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ABSTRACTThe performance of a rapid penicillin-binding protein 2a (PBP2a) detection assay, the Alere PBP2a culture colony test, was evaluated for identification of PBP2a-mediated beta-lactam resistance in human and animal clinical isolates of Staphylococcus intermediusgroup, Staphylococcus lugdunensis, and Staphylococcus schleiferi. The assay was sensitive and specific, with all PBP2a-negative and PBP2a-positive strains testing negative and positive, respectively.

ABSTRACTWe compared recovery of Staphylococcus aureusand methicillin-resistant S. aureus(MRSA) from nasal and groin swab specimens of 600 HIV-infected outpatients by selective and nonselective direct plating and broth enrichment. Swabs were collected at baseline, 6-month, and 12-month visits and cultured by direct plating to mannitol salt agar (MSA) and CHROMagar MRSA (CM) and overnight broth enrichment with subculture to MSA (broth). MRSA isolates were characterized by pulsed-field gel electrophoresis (PFGE), staphylococcal cassette chromosome mec(SCCmec) typing, and PCR for the Panton-Valentine leukocidin. At each visit, 13 to 15% of patients were colonized with MRSA and 30 to 33% were colonized with methicillin-susceptible S. aureus(MSSA). Broth, CM, and MSA detected 95%, 82%, and 76% of MRSA-positive specimens, respectively. MRSA recovery was significantly higher from broth than CM (P= 0.001) or MSA (P= 0.001); there was no significant difference in recovery between MSA and CM. MSSA recovery also increased significantly when using broth than when using MSA (P= 0.001). Among specimens collected from the groin, broth, CM, and MSA detected 88%, 54%, and 49% of the MRSA-positive isolates, respectively. Broth enrichment had a greater impact on recovery of MRSA from the groin than from the nose compared to both CM (P= 0.001) and MSA (P= 0.001). Overall, 19% of MRSA-colonized patients would have been missed with nasal swab specimen culture only. USA500/Iberian and USA300 were the most common MRSA strains recovered, and USA300 was more likely than other strain types to be recovered from the groin than from the nose (P= 0.05).

Numerous strategies are currently available for preparing liposomes, although no single method is ideal in every respect. Two methods for producing liposomes using compressed carbon dioxide in either its liquid or supercritical state were therefore investigated as possible alternatives to the conventional techniques currently used. The first technique used modified compressed carbon dioxide as a solvent system. The way in which changes in pressure, temperature, apparatus geometry and solvent flow rate affected the size distributions of the formulations was examined. In general, liposomes in the nano‐size range with an average diameter of 200 nm could be produced, although some micron‐sized vesicles were also present. Liposomes were characterized according to their hydrophobic drug‐loading capacity and encapsulated aqueous volumes. The latter were found to be higher than in conventional techniques such as high‐pressure homogenization. The second method used compressed carbon dioxide as an antisolvent to promote uniform precipitation of phospholipids from concentrated ethanolic solutions. Finely divided solvent‐free phospholipid powders of saturated lipids could be prepared that were subsequently hydrated to produce liposomes with mean volume diameters of around 5 μm.

Numerous strategies are currently available for preparing liposomes, although no single method is ideal in every respect. Two methods for producing liposomes using compressed carbon dioxide in either its liquid or supercritical state were therefore investigated as possible alternatives to the conventional techniques currently used. The first technique used modified compressed carbon dioxide as a solvent system. The way in which changes in pressure, temperature, apparatus geometry and solvent flow rate affected the size distributions of the formulations was examined. In general, liposomes in the nano-size range with an average diameter of 200 nm could be produced, although some micron-sized vesicles were also present. Liposomes were characterized according to their hydrophobic drug-loading capacity and encapsulated aqueous volumes. The latter were found to be higher than in conventional techniques such as high-pressure homogenization. The second method used compressed carbon dioxide as an antisolvent to promote uniform precipitation of phospholipids from concentrated ethanolic solutions. Finely divided solvent-free phospholipid powders of saturated lipids could be prepared that were subsequently hydrated to produce liposomes with mean volume diameters of around 5 μm.

The cannabinoid CB1 receptor transmembrane helix (TMH) 3−4−5−6 region includes an aromatic microdomain comprised of residues F3.25, F3.36, W4.64, Y5.39, W5.43, and W6.48. In previous work, we have demonstrated that aromaticity at position 5.39 in CB1 is crucial for proper function of CB1. Modeling studies reported here suggest that in the inactive state of CB1, the binding site of the CB1 inverse agonist/antagonist SR141716A is within the TMH3−4−5−6 aromatic microdomain and involves direct aromatic stacking interactions with F3.36, Y5.39, and W5.43, as well as hydrogen bonding with K3.28. Further, modeling studies suggest that in the active state of CB1, the CB agonist WIN55,212−2 binds in this same aromatic microdomain, with direct aromatic stacking interactions with F3.36, W5.43, and W6.48. In contrast, in the binding pocket model, the CB agonist anandamide binds in the TMH2−3−6−7 region in which hydrogen bonding and C−H···π interactions appear to be important. Only one TMH3 aromatic residue, F3.25, was found to be part of the anandamide binding pocket. To probe the importance of the TMH3−4−5−6 aromatic microdomain to ligand binding, stable transfected cell lines were created for single-point mutations of each aromatic microdomain residue to alanine. Improper cellular expression of the W4.64A was observed and precluded further characterization of this mutation. The affinity of the cannabinoid agonist CP55,940 was unaffected by the F3.25A, F3.36A, W5.43A, or W6.48A mutations, making CP55,940 an appropriate choice as the radioligand for binding studies. The binding of SR141716A and WIN55,212−2 were found to be affected by the F3.36A, W5.43A, and W6.48A mutations, suggesting that these residues are part of the binding site for these two ligands. Only the F3.25A mutation was found to affect the binding of anandamide, suggesting a divergence in binding site regions for anandamide from WIN55,212−2, as well as SR141716A. Taken together, these results support modeling studies that identify the TMH3−4−5−6 aromatic microdomain as the binding region of SR141716A and WIN55,212−2, but not of anandamide.

ABSTRACTOvine Johne's disease, or paratuberculosis, occurs in many countries. In Australia, surveillance using serology is used as part of a control program, but the testing regime is costly relative to its sensitivity. For this reason, culturing of Mycobacterium aviumsubsp. paratuberculosisin fecal samples pooled from a number of sheep was evaluated. Initially, the effect of pooling on the sensitivity of fecal culture was evaluated using samples from 20 sheep with multibacillary paratuberculosis and 20 sheep with paucibacillary paratuberculosis, each confirmed histologically. All multibacillary cases and 50% of paucibacillary cases were detected by culturing of feces at a pooling rate of 1 infected plus 49 uninfected sheep. In a pilot-scale study in 1997,M. aviumsubsp. paratuberculosiswas detected by pooled fecal culture on 93% of 27 infected farms which were identified originally based on history, clinical signs, and one or more rounds of testing using serologic and histopathologic examinations. Pooled fecal culture was compared with serologic examination for submissions from 335 farms where both tests had been conducted on the same sheep and was significantly more sensitive (P< 0.001). Computer simulation of random sampling indicated that the testing of 6 pools of 50 sheep would provide 95% confidence in detecting =2% prevalence of infection. The estimated laboratory cost of pooled fecal culture when applied as a flock test is approximately 30% that of serologic examination, and sample collection costs are lower. It is recommended that pooled fecal culture replace serologic examination for detection of M. aviumsubsp.paratuberculosisinfection at the flock level.

Rho plays a regulatory role in the formation of actin stress fibers and focal adhesions, and it is also involved in integrin-mediated signaling events. To study the role of Rho in alpha(v)beta(3)/gelsolin-dependent signaling, the HIV-Tat peptide, hemagglutinin (HA)-tagged Rho(Val-14) (constitutively active) and Rho(Asn-19) (dominant negative) were transduced into avian osteoclasts. Protein transduction by HA-Tat was highly efficient, and 90-100% of the cells were transduced with HA-tagged proteins. We demonstrate here that Rho(Val-14) transduction (100 nM) stimulated gelsolin-associated phosphatidylinositol 3-kinase activity, podosome assembly, stress fiber formation, osteoclast motility, and bone resorption, mimicking osteoclast stimulation by osteopontin/alpha(v)beta(3.) The effects of Rho(Val-14) transduction stimulation was time-dependent. C3 exoenzyme blocked the effects of Rho(Val-14) and induced podosome disassembly, loss of motility, and inhibition of bone resorption. Transduction of Rho(Asn-19) produced podosome disassembly, and blocked osteopontin stimulation. These data demonstrate that integrin-dependent activation of phosphoinositide synthesis, actin stress fiber formation, podosome reorganization for osteoclast motility, and bone resorption require Rho stimulation.

ObjectivesTo determine the frequency of excretion of Mycobacterium aviumsubsp paratuberculosisin Merino sheep with Johne's disease and to quantify excretion in a group of Merino sheep.

The human cannabinoid receptors, central cannabinoid receptor (CB1) and peripheral cannabinoid receptor (CB2), share only 44% amino acid identity overall, yet most ligands do not discriminate between receptor subtypes. Site-directed mutagenesis was employed as a means of mapping the ligand recognition site for the human CB2 cannabinoid receptor. A lysine residue in the third transmembrane domain of the CB2 receptor (K109), which is conserved between the CB1 and CB2 receptors, was mutated to alanine or arginine to determine the role of this charged amino acid in receptor function. The analogous mutation in the CB1 receptor (K192A) was found to be crucial for recognition of several cannabinoid compounds excluding (R)-(+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1, 4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN 55,212-2). In contrast, in human embryonic kidney (HEK)-293 cells expressing the mutant or wild-type CB2 receptors, we found no significant differences in either the binding profile of several cannabinoid ligands nor in inhibition of cAMP accumulation. We identified a high-affinity site for (-)-3-[2-hydroxyl-4-(1, 1-dimethylheptyl)phenyl]-4-[3-hydroxyl propyl] cyclohexan-1-ol (CP-55,940) in the region of helices 3, 6, and 7, with S3.31(112), T3.35(116), and N7.49(295) in the K109A mutant using molecular modeling. The serine residue, unique to the CB2 receptor, was then mutated to glycine in the K109A mutant. This double mutant, K109AS112G, retains the ability to bind aminoalkylindoles but loses affinity for classical cannabinoids, as predicted by the molecular model. Distinct cellular localization of the mutant receptors observed with immunofluorescence also suggests differences in receptor function. In summary, we identified amino acid residues in the CB2 receptor that could lead to subtype specificity.

The pulmonary residence time of polymyxin B has been shown to be substantially increased when administered as a liposomal formulation. The use of this system to improve the treatment of cystic fibrosis lung infections requires that the antimicrobial activity of polymyxin B is unaffected by the encapsulation process. To verify that activity against the target organism, Pseudomonas aeruginosa, was retained, the bactericidal activity and MICs of both free and encapsulated polymyxin B were determined. The roles of liposomal surface characteristics in determining interactions with bacterial cell surfaces were also investigated. Encapsulation of polymyxin B was reduced when the positively charged amphiphile, stearylamine (SA) was present, with entrapment efficiencies being lower than with neutral (egg phosphatidylcholine, EPC) or negatively charged (egg phosphatidylcholine:dicetylphosphate, EPC:DCP) formulations (EPC, 45.41% +/- 0.51%; EPC:DCP 9:1, 50.81% +/- 0.79%; EPC:SA 9:1, 31.92% +/- 2.08%, n x 3). The bactericidal activities were compared, and it was found that polymyxin B retained antimicrobial activity after encapsulation. At a polymyxin B concentration of 0.3 mg/L, both positively and negatively charged liposomal polymyxin B formulations, and free drug, killed all cells after 1 h. In contrast, neutral liposome formulations did not significantly decrease the surviving cell fraction. At 0.1 mg/L, fewer cells were killed, and all liposomal formulations produced a reduction in cell numbers, which was not significantly different from free drug. It was found that the MICs of liposomal formulations were attributable to the free drug concentration achieved through release of entrapped polymyxin B. Enhanced activity was seen only with positively charged EPC:SA liposomes and those containing distearoylphosphatidylcholine (DSPC) as the bulk phospholipid. This is likely to be the result of favourable electrostatic interactions and increased liposome:cell ratios respectively. In summary, liposome encapsulation of polymyxin B was not detrimental to antimicrobial activity, and liposome surface properties and release characteristics were important in determining interactions with bacterial cells.

In this study, we focused on the pharmacological characterization of cannabinoid receptor coupling to G protein-gated inwardly rectifying potassium (GIRK) channels. Cannabinoids were tested on Xenopus laevis oocytes coexpressing the CB(1) receptor and GIRK1 and GIRK4 channels (CB(1)/GIRK1/4) or the CB(2) receptor and GIRK1/4 channels (CB(2)/GIRK1/4). WIN 55,212-2 enhanced currents carried by GIRK channels in the CB(1)/GIRK1/4 and CB(2)/GIRK1/4 system; however, the CB(2) receptor did not couple efficiently to GIRK1/4 channels. In the CB(1)/GIRK1/4 system, WIN 55,212-2 was the most efficacious compound tested. CP 55,940 and anandamide acted as partial agonists. The rank order of potency was CP 55,940 > WIN 55,212-2 = anandamide. The CB(1)-selective antagonist SR141716A alone acted as a inverse agonist by inhibiting GIRK currents in oocytes expressing CB(1)/GIRK1/4, suggesting the CB(1) receptor is constitutively activated. A conserved aspartate residue, which was previously shown to be critical for G protein coupling in cannabinoid receptors, was mutated (to asparagine, D163N) and analyzed. Oocytes coexpressing CB(1)/GIRK1/4 or D163N/GIRK1/4 were compared. The potency of WIN 55, 212-2 at the mutant receptor was similar to wild type, but its efficacy was substantially reduced. CP 55,940 did not elicit currents in oocytes expressing D163N/GIRK1/4. In summary, it appears the CB(1) and CB(2) receptors couple differently to GIRK1/4 channels. In the CB(1)/GIRK1/4 system, cannabinoids evaluated demonstrated the ability to enhance or inhibit GIRK currents. Furthermore, a conserved aspartate residue in the CB(1) receptor is required for normal communication with GIRK channels in oocytes demonstrating the interaction between receptor and channels is G protein dependent.

ABSTRACTDefinitive diagnosis of Johne’s disease in ruminants depends on confirming the presence of the causative bacterium, Mycobacterium aviumsubsp. paratuberculosis, in tissues of the host. This is readily achieved in most ruminant species by culture. However, culture of clinical specimens from sheep in many countries has been unrewarding. Such a culture from sheep was achieved recently in Australia by using a radiometric culture medium. The aims of the present study were to evaluate the culture of M. aviumsubsp. paratuberculosisfrom sheep by using modified BACTEC 12B radiometric medium, to determine the sensitivity of culture in relation to histopathology, and to evaluate a range of solid media. Culture of M. aviumsubsp. paratuberculosisfrom sheep with Johne’s disease is a sensitive method of diagnosis: intestinal tissues from all 43 animals with multibacillary disease and all 22 animals with paucibacillary disease were culture positive, while 98% of feces from 53 animals with multibacillary disease and 48% of feces from 31 animals with paucibacillary disease were culture positive. Of sheep without histological evidence of Johne’s disease from infected flocks, intestinal tissue from 32% of 41 were culture positive, while feces from 17% of 41 were culture positive. Consequently, culture is recommended as the “gold standard” test for detection of ovine Johne’s disease. Of the wide range of solid media that were evaluated, only modified Middlebrook 7H10 and 7H11 agars, which were very similar in composition to modified BACTEC 12B medium, yielded growth of ovine strains of M. aviumsubsp.paratuberculosis. The sensitivity of detection of M. aviumsubsp. paratuberculosison solid media was slightly lower than that in modified BACTEC 12B radiometric medium. Both egg yolk and mycobactin J were essential additives for growth of ovine strains of M. aviumsubsp.paratuberculosisin both liquid and solid media.

Neutral and charged large vortexed multilamellar hydrogenated egg phosphatidylcholine liposomes containing the polycationic antibiotic polymyxin B (PXB) were characterized with respect to lipid-drug interactions (differential scanning calorimetry), surface charge (zeta potential analysis), size (photon correlation spectroscopy) and morphology (transmission electron microscopy). These physicochemical results will be used to develop a liposomal drug delivery system for PXB that may be useful in the clinical treatment of lung infection in cystic fibrosis patients. The liposomal morphology and membrane integrity of all the preparations were unaffected by associated antibiotic. Drug encapsulation increased in the order neutral = positive < negative, the negatively charged liposomes increasing drug association 2-fold. The phase transition temperature of neutral and positive liposomes was not significantly affected by PXB. Drug-loaded negative liposomes showed a decreased phase transition possibly due to attractive electrostatic interactions between drug and lipid that tended to increase drug penetration and destabilize the liposome bilayer. Drug loading did not affect liposome size. However, both empty and loaded negative liposomes were significantly smaller (approx. 1 μm) than neutral and positive (approx. 2.5 μm) liposomes. Increased encapsulation with negative liposomes is therefore due not to an increase in the entrapped volume but to electrostatic interactions.

Abstract:: The angular distribution of nucleons emitted in the 12C(γ,pn) reaction has been measured using tagged photons at the Mainz microtron MAMI. The variation of the reaction strength with the polar angles of the two emitted nucleons is reported for Eγ=120−150 MeV. The proton angular distribution peaks at more backward angles than the 2H(γ,p) differential cross section indicating a departure from the simple quasi-deuteron model of 2N photo-emission. The distribution shape is in reasonable agreement with microscopic theoretical models which include both π- and ρ-exchange.

The ability of Bdellovibrio bacteriovorus to relocalize the OmpF major outer membrane porins from its Escherichia coli prey to its own outer membranes is diminished in prey expressing smooth lipopolysaccharide (S-LPS). Since porins exist in the membrane complexed with LPS, we examined the LPS associated with relocalized porin to determine whether it had been acquired intact, mixed or replaced with Bdellovibrio LPS, or derivatized by the bdellovibrios. The relocalized trimers were found associated with the same LPS originally bound to them in the E. coli. The bulk-phase LPS from bdellovibrios grown on various chemotypes of rough prey was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine whether more than the trimer-bound LPS was acquired by the bdellovibrios. This analysis revealed bands of Bdellovibrio LPS matching the LPS chemotype of the prey. One or two other bands were identical in migration to the LPS of prey-independent mutants of B. bacteriovorus and represented bdellovibrio-synthesized LPS. The LPS of bdellovibrios grown on prey with radiolabeled lipid A showed radioactivity only in gel band positions identical with those of the prey's LPS. The amount of this prey-derived LPS was shown by enzyme-linked immunosorbent assay to reach a constant value during the purification of the bdellovibrios, and it represented approximately 25% of the total Bdellovibrio LPS. Immunoelectron microscopy confirmed the presence of prey-derived LPS on the cell surface of bdellovibrios, and no evidence could be found for bdellovibrio-induced modifications of the relocalized prey LPS.

ABSTRACTThe suitability of a radiometric culture medium consisting of BACTEC 12B with PANTA PLUS, mycobactin J, and egg yolk was evaluated for detection of Mycobacterium paratuberculosisin feces, mesenteric lymph nodes, and intestinal walls from cattle, sheep, and goats. In addition, a simple method that would enable the rapid identification of Mycobacterium paratuberculosisby IS900PCR in the primary cultures was sought so that subculture to secondary egg-free radiometric medium could be avoided. An ethanol extraction followed by differential centrifugation was used to separate M. paratuberculosisfrom PCR inhibitors in the primary culture. PCR was then undertaken with the pellet, after boiling to lyse the mycobacteria; if this test was negative, the DNA in the lysate was purified with guanidine thiocyanate and silica. Cultures of feces, ilea, and mesenteric lymph nodes from cattle, sheep, and goats known to have or suspected of having Johne’s disease yielded positive PCR results 1 to 7 weeks after inoculation. Similar results were obtained with soil and pasture samples that had been spiked withM. paratuberculosis. The results suggested that radiometric culture was more sensitive than histopathology in detectingM. paratuberculosisinfection in sheep and goats and more sensitive than culture on Herrold’s egg yolk medium for the detection of the infection in cattle. Of 259 individual PCR tests with samples from cultures with growth indices of =10,237 (91.5%) were positive, with only 28 (11.8%) requiring both ethanol and silica preparation to yield a positive result. Of the 22 negative PCR results for samples from cultures with growth indices of =10, 18 were for samples from cultures that had only just developed evidence of growth. PCR-positive cultures tended to remain PCR positive over successive weeks. Flexibility in the timing of the sampling for PCR is thus possible, facilitating batch processing of samples in large-scale disease control programs for ruminants.

Various commercially produced agglutination kits are widely used for the identification of Staphylococcus aureus. These kits detect the presence of protein A and/or clumping factor on S. aureus. The literature has shown that methicillin-resistant S. aureus (MRSA) isolates which are deficient in both clumping factor and protein A may be misidentified. Two products, Slidex and Staphaurex Plus, utilize specific anti-S. aureus antibodies, potentially giving them greater sensitivity compared to products without these antibodies. We report a prospective study designed to compare the performance characteristics of Fastaph, Slidex, Staphaurex, Staphaurex Plus, Staphyloslide, and the tube coagulase test for the identification of staphylococcal isolates. All discrepant isolates were tested with the Gen-Probe AccuProbe S. aureus test and were identified to the species level with conventional reference biochemicals. A total of 1,193 isolates were tested, including 33 MRSA and 423 methicillin-sensitive S. aureus isolates. The sensitivities and specificities of the tests, respectively, were as follows: Fastaph, 99.1 and 98.9%; Slidex, 99.6 and 96.4%; Staphaurex, 98.9 and 99.9%; Staphaurex Plus, 99.6 and 93.9%; Staphyloslide, 99.1 and 98.9%; and tube coagulase, 99.3 and 100%. Sensitivity was excellent for all of the products tested. The specificities of Fastaph, Staphaurex, and Staphyloslide were excellent, while Staphaurex Plus and Slidex demonstrated less optimal results.

We consider a two–dimensional complex holomorphic dynamical system. In particular, we use the singular point theory of C. H. Briot and J. C. Bouquet to establish the existence of complex holomorphic invariant manifolds of the system in the neighbourhood of an equilibrium point with two purely imaginary eigenvalues. Consequently, this enables us to establish the existence of isochronous centre families in the neighbourhood of the equilibrium point. The results are exhibited by application to the complex Takens–Bogdanov system.

Cytokine concentrations in plasma from patients transfused with packed erythrocytes contaminated with gram-negative bacilli were measured. Cytokine concentrations in posttransfusion plasma were significantly elevated. A difference in cytokine patterns between survivors and a nonsurvivor was observed.

Automation in Survey Processing(CBS‐Select 4), 1987 Centraal Bureau voor de Statistiek, Staatsuitgeverij xi+144 pp., Dfl 40,00 ISBN 0168‐4787

Background: Neonatal readmissions are an important group of hospital attendances. They have been used as a proxy measure for neonatal health and wellbeing and are associated with significant healthcare costs[1] as well as costs to the family. We evaluated neonatal readmissions in Nottingham, in which around 10000 babies are born p.a. We hypothesised that there is an emerging pattern of diagnoses requiring readmission.Methods: We analysed birth and admission data for all babies born between 1st December 2009 and 31st March 2010 who were subsequently readmitted to local hospitals within the first 28 days of life. Cases were attained by reviewing contemporaneous admission logs and birth records with subsequent case note review.Results: Of the 3286 babies born during this time, there were 168 admissions within the neonatal period of which 71 were excluded as interdepartmental transfers, repeated or planned admissions. In 25% of cases, infants were admitted following difficulty in establishing breastfeeding, of whom ten had hypernatraemic dehydration. Other common diagnoses were: jaundice, infections and bronchiolitis. Twenty infants were admitted with hypothermia associated with sepsis, failure to establish feeding or without clear cause.Conclusions: This study illustrates an emerging new distribution of diagnoses within those babies readmitted in the neonatal period with a significant number of infants readmitted with feeding difficulties, significant weight loss and dehydration. This work illustrates the need for future work on prevention of this now common issue.