Karen F. Fox

Intrajejunal levodopa infusion in Parkinson's disease: a pilot multicenter study of effects on nonmotor symptoms and quality of life.

Switching from oral medications to continuous infusion of levodopa/carbidopa gel reduces motor complications in advanced Parkinsons disease (PD), but effects on nonmotor symptoms (NMSs) are unknown. In this prospective open‐label observational study, we report the effects of intrajejunal levodopa/carbidopa gel infusion on NMS in PD based on standard assessments utilizing the nonmotor symptoms scale (NMSS) along with the unified Parkinsons disease rating scale (UPDRS 3 motor and 4 complications) and quality of life (QoL) using the Parkinsons disease questionnaire (PDQ‐8). Twenty‐two advanced PD patients (mean age 58.6 years, duration of disease 15.3 years) were followed for 6 months. A statistically significant beneficial effect was shown in six of the nine domains of the NMSS: cardiovascular, sleep/fatigue, attention/memory, gastrointestinal, urinary, and miscellaneous (including pain and dribbling) and for the total score of this scale (NMSST) paralleling improvement of motor symptoms (UPDRS 3 motor and 4 complications in “best on” state) and dyskinesias/motor fluctuations. In addition, significant improvements were found using the Parkinsons disease sleep scale (PDSS) and the PDQ‐8 (QoL). The improvement in PDQ‐8 scores correlated highly significantly with the changes in NMSST, whereas a moderately strong correlation was observed with UPDRS changes. This is the first demonstration that a levodopa‐based continuous dopaminergic stimulation is beneficial for NMS and health‐related quality of life in PD in addition to the reduction of motor fluctuations and dyskinesias.

Utility of 16S–23S rRNA spacer region methodology: how similar are interspace regions within a genome and between strains for closely related organisms?

Abstract Use of 16S–23S interspace region sequence variability, as a relatively new method, is becoming an important supplement to 16S rRNA sequencing as the standard for differentiating bacterial species. If interspace regions are as variable within a genome as between strains for closely related organisms, this limits the utility of the technique. Strains W23 and 168 represent two distinct genetic clusters within the species Bacillus subtilis . B. atrophaeus var. niger was selected as a member of a group of species closely related to B. subtilis . Comparison of the 10 rDNA operons, available from Genbank, for B. subtilis 168 shows three distinct types of interspace (ISR) regions. Two of the ten 16S–23S ISRs contain the sequences for isoleucine and alanine tRNA and are identical in sequence. The remaining eight ISRs lack tRNA sequences and have two distinct sizes. Variability among non-tRNA operons ranged from 97–100%. Counting the tRNA insert as one change, variability between tRNA and non-tRNA containing sequences ranged from 95.3–97%. The sequences of equivalent 16S–23S ribosomal operon interspace regions (ISRs) are highly conserved between W23 and 168 (99.9–100%). Thus the sequence differences between strains 168 and W23 are less than between multiple operons within 168. However, the sequence of an ISR from B. atrophaeus var. niger is quite distinct from any of the ISRs found in B. subtilis (range 88.2–91.6%). These observations are consistent with the previous suggestion that B . atrophaeus is distinct genetically from the B . subtilis sub-groups represented by W23 and 168 respectively. This is the first study to make sequence comparisons at the genome, strain and species level for the rRNA interspace region. Considerations of this issue will be important in using ISR methodology to differentiate other closely related bacterial species.

Discrimination among the B. Cereus group, in comparison to B. Subtilis, by structural carbohydrate profiles and ribosomal RNA spacer region PCR

Summary The B. cereus group (B. anthracis, B. thuringiensis and B. cereus) and B. subtilis were physiologically, molecularly and chemically characterized. Within the B. cereus group, strains grew anaerobically and were not lysozyme susceptible. B. anthracis strains were non-hemolytic unlike B. cereus or B. thuringiensis. Only B. thuringiensis produced parasporal bodies. The 16S / 23S rRNA spacer region was amplified giving 3 major products. The PCR products found in strains of B. subtilis were approximately 270, 400, and 430 nucleotides, with the corresponding bands in the B. cereus group at 250, 430, and 480. These patterns allowed B. subtilis to be differentiated from the other 3 species. Using gas chromatography-mass spectrometry, sugar profiles of vegetative cells were indistinguishable for B. cereus and B. thuringiensis. B. anthracis contained high levels of galactose which generally distinguished it from B. cereus/B. thuringiensis, while B. subtilis was distinguished from the B. cereus group by low mannosamine levels. Spore profiles differed from vegetative profiles in all 4 species. Like vegetative profiles, spore profiles were distinctive for B. cereus/B. thuringiensis, B. anthracis, and B. subtilis. B. cereus and B. thuringiensis spores both contained rhamnose, fucose, 2-O-methyl rhamnose and 3-O-methyl rhamnose, unlike B. anthracis spores which contained only rhamnose and 3-O-methyl rhamnose. B. subtilis strains were heterogeneous with some resembling B. anthracis and others B. cereus/B. thuringiensis, although B. subtilis strains typically contained quinovose. The B. cereus group can be easily distinguished from B. subtilis, however, differentiation within this group has always been problematic. Using carbohydrate profiling, B. anthracis is readily distinguished from B. cereus(B. thuringiensis. Additionally, changes in carbohydrate composition between vegetative cells and spores occurs in the B. cereus group and B. subtilis.

Restriction fragment length polymorphism of rRNA operons for discrimination and intergenic spacer sequences for cataloging of Bacillus subtilis sub-groups

Restriction fragment length polymorphism of rRNA operons (RFLP) and 16S-23S rRNA intergenic region (ISR) sequences of Bacillus subtilis subsp. subtilis, B. subtilis subsp. spizizenii, and B. atrophaeus were compared. ISR sequences of the B. subtilis subspecies were extremely similar (W23 versus 168 rrn H, J, G,W; 96.8%; rrn D, E; 98.4%; rrnB; 97.9%) and, therefore, not useful for their differentiation. However, RFLP of rRNA operons of the B. subtilis subspecies were distinct in terms of numbers and organization within the genome (e.g. the 168 sub-group generally contained 8.3- and 8.0-kb fragments absent in the W23 sub-group). The more distantly related B. atrophaeus was distinct from both B. subtilis subspecies in terms of ISR sequence and rRNA operon number and organization. RFLP of rRNA operons discriminates the two sub-groups of Bacillus subtilis that are indistinguishable by ISR sequence. However, ISR sequence defines the relatedness of B. subtilis to other species (e.g. B. atrophaeus) within the genus Bacillus.

Carbohydrates and glycoproteins of Bacillus anthracis and related bacilli: targets for biodetection.

The spore is the form released in a bioterrorism attack. There is a real need for definition of new targets for Bacillus anthracis that might be incorporated into emerging biodetection technologies. Particularly of interest are macromolecules found in B. anthracis that are (1) spore-specific, (2) readily accessible on the spore surface and (3) distinct from those present in related organisms. One of the few biochemical methods to identify the spores of B. anthracis is based on the presence of rhamnose and 3-O-methyl rhamnose as determined by gas chromatography-mass spectrometry. Related organisms additionally contain 2-O-methyl rhamnose and fucose. Carbohydrates and glycoproteins of the B. cereus group of organisms and the related B. subilis group are reviewed here. It is hypothesized that the spore-specific carbohydrate is a component of the newly described glycoprotein of the exosporium of B. anthracis. Further work to define the protein and carbohydrate components of the glycoprotein of B. anthracis could be highly useful in developing new technologies for rapid biodetection.

Identification of a Second Collagen-Like Glycoprotein Produced by Bacillus anthracis and Demonstration of Associated Spore-Specific Sugars

Certain carbohydrates (rhamnose, 3-O-methyl rhamnose, and galactosamine) have been demonstrated to be present in Bacillus anthracis spores but absent in vegetative cells. Others have demonstrated that these spore-specific sugars are constituents of the glycoprotein BclA. In the current work, spore extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A second collagen-like glycoprotein, BclB, was identified in B. anthracis. The protein moiety of this glycoprotein was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and the carbohydrate components by gas chromatography-mass spectrometry and tandem mass spectrometry. Spore-specific sugars were also demonstrated to be components of BclB.

Quantitative analysis of neutral and acidic sugars in whole bacterial cell hydrolysates using high-performance anion-exchange liquid chromatography–electrospray ionization tandem mass spectrometry

A procedure for analysis of a mixture of neutral and acidic sugars in bacterial whole cell hydrolysates using high-performance anion-exchange liquid chromatography-electrospray ionization tandem mass spectrometry (HPAEC-ESI-MS-MS) is described. Certain bacteria (including bacilli), grown under phosphate-limited conditions, switch from producing a teichoic acid (containing ribitol) to a teichuronic acid (characterized by glucuronic acid content). Bacterial cells were hydrolyzed with sulfuric acid to release sugar monomers. The solution was neutralized by extraction with an organic base. Hydrophobic and cationic contaminants (including amino acids) were removed using C18 and SCX columns, respectively. HPAEC is well established as a high-resolution chromatographic technique, in conjunction with a pulsed amperometric detector. Alternatively, for more selective detection, sugars (as M-H- ions) were monitored using ESI-MS. In HPAEC, the mobile phase contains sodium hydroxide and sodium acetate, which are necessary for chromatographic separation of mixtures of neutral and acidic sugars. Elimination of this high ionic content prior to entry into the ESI ion source is vital to avoid compromising sensitivity. This was accomplished using an on-line suppressor and decreasing post-column flow-rates from 1 ml to 50 microliters/min. In the selected ion monitoring mode, background (from the complex sample matrix as well as the mobile phase) was eliminated, simplifying chromatograms. Sugar identification was achieved by MS-MS using collision-induced dissociation.

The BclB Glycoprotein of Bacillus anthracis Is Involved in Exosporium Integrity

Anthrax is a highly fatal disease caused by the gram-positive, endospore-forming, rod-shaped bacterium Bacillus anthracis. Spores, rather than vegetative bacterial cells, are the source of anthrax infections. Spores of B. anthracis are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. Filaments of the hair-like nap are made up largely of a single collagen-like glycoprotein called BclA. A second glycoprotein, BclB, has been identified in the exosporium layer. The specific location of this glycoprotein within the exosporium layer and its role in the biology of the spore are unknown. We created a mutant strain of B. anthracis DeltaSterne that carries a deletion of the bclB gene. The mutant was found to possess structural defects in the exosporium layer of the spore (visualized by electron microscopy, immunofluorescence, and flow cytometry) resulting in an exosporium that is more fragile than that of a wild-type spore and is easily lost. Immunofluorescence studies also indicated that the mutant strain produced spores with increased levels of the BclA glycoprotein accessible to the antibodies on the surface. The resistance properties of the mutant spores were unchanged from those of the wild-type spores. A bclB mutation did not affect spore germination or kinetics of spore survival within macrophages. BclB plays a key role in the formation and maintenance of the exosporium structure in B. anthracis.

Gas chromatography-tandem mass spectrometry for trace detection of muramic acid, a peptidoglycan chemical marker, in organic dust

Abstract Muramic acid (MA) is a chemical marker for bacterial peptidoglycan. MA was analyzed (as its alditol acetate) at trace levels in organic dust using electron impact gas chromatography-tandem mass spectrometry (GC-MS-MS) and gas chromatography-mass spectrometry (GC-MS). GC-MS-MS in the multiple ion reaction mode (MRM) was demonstrated to exhibit considerably decreased background over GC-MS in the selected ion mode (SIM). MA was readily detected in air conditioning and house dust and extraneous peaks were not observed in chromatograms of plants or fungi in the MA region. GC-MS-MS has considerable potential as a tool for trace detection of muramic acid in organic dust and other complex matrices.

Total and Viable Airborne Bacterial Load in Two Different Agricultural Environments Using Gas Chromatography-Tandem Mass Spectrometry and Culture: A Prototype Study

Airborne exposure to bacterial components found in agricultural environments can lead to pulmonary inflammation. Total (viable and nonviable) bacterial load was monitored in a stable and a dairy by a new approach, gas chromatography-tandem mass spectrometry measurement of muramic acid, a component of gram positive and gram negative bacterial peptidoglycan. Also used to assess the gram negative bacterial load were 3-hydroxy fatty acids, markers of bacterial lipopolysaccharide. Culture, an established procedure for assessing the viable bacterial portion of airborne dust, served as a basis for comparison. The muramic acid and 3-hydroxy fatty acid concentrations (total C12:0, C14:0, and C16:0) showed a correlation with an R2 of 0.81. Dust and muramic acid levels also correlated. However, although relative muramic acid levels were lower in the stable than the dairy, colony forming units (CFU) were considerably higher in the stable. The total bacterial load (estimated from muramic acid values) for both the stable and dairy was also higher than would have been predicted from culture. These results suggest that nonculture based approaches and culture provide complementary but independent measurements of airborne biopollution.