The metabolic impact of extracellular nitrite on aerobic metabolism of Paracoccus denitrificans.

Nitrite, in equilibrium with free nitrous acid (FNA), can inhibit both aerobic and anaerobic growth of microbial communities through bactericidal activities that have considerable potential for control of microbial growth in a range of water systems. There has been much focus on the effect of nitrite/FNA on anaerobic metabolism and so, to enhance understanding of the metabolic impact of nitrite/FNA on aerobic metabolism, a study was undertaken with a model denitrifying bacterium Paracoccus denitrificans PD1222. Extracellular nitrite inhibits aerobic growth of P. denitrificans in a pH-dependent manner that is likely to be a result of both nitrite and free nitrous acid (pKa = 3.25) and subsequent reactive nitrogen oxides generated from the intracellular passage of FNA into P. denitrificans. Increased expression of a gene encoding a flavohemoglobin protein (Fhp) (Pden_1689) was observed in response to extracellular nitrite. Construction and analysis of a deletion mutant established Fhp to be involved in endowing nitrite/FNA resistance at high extracellular nitrite concentrations. Global transcriptional analysis confirmed nitrite-dependent expression of fhp and indicated that P. denitrificans expressed a number of stress response systems associated with protein, DNA and lipid repair. It is therefore suggested that nitrite causes a pH-dependent stress response that is due to the production of associated reactive nitrogen species, such as nitric oxide from the internalisation of FNA.

Nitrous Oxide Metabolism in Nitrate-Reducing Bacteria: Physiology and Regulatory Mechanisms.

Nitrous oxide (N2O) is an important greenhouse gas (GHG) with substantial global warming potential and also contributes to ozone depletion through photochemical nitric oxide (NO) production in the stratosphere. The negative effects of N2O on climate and stratospheric ozone make N2O mitigation an international challenge. More than 60% of global N2O emissions are emitted from agricultural soils mainly due to the application of synthetic nitrogen-containing fertilizers. Thus, mitigation strategies must be developed which increase (or at least do not negatively impact) on agricultural efficiency whilst decrease the levels of N2O released. This aim is particularly important in the context of the ever expanding population and subsequent increased burden on the food chain. More than two-thirds of N2O emissions from soils can be attributed to bacterial and fungal denitrification and nitrification processes. In ammonia-oxidizing bacteria, N2O is formed through the oxidation of hydroxylamine to nitrite. In denitrifiers, nitrate is reduced to N2 via nitrite, NO and N2O production. In addition to denitrification, respiratory nitrate ammonification (also termed dissimilatory nitrate reduction to ammonium) is another important nitrate-reducing mechanism in soil, responsible for the loss of nitrate and production of N2O from reduction of NO that is formed as a by-product of the reduction process. This review will synthesize our current understanding of the environmental, regulatory and biochemical control of N2O emissions by nitrate-reducing bacteria and point to new solutions for agricultural GHG mitigation.

Splenic injury as a rare complication of colonoscopy.

Injury resulting from colonoscopy is rare. Bleeding and perforation are the most serious complications. The case history is described of a patient who developed splenic rupture following screening colonoscopy with associated polypectomy.

All stressed out. Salmonella pathogenesis and reactive nitrogen species.

Bacterial pathogens must overcome a range of challenges during the process of infecting their host. The ability of a pathogen to sense and respond appropriately to changes in host environment is vital if the pathogen is to succeed. Mammalian defense strategies include the use of barriers like skin and epithelial surfaces, the production of a chemical arsenal, such as stomach acid and reactive oxygen and nitrogen species, and a highly coordinated cellular and humoral immune response. Salmonella serovars are significant human and animal pathogens which have evolved several mechanisms to overcome mammalian host defense. Here we focus on the interplay which occurs between Salmonella and the host during the infection process, with particular emphasis on the complex bacterial response to reactive nitrogen species produced by the host. We discuss recent advances in our understanding of the key mechanisms which confer bacterial resistance to nitrogen species, which in response to nitric oxide include the flavohemoglobin, HmpA, the flavorubredoxin, NorV, and the cytochrome c nitrite reductase, NrfA, whilst in response to nitrate include a repertoire of nitrate reductases. Elucidating the precise role of different aspects of microbial physiology, nitrogen metabolism, and detoxification during infection will provide valuable insight into novel opportunities and potential targets for the development of therapeutic approaches.

Delayed diagnosis of infarcted small bowel due to right-sided Bochdalek hernia.

We report a case of an adult patient with small bowel infarction, secondary to a strangulated Bochdalek hernia.

The effect of poloxamer viscosity on liquid-filling of solid dispersions in hard gelatin capsules.

Preliminary studies were undertaken on poloxamers to investigate their suitability for liquid-fill formulations for hard gelatin capsules. Poloxamers with viscosity in the range (0.32-2.8 Pa s) and melting point 48-58 degrees C were used as the continuous phase, with alpha-lactose monohydrate of negligible solubility in the molten poloxamers, as a model insoluble disperse phase. Physicochemical characterization by rheology, melt solidification and moisture uptake indicated that poloxamers were suitable excipients for liquid-filling in hard gelatin capsules. 10% w/w lactose/poloxamer dispersions were thixotropic and shear thinning and exhibited good capsule-filling properties, disperse-phase uniformity and satisfactory apparent viscosity at 70 degrees C.

Influence of rheological behaviour of particulate/polymer dispersions on liquid-filling characteristics for hard gelatin capsules.

Lactose/poloxamer dispersions were prepared by mixing under vacuum to achieve a de-aerated mix with good capsule filling properties and disperse phase uniformity at 70 degrees C. Satisfactory capsule filling of molten dispersions was achieved up to a limiting concentration of disperse phase, dependent on particle size distribution and continuous phase viscosity. Lactose/poloxamer dispersions exhibited thixotropic shear thinning behaviour with an abrupt increase in apparent viscosity above a limiting concentration of disperse phase. There was a good correlation between satisfactory filling of molten dispersions into capsules and apparent viscosity of the formulation, whereas, the pronounced increase in apparent viscosity resulted in unsatisfactory filling above a critical concentration of disperse phase. The rheological data was analysed in detail using empirical models and also used to identify capsule filling problems at extrudate shear rates for flow from hopper to pump (12 s(-1)) and from nozzle to capsule (340 s(-1)).

Gastric colonisation, intestinal permeability and septic morbidity in acute pancreatitis.

INTRODUCTION: Bacterial translocation (BT) may represent an important cause of septic morbidity in patients with acute pancreatitis. We have previously demonstrated an association between BT, septic morbidity and colonisation of the proximal GI tract. Alterations in intestinal permeability (IP) may also predispose to BT. The aim of this study was to assess the extent of gastric colonisation, measure IP in patients with acute pancreatitis and relate these to both disease severity and septic complications. METHODS: Gastric colonisation was determined by culturing a sample of nasogastric aspirate, and IP was measured using a dual sugar probe technique (lactulose/rhamnose test). Disease severity was assessed according to the modified Glasgow (Imrie) criteria. All septic complications were recorded prospectively. RESULTS: A total of 59 patients were studied (M:F ratio 32:27, median age 66 years, range 18-89), 24 (31%) of whom had severe disease. A nasogastric aspirate was obtained in 56 patients. There was a significantly higher incidence of colonisation with potentially pathogenic enteric bacteria in patients with severe disease compared to those with mild disease (57 vs. 6%, p < 0.001). Septic morbidity occurred in 29% of severe patients and 11% mild patients (p = 0.17). 33% of patients colonised with enteric organisms developed sepsis, compared to 16% with no enteric bacteria in the NG aspirate (p = 0.34). Enteric bacteria caused 77% of the septic complications. Intestinal permeability was neither associated with disease severity nor was it predictive of septic morbidity. CONCLUSIONS: There is significantly higher incidence of gastric colonisation with enteric bacteria in patients with severe acute pancreatitis, but no difference in IP. Enteric bacteria were implicated in the majority of septic complications. These findings support the gut origin of sepsis hypothesis in acute pancreatitis.

Quantifying electrostatic interactions in pharmaceutical solid systems.

Triboelectrification of pharmaceutical powders with stainless steel and polymer contact surfaces was investigated. alpha-Lactose monohydrate, from 90 to 125 up to 355-500 microm, was used to quantify electrostatic interactions with negligible powder adhesion to the contact surface. Size fractions down to 53-75 microm alone and in binary mixtures with <10 microm lactose or micronized salbutamol were used to investigate triboelectrification with powder adhered to the contact surface. Triboelectrification was performed in a cyclone charger fitted with interchangeable contact surfaces of steel and polymers, representing the surfaces of pharmaceutical processing and manufacturing equipment, packaging materials and components of dry powder inhaler devices. The results for single component powders showed charge acquisition was inversely related to particle size, where contact surface contamination was negligible. However, with particulate contamination, triboelectrification was more complex due to particle collisions with clean and contaminated contact surfaces. Analysis of adhered and non-adhered powder provided information about changes in composition of two component powders during triboelectrification. Particle size and chemical analyses showed that composition changes of mixtures may be related to powder/contact surface affinity and interparticulate forces for separation of components in a cohesive mix during triboelectrification.

Triboelectrification of spray-dried lactose prepared from different feedstock concentrations.

Powder systems may acquire electrostatic charge during various pharmaceutical processing operations and may give rise to difficulties in handling and powder flow, mainly due to adhesion/cohesion effects. We have investigated the electrostatic charging of spray-dried lactose prepared from different feedstock concentrations using a laboratory spray-dryer. Triboelectrification of the spray-dried lactose samples was effected through contact with the stainless steel surface of either a mixing vessel or a cyclone separator. Results from both techniques showed differences in charge accumulation and particle-steel adhesion between the spray-dried lactose samples. As the feedstock concentration used to produce the spray-dried lactose was increased in the range 10-50% w/v, the mean charge on the lactose decreased from -20.8 to -1.3 nC g(-1) and -54.9 to -4.1 nC g(-1) for the mixing vessel and cyclone separator, respectively, with a corresponding decrease in adhesion. In addition, as the feedstock concentration was increased from 10 to 50% w/v, decreases were obtained in surface area values (1.06 to 0.56 m2 g(-1)), pore diameter (198.7 to 83.5 microm) and pore volume (1.09 to 0.75 cm3 g(-1)), and together with differences in crystal form correlated with the charge and adhesion results. The results suggested that the feedstock concentration could have a considerable influence on the charging and adhesional properties of spray-dried lactose. This may have relevance during pharmaceutical processing and manufacturing operations.

Effect of contamination of pharmaceutical equipment on powder triboelectrification.

Triboelectrification of pharmaceutical powders may cause problems during processing and manufacture due to adhesion/cohesion effects. The aim of this work was to investigate the role of adhered particles and moisture as contact surface contaminants on the electrostatic charging of size fractionated lactose, following contact with a surface, i.e. stainless steel, typically used in pharmaceutical process and manufacturing operations. Replicated experimental runs without cleaning the contact surface showed a successive decrease in the net electronegative charge due to adhered lactose particles. Removal of these contaminating particles by different cleaning methods had a considerable effect on the charge after triboelectrification. The charge on the lactose samples was found to decrease when humidity in the cyclone apparatus was increased from 2 to 100% relative humidity. These results clearly demonstrate that moisture, particulate contamination and method of cleaning of processing equipment during pharmaceutical manufacturing operations may influence the electrostatic behaviour of powders.

Comparison of surface modification and solid dispersion techniques for drug dissolution.

Surface modification and solid dispersion formulations using hydrophilic excipients can significantly alter the dissolution behaviour of hydrophobic drug materials. The effect of these techniques used individually and in combination on the dissolution properties of the hydrophobic drug, phenylbutazone (PB), are compared. PB was treated with a poloxamer, Synperonic((R)) F127 by an adsorption method. Solid dispersions (10 and 20% w/w) were prepared with untreated PB or PB previously modified with Synperonic((R)) F127 (PBT) in molten F127. Dissolution tests of capsule formulations of PB, PBT and solid dispersion formulations, in pH 6.4 buffer at 37+/-0.5 degrees C demonstrated that after 140 min, release of PB was 16.7%, but 71.4% from the solid dispersion, whereas from the PBT formulation 85.6% was released. The Synperonic((R)) F127 content of PBT was only 0.05% of that in the solid dispersion formulation which suggests that it is the nature of the drug polymer contact rather than the amount of polymer which is more critical in influencing dissolution behaviour. Comparison of PBT and the 10% w/w solid dispersion of PBT in F127 showed similar amounts of drug in solution after 140 min. However there was a significantly higher release rate for PBT. Both formulation techniques offer significant improvements in drug release over untreated PB, and a combination of techniques changes the rate but not the extent of release in comparison with the surface modification technique alone.

The contribution of nurses' perceptions and actions in defining scope and stabilising professional boundaries of nursing practice.

This paper reports on the findings of a naturalistic inquiry study that explored the scope and boundaries of nursing practice. Findings from interview and observation data suggest that nurses negotiate and adjust professional boundaries on an individual, case-by-case basis, thereby managing the scope of their practice as they see it in that circumstance. The strategies they used are presented in four major categories: 1) maintaining a comfort zone, 2) expanding into safe territory, 3) moving into the grey zone and 4) stepping over the line. Findings show that nurses' efforts to maintain the comfort zone serve to perpetuate the status quo and may threaten holistic care. Expanding nursing actions to include functional roles such as coordinating care, sharing information, advocating (for patients), collaborating and innovating offers the profession critical building blocks for defining the scope of nursing practice. Clarifying the grey zone (or overlapping territory) is an essential task for the profession in determining the boundaries of nursing practice. The data revealed that, partly due to the ambiguity of the grey zone, nurses may step over the line into medical decision-making and outside the legal sanctions for the professional nursing role. The implications of this study highlight the need for nursing to define its scope of practice and in so doing stabilise professional boundaries.

Surface modification and electrostatic charge of polystyrene particles.

Particle surface modification by poloxamer adsorption can significantly alter the electrostatic charge, adhesion behaviour and consequently handling properties of a material. The charge reduction on polystyrene spheres achieved by this modification technique is dependent on the concentration, molecular weight and conformation of poloxamer at the particle surface. Adsorption isotherms of poloxamers on polystyrene particles follow a Langmuir profile and there is an apparent correlation between the extent of adsorption and ability of poloxamer to reduce electrostatic charge. Surface analysis techniques, X-ray photoelectron spectroscopy and Time of Flight Secondary Ion Mass Spectrometry have generated data on the thickness of the adsorbed poloxamer layer and provided evidence to suggest that the polypropylene oxide component of the poloxamer adsorbs to the polystyrene surface and there is a polyethylene oxide rich outer surface which may influence the charge alteration.

DNA variation in a 5-Mb region of the X chromosome and estimates of sex-specific/type-specific mutation rates.

We describe a new approach for the study of human genome variation, based on our solid-phase fluorescence chemical mismatch-cleavage method. Multiplex screening rates >/=80 kb/36-lane gels are achieved, and accuracy of mismatch location is within +/-2 bp. The density of differences between DNA from any two humans is sufficiently low, and the estimate of their position is accurate enough, to avoid sequencing of most polymorphic sites when defining their allelic state. Furthermore, highly variable sequences, such as microsatellites, are distinguished easily, so that separate consideration can be given to loci that do and do not fit the definition of infinite mutation sites. We examined a 5-Mb region of Xq22 to define the haplotypes of 23 men (9 Europeans, 9 Ashkenazim, and 5 Pygmies) by reference to DNA from one Italian man. Fifty-eight 1.5-kb segments revealed 102 segregating sites. Seven of these are shared by all three groups, two by Pygmies and Europeans, two by Pygmies and Ashkenazim, and 19 by Ashkenazim and Europeans. Europeans are the least polymorphic, and Pygmies are the most polymorphic. Conserved allelic associations were recognizable within 40-kb DNA segments, and so was recombination in the longer intervals separating such segments. The men showed only three segregating sites in a 16.5-kb unique region of the Y chromosome. Divergence between X- and Y-chromosome sequences of humans and chimpanzees indicated higher male mutation rates for different types of mutations. These rates for the X chromosomes were very similar to those estimated for the X-linked factor IX gene in the U.K. population.

Modification of electrostatic charge on inhaled carrier lactose particles by addition of fine particles.

Triboelectrification affects particle adhesion and agglomeration and hence the formulation, manufacture, and use of dry powder inhaler (DPI) devices. Electrostatic charge measurement of two component mixes of spray-dried or crystalline lactose fine particles (< 10 microns) 0, 5, 10, 15, 20, and 30% w/w with spray-dried or crystalline lactose 63-90 microns, respectively, has been undertaken using a system incorporating pneumatic transport of the mixed powders to a stainless steel cyclone charging device. The magnitude of charge on the mixes was shown to decrease with increased fine particle content, and there was no significant difference in charge for each concentration between spray-dried and crystalline lactose. Both the variation of charge and powder adhesion to the cyclone surface increased with increase in fine particle content. The proportion of fine particles in carrier systems in DPIs may thus have an important role where triboelectrification is involved.

Rheology and filling characteristics of particulate dispersions in polymer melt formulations for liquid fill hard gelatin capsules.

The rheology and capsule filling properties of molten excipients, Dynafill, Dynasan-114. Lutrol-F68, and polyethylene glycols (PEG) 6000, 8000, 10,000, and 20,000 have been investigated. Lactose (alpha-monohydrate) was selected as a model particulate solid with low solubility in PEG in order to investigate the effects of disperse phase particle size, concentration, and PEG molecular weight on rheology and capsule filling properties of these systems. All excipients behaved as Newtonian fluids between 65 and 90 degrees C, which was chosen as a possible temperature range for liquid filling of hard gelatin capsules. The excipients, apart from Dynasan-114 and PEG 20,000, showed satisfactory capsule filling properties at 70 degrees C using a semi-automatic filling machine. Dynasan-114 (viscosity = 0.012 Pa.s at 70 degrees C) leaked from the seals between the hopper and pump of the filling machine, whereas PEG 20,000 (viscosity = 24 Pa.s at 70 degrees C) showed bridging of the molten polymer between successive capsule bodies during the filling process. The effect of disperse phase (lactose) particle size and concentration, and continuous phase (PEG) molecular weight on the apparent viscosity and filling properties of the non-Newtonian dispersions were investigated at 70 degrees C. Satisfactory filling of the dispersions was achieved at 70 degrees C up to a limiting concentration of disperse phase which was dependent upon disperse phase particle size and continuous phase molecular weight, and corresponded to a pronounced increase in apparent viscosity of the dispersion.