A Combined Enrichment and Aptamer Pulldown Assay for Francisella tularensis Detection in Food and Environmental Matrices

Francisella tularensis, a Gram-negative bacterium and causative agent of tularemia, is categorized as a Class A select agent by the Centers for Disease Control and Prevention due to its ease of dissemination and ability to cause disease. Oropharyngeal and gastrointestinal tularemia may occur due to ingestion of contaminated food and water. Despite the concern to public health, little research is focused on F. tularensis detection in food and environmental matrices. Current diagnostics rely on host responses and amplification of F. tularensis genetic elements via Polymerase Chain Reaction; however, both tools are limited by development of an antibody response and limit of detection, respectively. During our investigation to develop an improved culture medium to aid F. tularensis diagnostics, we found enhanced F. tularensis growth using the spent culture filtrate. Addition of the spent culture filtrate allowed for increased detection of F. tularensis in mixed cultures of food and environmental matrices. Ultraperformance liquid chromatography (UPLC)/MS analysis identified several unique chemicals within the spent culture supernatant of which carnosine had a matching m/z ratio. Addition of 0.625 mg/mL of carnosine to conventional F. tularensis medium increased the growth of F. tularensis at low inoculums. In order to further enrich F. tularensis cells, we developed a DNA aptamer cocktail to physically separate F. tularensis from other bacteria present in food and environmental matrices. The combined enrichment steps resulted in a detection range of 1–10⁶ CFU/mL (starting inoculums) in both soil and lettuce backgrounds. We propose that the two-step enrichment process may be utilized for easy field diagnostics and subtyping of suspected F. tularensis contamination as well as a tool to aid in basic research of F. tularensis ecology.     

Oral microbial communities in sickness and in health

The relationship between humans and their oral microflora begins shortly after birth and lasts a lifetime. Up until fairly recently, the associations between the host and oral bacteria were considered in terms of a multiplicity of single species interactions. However, it is becoming more apparent that the oral microbes comprise a complex community, and that oral health or disease depends on the interface between the host and the microbial community as a whole. Although it is important to continue studies of the pathogenic properties of specific microbes, these are relevant only in the context of the properties of the community within which they reside. Understanding the microbial communities that drive sickness or health is a key to combating human oral diseases.     

Functional equivalence of monomeric and dimeric forms of purified acetylcholine receptors from Torpedo californica in reconstituted lipid vesicles

Acetylcholine receptors from Torpedo californica electric organ were solubilized and purified under conditions which prevent inactivation of the agonist-regulated cation channels. The dimer form of the receptors was preserved during purification. Treatment with reducing agents converted dimers into monomers. Receptor monomers and dimers were separately reconstituted into soybean lipid vesicles by the cholate dialysis technique. Reconstituted monomers and dimers were functionally equivalent with respect to their carbamylcholine-induced dose-dependent uptake of 22Na+, the total flux of 22Na+ per receptor during the permeability response, and the occurrence of desensitization. Evidence against non-covalent association of monomers to produce dimeric functional units was obtained using glutaraldehyde as a crosslinking agent. These results show that both the acetylcholine-binding sites and the agonist-regulated cation-specific channel are contained within the alpha 2 beta gamma delta subunit structure of the acetylcholine receptor monomer.     

Sacrificial cell death and trichome breakage in an oscillatoriacean blue-green alga: the role of murein

Summary Trichomes of Microcoleus vaginatus, a motile blue-green alga of the family Oscillatoriaceae, were studied by light and electron microscopy in an effort to determine the sites of trichome breakage during production of hormogonia.According to the evidence presented herein, transcellular breakage of trichomes is the only mechanism of hormogonium production in M. vaginatus. Tearing of the murein sacculus appears to be necessary and sufficient for transcellular breakage to ensue. As Fuhs and earlier investigators have correctly claimed, this process always involves the death of the cell whose wall is torn.When trichomes of M. vaginatus break across cells to produce hormogonia, the murein sacculus usually tears along a circumferential set of junctional pores. This particular mechanism of trichome breakage is not universal among members of the family Oscillatoriaceae.This report is based on a thesis submitted in partial fulfillment of the requirements for a Ph. D. degree in Biology at Harvard University.     

Orally active achiral N-hydroxyformamide inhibitors of ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) for the treatment of osteoarthritis

A new achiral class of N-hydroxyformamide inhibitor of both ADAM-TS4 and ADAM-TS5, 2 has been discovered through modification of the complex P1 group present in historical inhibitors 1. This structural change improved the DMPK properties and greatly simplified the synthesis whilst maintaining excellent cross-MMP selectivity profiles. Investigation of structure-activity and structure-property relationships in the P1 group resulted in both ADAM-TS4 selective and mixed ADAM-TS4/5 inhibitors. This led to the identification of a pre-clinical candidate with excellent bioavailability across three species and predicting once daily dosing kinetics.     

Asparagine deamidation perturbs antigen presentation on class II major histocompatibility complex molecules

Post-translational protein modifications can be recognized by B and T lymphocytes and can potentially make "self"-proteins appear foreign to the immune system. Such modifications may directly affect major histocompatibility complex-restricted T cell recognition of processed peptides or may perturb the processing events that generate such peptides. Using the tetanus toxin C fragment protein as a test case, we show that spontaneous deamidation of asparagine residues interferes with processing by the enzyme asparagine endopeptidase (AEP) and contributes to diminished antigen presentation. Deamidation inhibits AEP action either directly, when asparagine residues targeted by AEP are modified, or indirectly, when adjacent Asn residues are deamidated. Thus, deamidation of long-lived self-proteins may qualitatively or quantitatively affect the spectrum of self-peptides displayed to T cells and may thereby contribute to the onset or exacerbation of autoimmune disease.     

Disproportionate allocation of mineral nutrients and carbon between vegetative and reproductive structures in Banksia hookeriana

We compared above-ground allocation patterns in mature shrubs of Banksia hookeriana from three 13-year-old populations, growing on nutrient-impoverished sands to determine whether C (dry mass) could be a substitute for mineral nutrients (N, P, K, Ca, Mg and NA). The percentage of reproductive structures to total above-ground growth (reproductive effort; RE) was integrated over nine successive reproductive cycles. Only 0.5% of above-ground dry mass was allocated to seeds compared with 31% to total RE. Allocations of N (24%) and P (48%) to seeds, and N (44%) and P (65%) to RE were much higher. Allocations of K, Ca, Mg and Na to seeds (<1–3%), and RE (21–35%) were closer to that of dry mass. Relative allocation (RA) is defined as the proportion of a nutrient element allocated to a structure relative to its dry mass. RA of P to seeds was 91 and N was 44, but for K, Ca, Mg and Na ranged from only 6 for K to<1 for Na. Thus P, and to a lesser extent N, provide a much more sensitive measure of the relative cost of reproduction than C in this nutrient-limited system.     

Signaling system in Porphyromonas gingivalis based on a LuxS protein

The luxS gene of quorum-sensing Vibrio harveyi is required for type 2 autoinducer production. We identified a Porphyromonas gingivalis open reading frame encoding a predicted peptide of 161 aa that shares 29% identity with the amino acid sequence of the LuxS protein of V. harveyi. Conditioned medium from a late-log-phase P. gingivalis culture induced the luciferase operon of V. harveyi, but that from a luxS insertional mutant did not. In P. gingivalis, the expression of luxS mRNA was environmentally controlled and varied according to the cell density and the osmolarity of the culture medium. In addition, differential display PCR showed that the inactivation of P. gingivalis luxS resulted in up-regulation of a hemin acquisition protein and an arginine-specific protease and reduced expression of a hemin-regulated protein, a TonB homologue, and an excinuclease. The data suggest that the luxS gene in P. gingivalis may function to control the expression of genes involved in the acquisition of hemin.