Exploring a route to a selective and sensitive portable system for explosive detection- swab spray ionisation coupled to of high-field assisted waveform ion mobility spectrometry (FAIMS).

Paper spray mass spectrometry is a rapid and sensitive tool for explosives detection but has so far only been demonstrated using high resolution mass spectrometry, which bears too high a cost for many practical applications. Here we explore the potential for paper spray to be implemented in field applications with portable mass spectrometry. This involved (a) replacing the paper substrate with a swabbing material (which we call "swab spray") for compatibility with standard collection materials; (b) collection of explosives from surfaces; (c) an exploration of interferences within a ±â€¯0.5 m/z window; and (d) demonstration of the use of high-field assisted waveform ion mobility spectrometer (FAIMS) for enhanced selectivity. We show that paper and Nomex® are viable collection materials, with Nomex providing cleaner spectra and therefore greater potential for integration with portable mass spectrometers. We show that sensitive detection using swab spray will require a mass spectrometer with a mass resolving power of 4000 or more. We show that by coupling the swab spray ionisation source with FAIMS, it is possible to reduce background interferences, thereby facilitating the use of a low resolving power (e.g. quadrupole) mass spectrometer.

Direct monitoring of pulmonary disease treatment biomarkers using plasmonic gold nanorods with diffusion-sensitive OCT.

The solid concentration of pulmonary mucus (wt%) is critical to respiratory health. In patients with respiratory disease, such as Cystic Fibrosis (CF) and Chronic Obstructive Pulmonary Disorder (COPD), mucus hydration is impaired, resulting in high wt%. Mucus with high wt% is a hallmark of pulmonary disease that leads to obstructed airways, inflammation, and infection. Methods to measure mucus hydration in situ and in real-time are needed for drug development and personalized therapy. We employed plasmonic gold nanorod (GNR) biosensors that intermittently collide with macromolecules comprising the mucus mesh as they self-diffuse, such that GNR translational diffusion (DT) is sensitive to wt%. GNRs are attractive candidates for bioprobes due to their anisotropic optical scattering that makes them easily distinguishable from native tissue using polarization-sensitive OCT. Using principles of heterodyne dynamic light scattering, we developed diffusion-sensitive optical coherence tomography (DS-OCT) to spatially-resolve changing DT in real-time. DS-OCT enables, for the first time, direct monitoring of changes in nanoparticle diffusion rates that are sensitive to nanoporosity with spatial and temporal resolutions of 4.7 µm and 0.2 s. DS-OCT therefore enables us to measure spatially-resolved changes in mucus wt% over time. In this study, we demonstrate the applicability of DS-OCT on well-differentiated primary human bronchial epithelial cells during a clinical mucus-hydrating therapy, hypertonic saline treatment (HST), to reveal, for the first time, mucus mixing, cellular secretions, and mucus hydration on the micrometer scale that translate to long-term therapeutic effects.

Effects of inoculum, pH, and cations on the in vitro activity of fidaxomicin (OPT-80, PAR-101) against Clostridium difficile.

The effects of the inoculum, pH, cation concentrations, and different lots of commercial media on the in vitro susceptibility of Clostridium difficile to fidaxomicin were examined. Of the factors evaluated, only pH alterations influenced the activity of fidaxomicin against C. difficile, noticeably reducing its activity at higher pH (> or =7.9).

Typing and susceptibility of bacterial isolates from the fidaxomicin (OPT-80) phase II study for C. difficile infection.

BACKGROUND: Clostridium difficile infection (CDI) has been increasing in incidence and severity in recent years, coincident with the spread of a "hypervirulent" strain, REA type BI (ribotype 027, PFGE NAP 1). Exacerbating the problem has been the observation that metronidazole may be showing decreased effectiveness, particularly in the more severe cases. Fidaxomicin is an 18-membered macrocycle currently in phase 3 trials for the treatment of C. difficile infection (CDI). An open-label, phase II study in CDI patients has been completed and the clinical results published. C. difficile organisms were isolated from patient stool specimens and typed by restriction endonuclease analysis (REA) in order to determine the frequency and susceptibility of the C. difficile isolates and their response to treatment. METHODS: Fecal samples were plated on CCFA agar for isolation of C. difficile. These isolates were tested for susceptibility to fidaxomicin, vancomycin, and metronidazole using CLSI agar dilution methods and were typed by REA. RESULTS: C. difficile was isolated from 38 of 49 subjects and 16 (42%) were the epidemic C. difficile BI group. The BI strain was distributed approximately equally in the three dosing groups. Overall antibiotic susceptibilities were consistent with the previously reported MIC(90) values for the three antibiotics tested, but the MIC(90) of BI strains was two dilutions higher than non-BI strains for metronidazole and vancomycin (for both antibiotics, MIC(90) was 2 microg/mL vs. 0.5 microg/mL, P<0.01 for metronidazole, P=NS for vancomycin). Clinical cure for BI isolates (11/14, 79%) was not significantly different from non-BI isolates (21/22, 95%). CONCLUSION: These results underscore the high prevalence of the BI epidemic strain and demonstrate that mild to moderate CDI infection as well as severe disease can be caused by these strains. Fidaxomicin cure rates for subjects with BI and with non-BI strains are similar, although the small numbers of subjects preclude a robust statistical comparison.

Safety, tolerance, and pharmacokinetic studies of OPT-80 in healthy volunteers following single and multiple oral doses.

Current therapies for Clostridium difficile infection (CDI) are encumbered by treatment failures and recurrences. Due to its high in vitro activity against C. difficile but low activity against the typical intestinal flora, minimal absorption, and durable cure in the hamster model of C. difficile infection, OPT-80 was considered for clinical development as a therapy for CDI. This trial consisted of two phases. Four single oral doses of OPT-80 (100, 200, 300, and 450 mg) were administered in a crossover manner to 16 healthy volunteers in a double-blind, placebo-controlled phase 1A study; a 1- to 2-week washout interval separated the treatments. In the double-blind phase 1B study, 24 healthy subjects were randomized to receive OPT-80 (150, 300, or 450 mg) or placebo for 10 days. In both studies, OPT-80's safety and tolerability were evaluated and the concentrations of OPT-80 and its primary metabolite (OP-1118) in plasma and feces were determined. OPT-80 levels in the urine were also analyzed for the phase 1A study. In both the single-dose and the multiple-dose studies, OPT-80 was well tolerated by all subjects in all dose groups. Maximal plasma concentrations were near or below the limit of quantification (5 ng/ml) across the dose range; urine concentrations were below the detection limit. The fecal total recovery of OPT-80 plus its major metabolite, OP-1118, approximated 100%. The tolerability, high fecal concentration, and low systemic exposure data from these studies support the further clinical development of OPT-80 as an oral therapy for CDI.

The effect of ascorbic acid and L-histidine therapy on acute mammary inflammation in dairy cattle.

Ascorbic acid and L-histidine were investigated as antioxidant therapies for acute mammary inflammation. Mastitis was induced in eight nonpregnant Holstein cows by intramammary infusion of endotoxin. Treatments were administered in a 4 x 4 Latin square crossover design with 1-wk periods between challenges with endotoxin. Four individual treatments, control, ascorbic acid only, L-histidine only, and ascorbic acid plus L-histidine, were applied. Two doses of 25 g of ascorbic acid administered intravenously at 3- and 5-h postendotoxin challenge increased milk production recovery (9% higher, P < 0.02) and tended to reduce the extent of rumen stasis. Two doses of 25 g of L-histidine similarly administered decreased plasma antioxidant activities 5.5% (P < 0.05). However, ascorbic acid and L-histidine had no effects on rectal temperature, heart rate, respiratory rate, and dry matter intake. The data suggested that ascorbic acid provided some potential benefit for recovery from acute mammary inflammation in dairy cattle.

Diversity of Salmonella serotypes in cull (market) dairy cows at slaughter.

OBJECTIVE: To determine the diversity of Salmonella serotypes isolated from a large population of cull (market) dairy cows at slaughter. DESIGN: Cross-sectional study. SAMPLE POPULATION: Salmonella organisms isolated from the cecal-colon contents of 5,087 market dairy cows. PROCEDURE: During winter and summer 1996, cecal-colon contents of cull dairy cows at slaughter were obtained from 5 US slaughter establishments. Specimens were subjected to microbiologic culturing for Salmonella spp at 1 laboratory. Identified isolates were compared with Salmonella isolation lists published by the Centers for Disease Control and Prevention (CDC) and the National Veterinary Services Laboratory (NVSL) for approximately the same period. The Simpson diversity index was used to calculate the likelihood that Salmonella isolates selected randomly by establishment were different. RESULTS: Of 58 Salmonella serotypes identified, Salmonella ser. Montevideo was the most prevalent. Two of the top 10 CDC serotypes identified from in 1996, Salmonella ser. Typhimurium and S Montevideo, appeared on our top 10 list; 8 of the top 10 were found on NVSL listings. Thirty-one of 59 S. Typhimurium isolates were identified as DT104 and found at a west slaughter establishment, 30 during the winter and 1 during the summer. The greatest diversity of serotypes was at a southeast establishment during the summer; the least diversity was at a central establishment in the winter. CONCLUSIONS AND CLINICAL RELEVANCE: 58 Salmonella serotypes were isolated from market dairy cows at slaughter and could pose a threat for food-borne illness. Salmonella Montevideo was the most frequently isolated serotype and may contribute substantially to salmonellosis in dairy cattle.

Prevalence of Salmonella spp in cull (market) dairy cows at slaughter.

OBJECTIVE: To determine the prevalence of Salmonella spp in the cecal-colon contents of cull (market) dairy cows at slaughter because of potential public health ramifications. DESIGN: Survey study. SAMPLE POPULATION: Cecal-colon contents collected from 5,087 cull (market) dairy cows at slaughter at 5 slaughter establishments across the United States. PROCEDURE: During 2 periods of the year, winter (January and February) and summer (July through September), 5 cull (market) cow slaughter establishments in the United States--west (WE), southeast (SEE), central (CE), north central (NCE), and south central (SCE)--establishments were visited, and cecal-colon contents of cull dairy cows were obtained at the time of slaughter. Samples were examined by microbiologic culture at a single laboratory for Salmonella spp. RESULTS: Salmonella spp were detected in 23.1% of cecal-colon content samples from cull dairy cows across the 5 slaughter establishments. The highest site prevalence (54.5%) was detected at the WE during the summer period, whereas the lowest was found at the CE during the summer (4.3%) and at the NCE during the winter (4.5%). Considerable variation in the daily prevalence of Salmonella spp was found, particularly at the WE and the SCE. Salmonella spp were isolated from 93% of cecal-colon contents collected on a summer day at the WE. CONCLUSIONS AND CLINICAL RELEVANCE: Results strongly suggest that there is a high prevalence of Salmonella spp in cull dairy cows at slaughter, which could burden Hazard Analysis Critical Control Point programs implemented in slaughter establishments. Procedures to reduce Salmonella load at the dairy farm and during transport to slaughter could reduce the risk of spread during the slaughter process.

Toward automated synthesis of oligosaccharides and glycoproteins.

The discovery of previously unknown functions associated with carbohydrates and the study of their structure-function relations are of current interest in carbohydrate chemistry and biology. Progress in this area is, however, hampered by the lack of convenient and effective tools for the synthesis and analysis of oligosaccharides and glycoconjugates. Development of automated synthesis of such materials is necessary to facilitate research in this field. This review describes recent advances in carbohydrate synthesis, with particular focus on developments that have potential application to the automated synthesis of oligosaccharides, glycopeptides, and glycoproteins.

Synthesis of solid-supported mirror-image sugars: a novel method for selecting receptors for cellular-surface carbohydrates.

We introduced a novel method, through mirror-image phage display, for the identification of high-affinity D-peptides to target specific cell-surface carbohydrates. Both 3-deoxy-alpha-L-manno-2-octulosonic acid (L-KDO) and L-sialic acid and an L-sialo-disaccharide have been synthesized and attached to a solid support for selection of high-affinity peptide binders displayed on phages. Our initial studies in this effort produce single-chain Fab sequences and dodecapeptides that bind to sialic acid and KDO with nanomolar and high micromolar affinity.

Natural electrophoresis of norepinephrine and ascorbic acid.

The electric field produced by cell membranes, extending only a few nanometers, is 1000 times stronger than the electric fields required to produce dissociation of molecular complexes. Using the complex formed by norepinephrine (NE) and ascorbic acid (AA), we have demonstrated the quantitative binding of AA to NE, the use of capillary electrophoresis to measure quantitative binding of nonelectrolyte complexes, the determination of a dissociation constant (Kd) from electric field-dissociation constants (Ke), and a model for natural dissociation of the NE-AA complex due to the electric field generated by a cell membrane. NE-AA dissociation constants show little effect of NE concentration or pH changes. NE-related compounds also bind AA: epinephrine > norepinephrine > tyrosine > histamine > phenylalanine. Serotonin does not bind AA. Phosphorylated AA and glucose also bind NE at 0.05 and 0.08 of the AA binding, respectively. Natural electrophoresis of molecular complexes allows compounds to travel through the body in a protected state and still be available for physiological activity upon reaching a membrane.

Nurses' knowledge of palliative care in the Australian Capital Territory.

Australia has a growing number of specialist palliative care services. As they expanded in the Australian Capital Territory, a working party was established to discuss issues associated with palliative care. One activity authorized by this committee was a survey of nurses' knowledge of palliative care. The Palliative Care Quiz for Nurses (Ross et al, 1996) was adapted with permission for this survey: 455 registered and enrolled nurses were surveyed; 247 (54%) participants returned completed questionnaires. The overall mean score for the Palliative Care Quiz was 12.4 of a possible 20; the mean scores were 13.2 for registered nurses and 10.6 for enrolled nurses. Nurses with some oncology or palliative care experience scored significantly higher than others. Nurses with more work experience as measured by working years also attained significantly higher scores. Analysis and examination of correct items suggest that nurses have acquired basic knowledge through experience. However, as other studies have suggested, there is also a lack of knowledge of complex symptoms found in palliative care patients.

Differential coupling of smooth and skeletal muscle pyruvate kinase to creatine kinase.

The interaction of pyruvate kinase from skeletal (SKPK) and smooth (SMPK) muscle with MM-creatine kinase (MMCK) and BB-creatine kinase (BBCK) was assessed using temporal absorbance changes, variations in absorbance at different wavelengths, concentration dependence, association in an electric field, and PK kinetic activity. SKPK exhibits a time course of absorbance increase in the presence of MMCK with a time constant of 29.5 min. This increase occurs at all wavelength from 240 to 1000 nm. At 195 nm, the combination of SKPK and MMCK produces a decrease in absorption with electric fields of both 0 and 204 V/cm. The change in SKPK-MMCK is saturable. SKPK activity is significantly increased by the presence of MMCK in solutions of 0-32% ethanol. These results indicate specific SKPK-MMCK interaction. SMPK and BBCK did not exhibit similar coupling when the BBCK concentration dependence of absorbance or SMPK activity in solutions of 0-32% ethanol was determined. Both MMCK and BBCK increased SKPK activity; neither MMCK nor BBCK increased SMPK activity. The ability to form diazymatic complexes with creatine kinase appears to reside in SKPK. This coupling may account for the increased flux through PK without significant substrate changes seen during skeletal muscle activation. This coupling will not occur in smooth muscle.

Carbohydrate Mimetics: A New Strategy for Tackling the Problem of Carbohydrate-Mediated Biological Recognition.

Useful strategies for the design of molecules to mimic carbohydrates have been developed over the past few years. Mimics of the target may contain new functional groups, a new scaffold, or both (in the schematic representation the natural ligand is shown on the left and the modified version on the right). Many examples of successful carbohydrate mimetics that interfere with sugar-protein and sugar-nucleic acid interactions are known.

Breaking the low barrier hydrogen bond in a serine protease.

The serine protease subtilisin BPN' is a useful catalyst for peptide synthesis when dissolved in high concentrations of a water-miscible organic co-solvent such as N,N-dimethylformamide (DMF). However, in 50% DMF, the k(cat) for amide hydrolysis is two orders of magnitude lower than in aqueous solution. Surprisingly, the k(cat) for ester hydrolysis is unchanged in 50% DMF. To explain this alteration in activity, the structure of subtilisin 8397+1 was determined in 20, 35, and 50% (v/v) DMF to 1.8 A resolution. In 50% DMF, the imidazole ring of His64, the central residue of the catalytic triad, has rotated approximately 180 degrees around the Cbeta-Cgamma bond. Two new water molecules in the active site stabilize the rotated conformation. This rotation places His64 in an unfavorable geometry to interact with the other members of the catalytic triad, Ser221 and Asp32. NMR experiments confirm that the characteristic resonance due to the low barrier hydrogen bond between the His64 and Asp32 is absent in 50% DMF. These experiments provide a clear structural basis for the change in activity of serine proteases in organic co-solvents.

Effects of milk somatic cell count on cottage cheese yield and quality.

Eight Holstein cows in midlactation were selected for low milk somatic cell count (SCC) and the absence of the pathogens that cause mastitis. Milk collection and cottage cheese manufacture from low SCC milk were replicated on each of 4 d (control period). Each cow was infused with 1000 cfu of Streptococcus agalactiae. One week after infusion, milk from the same eight cows was collected and commingled. On each of 4 d, cottage cheese was made from milk with high SCC (treatment period). A mass-balance protocol, accounting for protein and total solids, was used to determine recoveries in whey, wash water, and uncreamed curd. Actual yields, yields adjusted for composition, and theoretical yields of uncreamed curd were calculated. Mean milk SCC for the periods with the low SCC (control) and the high SCC (treatment) were 83 x 10(3) and 872 x 10(3) cells/ml, respectively. The recovery of protein in the uncreamed curd was higher during the low SCC period than during the high SCC period (75.85% vs. 74.35%). High SCC and the associated higher proteolytic activity caused higher protein loss in the whey and wash water and more curd fines. The percentage of total solids recovery in uncreamed curd was higher for high SCC milk because the lactose content of the high SCC milk was 0.27% lower than that of the low SCC milk. The moisture content of the curd was higher for the high SCC milk (82.75% vs. 83.81%). Proteolysis during refrigerated storage was faster in cottage cheese made from high SCC milk. The yield efficiency of uncreamed curd, adjusted for composition based on 81% moisture, was 4.34% lower for the cottage cheese curd made from high SCC milk.

Enzyme action in glycoprotein synthesis.

Just a few decades ago, the saccharides bound to glycoproteins were considered little more than an irritation. They increased the difficulty of purifying and characterizing proteins, making proteins run as several bands on gels and smearing them on columns. They were considered a nuisance and were typically cleaved away to reveal the 'important part', the protein moiety, for structural (e.g. via X-ray crystallography or nuclear magnetic resonance) and functional studies. We now realize that that the saccharide is often as important as the protein itself, and that glycosylation can have many effects on the function, structure, physical properties and targeting of a protein. There are a myriad of reviews and books on this subject, reflecting the nearly overwhelming number of articles in print discussing saccharide structures, glycoprotein processing enzymes and the biological implication of glycosylation. This review discusses, in turn, the extent and biological relevance of glycosylation; the structures observed; how glycosylated proteins are formed in vivo; the clinical relevance of glycosylation, in terms of the correlations between disease states and unusual glycosylation patterns; and, finally, the molecules, both natural and synthetic, that can be used to study the role of carbohydrates in glycoprotein structure and function or to disrupt various carbohydrate recognition processes and enzymatic reactions in the glycoprotein synthetic pathway.

The murine homolog of the interleukin-8 receptor CXCR-2 is essential for the occurrence of neutrophilic inflammation in the air pouch model of acute urate crystal-induced gouty synovitis.

OBJECTIVE: Acute neutrophil-dependent inflammation is central to acute gout. Urate crystals induce different classes of neutrophil chemotaxins, including certain chemokines (e.g., interleukin-8 [IL-8], growth-related oncogene alpha [GROalpha]) that share CXCR-2 as a receptor. This study was undertaken to assess the role of CXCR-2 ligands in a model of acute gout. METHODS: Urate crystals were injected into subcutaneous air pouches in mice that expressed or lacked the murine CXCR-2 homolog (mIL-8RH), and the development of neutrophilic inflammation was assessed. RESULTS: In normal mice, urate crystals induced a 10-fold increase (P < 0.01) in pouch fluid leukocytes (principally neutrophils) at 4 hours. Leukocytes adhered to the pouch lining, where crystals, the mIL-8RH ligand KC/GROalpha, and cells bearing mIL-8RH were abundant. In mIL-8RH(-/-) mice, urate crystals induced a proteinaceous leukocyte-poor exudate at 4 hours, despite crystal-induced activation of resident cells (documented by KC/GROalpha expression). CONCLUSION: Chemokines that bind the IL-8 receptor CXCR-2 are essential for the development of acute neutrophilic inflammation in response to urate crystals in the subcutaneous air pouch model.