Christopher J. Woolverton

Lyotropic Chromonic Liquid Crystals for Biological Sensing Applications

ABSTRACT We describe director distortions in the nematic liquid crystal (LC) caused by a spherical particle with tangential surface orientation of the director and show that light transmittance through the distorted region is a steep function of the particles size. The effect allows us to propose a real-time microbial sensor based on a lyotropic chromonic LC (LCLC) that detects and amplifies the presence of immune complexes. A cassette is filled with LCLC, antibody, and antigen-bearing particles. Small and isolated particles cause no macroscopic distortions of the uniformly aligned LCLC. Upon antibody-antigen binding, the growing immune complexes disrupt the uniformity of the director and cause detectable optical transmittance between crossed polarizers.

Lyotropic liquid crystal as a real‐time detector of microbial immune complexes

Aims:  To design a simple method for the detection of microbe–immune complexes exploiting the optical and elastic properties of a biocompatible liquid crystalline material.

Inactivation of Bacillus Endospores in Envelopes by Electron Beam Irradiation

ABSTRACT The anthrax incidents in the United States in the fall of 2001 led to the use of electron beam (EB) processing to sanitize the mail for the U.S. Postal Service. This method of sanitization has prompted the need to further investigate the effect of EB irradiation on the destruction of Bacillus endospores. In this study, endospores of an anthrax surrogate, B. atrophaeus, were destroyed to demonstrate the efficacy of EB treatment of such biohazard spores. EB exposures were performed to determine (i) the inactivation of varying B. atrophaeus spore concentrations, (ii) a D10 value (dose required to reduce a population by 1 log10) for the B. atrophaeus spores, (iii) the effects of spore survival at the bottom of a standardized paper envelope stack, and (iv) the maximum temperature received by spores. A maximum temperature of 49.2°C was reached at a lethal dose of ∼40 kGy, which is a significantly lower temperature than that needed to kill spores by thermal effects alone. A D10 value of 1.53 kGy was determined for the species. A surface EB dose between 25 and 32 kGy produced the appropriate killing dose of EB between 11 and 16 kGy required to inactivate 8 log10 spores, when spore samples were placed at the bottom of a 5.5-cm stack of envelopes.

Antimicrobial Effects of Virgin Coconut Oil and Its Medium-Chain Fatty Acids on Clostridium difficile

Clostridium difficile is the leading cause of hospital-acquired antibiotic-associated diarrhea worldwide; in addition, the proliferation of antibiotic-resistant C. difficile is becoming a significant problem. Virgin coconut oil (VCO) has been shown previously to have the antimicrobial activity. This study evaluates the lipid components of VCO for the control of C. difficile. VCO and its most active individual fatty acids were tested to evaluate their antimicrobial effect on C. difficile in vitro. The data indicate that exposure to lauric acid (C12) was the most inhibitory to growth (P<.001), as determined by a reduction in colony-forming units per milliliter. Capric acid (C10) and caprylic acid (C8) were inhibitory to growth, but to a lesser degree. VCO did not inhibit the growth of C. difficile; however, growth was inhibited when bacterial cells were exposed to 0.15-1.2% lipolyzed coconut oil. Transmission electron microscopy (TEM) showed the disruption of both the cell membrane and the cytoplasm of cells exposed to 2 mg/mL of lauric acid. Changes in bacterial cell membrane integrity were additionally confirmed for VCO and select fatty acids using Live/Dead staining. This study demonstrates the growth inhibition of C. difficile mediated by medium-chain fatty acids derived from VCO.

Novel delivery systems for coagulation proteins

Summary. Long‐term haemophilia prophylaxis with clotting factors administered by alternative delivery modes requires stable liquid formulations of these factors. We developed an aqueous‐formulated human coagulation factor IX (hCFIX) with in vitro half‐life (T1/2) of 6 weeks at 37°C and 18 months at 4°C. Upon bolus subcutaneous (SC) injection in animals, hCFIX had a bioavailability of up to 16% compared to intravenous (IV) dose. When delivered by SC implanted pumps, hCFIX attained >2% of normal human levels in the animal plasma. Hydrogels of hCFIX in a chitosan derivative, N,O‐carboxymethyl chitosan (NOCC), released hCFIX slowly in vitro, and when injected SC, gave prolonged plasma levels over those obtained by bolus IV or SC injection. Freeze‐dried human coagulation factor VIII (hCFVIII) formulated in non‐aqueous solvents had in vitro T1/2 up to 80 days at 37°C.

Electron beam irradiation dose dependently damages the bacillus spore coat and spore membrane.

Effective control of spore-forming bacilli begs suitable physical or chemical methods. While many spore inactivation techniques have been proven effective, electron beam (EB) irradiation has been frequently chosen to eradicate Bacillus spores. Despite its widespread use, there are limited data evaluating the effects of EB irradiation on Bacillus spores. To study this, B. atrophaeus spores were purified, suspended in sterile, distilled water, and irradiated with EB (up to 20 kGy). Irradiated spores were found (1) to contain structural damage as observed by electron microscopy, (2) to have spilled cytoplasmic contents as measured by spectroscopy, (3) to have reduced membrane integrity as determined by fluorescence cytometry, and (4) to have fragmented genomic DNA as measured by gel electrophoresis, all in a dose-dependent manner. Additionally, cytometry data reveal decreased spore size, increased surface alterations, and increased uptake of propidium iodide, with increasing EB dose, suggesting spore coat alterations with membrane damage, prior to loss of spore viability. The present study suggests that EB irradiation of spores in water results in substantial structural damage of the spore coat and inner membrane, and that, along with DNA fragmentation, results in dose-dependent spore inactivation.

Subverting bacterial resistance using high dose, low solubility Antibiotics in fibrin

SummaryAntibiotics (ABs) delivered from fibrin were evaluated for control of multi-drug resistant (MDR)Staphylococcus aureus. ABs having low aqueous solubility (≤1 mg/ml) were encapsulated by fibrin (composed of fibrinogen, thrombin, Factor XIIIa and calcium chloride) and examined. Electron microscopy revealed fibrin-caged, tetracycline crystals that were 0.26 to 2.8 μm in size and bound within the reticular matrix. Antibiograms documented thatS. aureus ATCC 27659 was resistant to erythromycin (ERY), penicillin G (PEN), streptomycin (STR), sulfamethoxazole-trimethoprim (SXT) and tetracycline (TET). However, low solubility formulations of STR (10 mg/ml) or SXT (0.5 mg/ml), delivered from fibrin and evaluated by the agar disk diffusion assay, produced zones of growth inhibition after 18–24h at 37°Cin vitro, indicating renewed susceptibility ofS. aureus ATCC 27659 to these ABs. ERY, PEN and TET were unable to overcome resistance at concentrations up to 10 mg/ml.In vivo, intraperitoneal (i.p.) injection of 150 mg/kg STR delivered from fibrin resulted in 100% survival of rats with MDRS. aureus peritonitis as compared with control rats receiving i.p. STR (150 mg/kg) in 0.9% saline. The results demonstrate that some low solubility ABs delivered from fibrin are efficacious in controlling infection mediated by MDRS. aureus.

The immunological and metabolic responses to exercise of varying intensities in normoxic and hypoxic environments.

Blegen, M, Cheatham, C, Caine-Bish, N, Woolverton, C, Marcinkiewicz, J, and Glickman, E. The immunological and metabolic responses to exercise of varying intensities in normoxic and hypoxic environments. J Strength Cond Res 22(5): 1638-1644, 2008-The purpose of this study was to determine the effects of varying intensities of exercise in normoxic and hypoxic environments on selected immune regulation and metabolic responses. Using a within-subjects design, subjects performed maximal tests on a cycle ergometer in both normoxic (PiO2 = 20.94%) and hypoxic (PiO2 = 14.65%) environments to determine &OV0312;O2max. On separate occasions, subjects then performed four randomly assigned, 1-hour exercise bouts on a cycle ergometer (two each in normoxic and hypoxic environments). The hypoxic environment was created by reducing the O2 concentration of inspired air using a commercially available hypoxic chamber. The intensities for the exercise bouts were predetermined as 40 and 60% of their normoxic &OV0312;O2max for the normoxic exercise bouts and as 40 and 60% of their hypoxic &OV0312;O2max for the hypoxic exercise bouts. Blood samples were collected preexercise, postexercise, 15 minutes postexercise, 2 hours postexercise, and 24 hours postexercise for the determination of interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), glucose, glycerol, free fatty acids, epinephrine, norepinephrine, and cortisol. There were no significant differences (p < 0.05) between condition or intensity for IL-1 or TNF-α. Significant differences (p < 0.05) between intensities were demonstrated for epinephrine, norepinephrine, and cortisol (p < 0.05). A significant difference was identified between normoxic and hypoxic environments with respect to nonesterifed fatty acids (0.45 ± 0.37 vs. 0.58 ± 0.31 mEq·L−1, respectively; p = 0.012). During prolonged exercise at 40 and 60% of their respective &OV0312;O2max values, hypoxia did not seem to dramatically alter the response of the selected immune system or metabolic markers. Exercise training that uses acute hypoxic environments does not adversely affect immune regulation system status and may be beneficial for those individuals looking to increase endurance performance.

Effects of remediation on the bacterial community of an acid mine drainage impacted stream

Acid mine drainage (AMD) represents a global threat to water resources, and as such, remediation of AMD-impacted streams is a common practice. During this study, we examined bacterial community structure and environmental conditions in a low-order AMD-impacted stream before, during, and after remediation. Bacterial community structure was examined via polymerase chain reaction amplification of 16S rRNA genes followed by denaturing gradient gel electrophoresis. Also, bacterial abundance and physicochemical data (including metal concentrations) were collected and relationships to bacterial community structure were determined using BIO-ENV analysis. Remediation of the study stream altered environmental conditions, including pH and concentrations of some metals, and consequently, the bacterial community changed. However, remediation did not necessarily restore the stream to conditions found in the unimpacted reference stream; for example, bacterial abundances and concentrations of some elements, such as sulfur, magnesium, and manganese, were different in the remediated stream than in the reference stream. BIO-ENV analysis revealed that changes in pH and iron concentration, associated with remediation, primarily explained temporal alterations in bacterial community structure. Although the sites sampled in the remediated stream were in relatively close proximity to each other, spatial variation in community composition suggests that differences in local environmental conditions may have large impacts on the microbial assemblage.

Applying automatically derived gene-groups to automatically predict and refine metabolic pathways

This paper describes an automated technique to predict integrated pathways and refine existing metabolic pathways using the information of automatically derived, functionally similar gene-groups and orthologs (functionally equivalent genes) derived by the comparison of complete microbial genomes archived in GenBank. The described method integrates automatically derived orthologous and homologous gene-groups (http://www.mcs.kent.edu//spl sim/arvind/orthos.html) with the biochemical pathway template available at the KEGG database (http://www.genome.ad.jp), the enzyme information derived from the SwissProt enzyme database (http:// expasys.hcuge.ch/), and the Ligand database (http://www.genome.ad.jp). The technique refines existing pathways (based upon the network of reactions of enzymes) by associating corresponding nonenzymatic and regulatory proteins to enzymes and operons and by identifying substituting homologs. The technique is suitable for building and refining integrated pathways using evolutionary diverse organisms. A methodology and the corresponding algorithm are presented. The technique is illustrated by comparing the genomes of E coli and B. subtilis with M. tuberculosis. The findings about integrated pathways are briefly discussed.