commercial antibacterial catheters. Our data suggest that PDMS-HNT composites have potential as a coating material for anti-bacterial invasive devices and in the prevention of institutional-acquired infections. Keywords: antibacterials, halloysite, medical devices, nanocomposites, PDMS

Bacterial spores possess a series of concentrically arranged protective structures that contribute to dormancy, survival and, ultimately, germination. One of these structures, the coat, is present in all spores. InBacillus anthracis, however, the spore is surrounded by an additional, poorly understood, morphologically complex structure called the exosporium. Here, we characterize three previously discovered exosporium proteins called ExsFA (also known as BxpB), ExsFB (a highly related paralogue ofexsFA/bxpB) and IunH (similar to an inosine–uridine-preferring nucleoside hydrolase). We show that in the absence of ExsFA/BxpB, the exosporium protein BclA accumulates asymmetrically to the forespore pole closest to the midpoint of the sporangium (i.e. the mother-cell-proximal pole of the forespore), instead of uniformly encircling the exosporium. ExsFA/BxpB may also have a role in coat assembly, as mutant spore surfaces lack ridges seen in wild-type spores and have a bumpy appearance. ExsFA/BxpB also has a modest but readily detected effect on germination. Nonetheless, anexsFA/bxpBmutant strain is fully virulent in both intramuscular and aerosol challenge models in Guinea pigs. We show that the pattern of localization of ExsFA/BxpB–GFP is a ring, consistent with a location for this protein in the basal layer of the exosporium. In contrast, ExsFB–GFP fluorescence is a solid oval, suggesting a distinct subcellular location for ExsFB–GFP. We also used these fusion proteins to monitor changes in the subcellular locations of these proteins during sporulation. Early in sporulation, both fusions were present throughout the mother cell cytoplasm. As sporulation progressed, GFP fluorescence moved from the mother cell cytoplasm to the forespore surface and formed either a ring of fluorescence, in the case of ExsFA/BxpB, or a solid oval of fluorescence, in the case of ExsFB. IunH–GFP also resulted in a solid oval of fluorescence. We suggest the interpretation that at least some ExsFB–GFP and IunH–GFP resides in the region between the coat and the exosporium, called the interspace.

Bacterial spores are encased in a multilayered proteinaceous shell, called the coat. In many Bacillus spp., the coat protects against environmental assault and facilitates germination. In Bacillus anthracis, the spore is the etiological agent of anthrax, and the functions of the coat likely contribute to virulence. Here, we characterize a B. anthracis spore protein, called Cotbeta, which is encoded only in the genomes of the Bacillus cereus group. We found that Cotbeta is synthesized specifically during sporulation and is assembled onto the spore coat surface. Our analysis of a cotbeta null mutant in the Sterne strain reveals that Cotbeta has a role in determining coat-surface morphology but does not detectably affect germination. In the fully virulent Ames strain, a cotbeta null mutation has no effect on virulence in a murine model of B. anthracis infection.

A major factor contributing to the failure of orthopedic and orthodontic implants is post-surgical infection. Coating metallic implant surfaces with anti-microbial agents has shown promise but does not always prevent the formation of bacterial biofilms. Furthermore, breakdown of these coatings within the human body can cause release of the anti-microbial drugs in an uncontrolled or unpredictable fashion. In this study, we used a calcium alginate and calcium phosphate cement (CPC) hydrogel composite as the base material and enriched these hydrogels with the anti-microbial drug, gentamicin sulfate, loaded within a halloysite nanotubes (HNTs). Our results demonstrate a sustained and extended release of gentamicin from hydrogels enriched with the gentamicin-loaded HNTs. When tested against the gram-negative bacteria, the hydrogel/nanoclay composites showed a pronounced zone of inhibition suggesting that anti-microbial doped nanoclay enriched hydrogels can prevent the growth of bacteria. The release of gentamicin sulfate for a period of five days from the nanoclay-enriched hydrogels would supply anti-microbial agents in a sustained and controlled manner and assist in preventing microbial growth and biofilm formation on the titanium implant surface. A pilot study, using mouse osteoblasts, confirmed that the nanoclay enriched surfaces are also cell supportive as osteoblasts readily, proliferated and produced a type I collagen and proteoglycan matrix.

The outermost proteinaceous layer of bacterial spores, called the coat, is critical for spore survival, germination, and, for pathogenic spores, disease. To identify novel spore coat proteins, we have carried out a preliminary proteomic analysis of Bacillus subtilis and Bacillus anthracis spores, using a combination of standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and improved two-dimensional electrophoretic separations, followed by matrix-assisted laser desorption ionization-time of flight and/or dual mass spectrometry. We identified 38 B . subtilis spore proteins, 12 of which are known coat proteins. We propose that, of the novel proteins, YtaA, YvdP, and YnzH are bona fide coat proteins, and we have renamed them CotI, CotQ, and CotU, respectively. In addition, we initiated a study of coat proteins in B . anthracis and identified 11 spore proteins, 6 of which are candidate coat or exosporium proteins. We also queried the unfinished B . anthracis genome for potential coat proteins. Our analysis suggests that the B . subtilis and B . anthracis coats have roughly similar numbers of proteins and that a core group of coat protein species is shared between these organisms, including the major morphogenetic proteins. Nonetheless, a significant number of coat proteins are probably unique to each species. These results should accelerate efforts to develop B . anthracis detection methods and understand the ecological role of the coat.

A polymer nanostructured Fabry–Perot interferometer (FPI) based biosensor is reported. Different from a conventional FPI, the nanostructured FPI has a layer of Au-coated nanopores inside its cavity. The Au-coated nanostructure layer offers significant enhancement of optical transducing signals due to the localized surface plasmon resonance effect and also due to the significantly increased sensing surface area, which is up to at least two orders of magnitude larger than that of a conventional FPI-based biosensor. Using this technical platform, the immobilization of captures proteins (protein A) on the nanostructure layer and their binding with immunoglobulin G (IgG) has been monitored in real time, resulting in the shift of the interference fringes of the optical transducing signals. Current results show that the limit-of-detection of the biosensor should be lower than 10 pg/mL for IgG-protein A binding.

This paper reports a novel label-free biosensor based on a nanostructured Fabry-Perot interferometer (FPI) for the first time. Au-coated nanostructure layer inside the FPI offers significantly enhanced optical interference signals due to localized surface Plasmon resonance (SPR) effect. Immobilization of capture protein, Protein A, on the nanostructure layer and its binding with immunoglobulin G (IgG) inside the FPI are monitored in real time, resulting in interference fringes shift of the signals. Fourier transform (FT) is performed on the measured reflectance interference spectrum. Only a single peak whose X-axis position is the effective optical thickness (EOT) of nanostructured layer is presented for each spectrum, which simplifies the quantified analysis of the signals. Current results show that the detection-of-limit (DOL) of the biosensor should be much lower than 5ng/ml.

A polymer-based micromachined Fabry-Perot interferometer (µFPI) with embedded nanostructures in its cavity, called nanostructured-FPI, is reported. The nanostructures inside the cavity are a layer of Au-coated nanopores. As a refractive-index sensitive optical sensor, it offers the following advantages over a traditional µFPI for label-free biosensing applications, including increased sensing surface area, extended penetration depth of the excitation light and amplified optical transducing signals. For a nanostructured-FPI with nanopore size of 50 nm in diameter and the gap size of FPI cavity of 50 µm, measurements find that it has ~20 times improvement in free spectral range (FSR), ~2 times improvement in finesse and ~4 times improvement in contrast of optical transducing signals over a traditional µFPI even without any device performance optimization. Several chemicals have also been evaluated using this device. Fourier transform has been performed on the measured optical signals to facilitate the analysis of the transducing signals.

A white-light source operated polymer-based micromachined Fabry-Perot Interferometer (µFPI) with embedded nanostructures in its cavity is reported for the first time. The nanostructures inside the cavity are Au-coated nanopore-plasmonic substrate, forming an Au-nanopore-FPI. It offers at least three advantages over a traditional μFPI including the increased sensing surface area, the extended penetration depth of the excitation light and the tremendous amplification of the optical transducing signal due to the localized surface Plasmon resonance (L-SPR) effect. Measurements have been carried out on the prototype devices. It shows tremendous enhancement of output signal intensity, sensitivity and contrast for the Au-nanopore-µFPI compared to traditional µFPI. Several chemicals have been evaluated using these devices.

Bacillus spp. and Clostridium spp. form a specialized cell type, called a spore, during a multistep differentiation process that is initiated in response to starvation. Spores are protected by a morphologically complex protein coat. The Bacillus anthracis coat is of particular interest because the spore is the infective particle of anthrax. We determined the roles of several B. anthracis orthologues of Bacillus subtilis coat protein genes in spore assembly and virulence. One of these, cotE , has a striking function in B. anthracis : it guides the assembly of the exosporium, an outer structure encasing B. anthracis but not B. subtilis spores. However, CotE has only a modest role in coat protein assembly, in contrast to the B. subtilis orthologue. cotE mutant spores are fully virulent in animal models, indicating that the exosporium is dispensable for infection, at least in the context of a cotE mutation. This has implications for both the pathophysiology of the disease and next-generation therapeutics. CotH, which directs the assembly of an important subset of coat proteins in B. subtilis , also directs coat protein deposition in B. anthracis . Additionally, however, in B. anthracis , CotH effects germination; in its absence, more spores germinate than in the wild type. We also found that SpoIVA has a critical role in directing the assembly of the coat and exosporium to an area around the forespore. This function is very similar to that of the B. subtilis orthologue, which directs the assembly of the coat to the forespore. These results show that while B. anthracis and B. subtilis rely on a core of conserved morphogenetic proteins to guide coat formation, these proteins may also be important for species-specific differences in coat morphology. We further hypothesize that variations in conserved morphogenetic coat proteins may play roles in taxonomic variation among species.

Educators must often measure the effectiveness of their instruction. We designed, developed, and preliminarily evaluated a multiple-choice assessment tool that requires students to apply what they have learned to evaluate scientific abstracts. This examination methodology offers the flexibility to both challenge students in specific subject areas and develop the critical thinking skills upper-level classes and research require. Although students do not create an end product (performance), they must demonstrate proficiency in a specific skill that scientists use on a regular basis: critically evaluating scientific literature via abstract analysis, a direct measure of scientific literacy. Scientific abstracts from peer-reviewed research articles lend themselves to in-class testing, since they are typically 250 words or less in length, and their analysis requires skills beyond rote memorization. To address the effectiveness of particular courses, in five different upper-level courses (Ecology, Genetics, Virology, Pathology, and Microbiology) we performed pre- and postcourse assessments to determine whether students were developing subject area competence and if abstract-based testing was a viable instructional strategy. Assessment should cover all levels in Bloom’s hierarchy, which can be accomplished via multiple-choice questions (2). We hypothesized that by comparing the mean scores of pre- and posttest exams designed to address specific tiers of Bloom’s taxonomy, we could evaluate the effectiveness of a course in preparing students to demonstrate subject area competence. We also sought to develop general guidelines for preparing such tests and methods to identify test- and course-specific problems.

A white-light source operated polymer-based micromachined Fabry-Perot biochemical sensor is reported. As a refractive-index sensitive optical sensor, its transducing signal varies upon the changes of the effective refractive index in the Fabry-Perot cavity. This sensor is fabricated from PDMS and glass. More specifically, this sensor is a micromachined Fabry-Perot interferometer (microFPI) and is fabricated by bonding a glass substrate and the soft-lithographically patterned PDMS. Several biochemicals have been detected with the microFPI biochemical sensors. Measurements show that rabbit IgG at a concentration of as low as 5 to 50 ng/ml can be detected even without any performance optimization of the devices.