Fire safety and emergency evacuation guidelines for intensive care units and operating theatres: for use in the event of fire, flood, power cut, oxygen supply failure, noxious gas, structural collapse or other critical incidents: Guidelines from the Association of Anaesthetists and the Intensive Care Society.

The need to evacuate an ICU or operating theatre complex during a fire or other emergency is a rare event but one potentially fraught with difficulty: Not only is there a risk that patients may come to harm but also that staff may be injured and unable to work. Designing newly-built or refurbished ICUs and operating theatre suites is an opportunity to incorporate mandatory fire safety features and improve the management and outcomes of such emergencies: These include well-marked manual fire call points and oxygen shut off valves (area valve service units); the ability to isolate individual zones; multiple clear exit routes; small bays or side rooms; preference for ground floor ICU location and interconnecting routes with operating theatres; separate clinical and non-clinical areas. ICUs and operating theatre suites should have a bespoke emergency evacuation plan and route map that is readily available. Staff should receive practical fire and evacuation training in their clinical area of work on induction and annually as part of mandatory training, including 'walk-through practice' or simulation training and location of manual fire call points and fire extinguishers, evacuation routes and location and operation of area valve service units. The staff member in charge of each shift should be able to select and operate fire extinguishers and lead an evacuation. Following an emergency evacuation, a network-wide response should be activated, including retrieval and transport of patients to other ICUs if needed. A full investigation should take place and ongoing support and follow-up of staff provided.

Genome-wide transposon mutagenesis identifies a role for host neuroendocrine stress hormones in regulating the expression of virulence genes in Salmonella.

Bacterial sensing of environmental signals plays a key role in regulating virulence and mediating bacterium-host interactions. The sensing of the neuroendocrine stress hormones epinephrine (adrenaline) and norepinephrine (noradrenaline) plays an important role in modulating bacterial virulence. We used MudJ transposon mutagenesis to globally screen for genes regulated by neuroendocrine stress hormones in Salmonella enterica serovar Typhimurium. We identified eight hormone-regulated genes, including yhaK, iroC, nrdF, accC, yedP, STM3081, and the virulence-related genes virK and mig14. The mammalian alpha-adrenergic receptor antagonist phentolamine reversed the hormone-mediated effects on yhaK, virK, and mig14 but did not affect the other genes. The beta-adrenergic receptor antagonist propranolol had no activity in these assays. The virK and mig14 genes are involved in antimicrobial peptide resistance, and phenotypic screens revealed that exposure to neuroendocrine hormones increased the sensitivity of S. Typhimurium to the antimicrobial peptide LL-37. A virK mutant and a virK mig14 double mutant also displayed increased sensitivity to LL-37. In contrast to enterohemorrhagic Escherichia coli (EHEC), we have found no role for the two-component systems QseBC and QseEF in the adrenergic regulation of any of the identified genes. Furthermore, hormone-regulated gene expression could not be blocked by the QseC inhibitor LED209, suggesting that sensing of hormones is mediated through alternative signaling pathways in S. Typhimurium. This study has identified a role for host-derived neuroendocrine stress hormones in downregulating S. Typhimurium virulence gene expression to the benefit of the host, thus providing further insights into the field of host-pathogen communication.

The efficiency of ozonated water from a water treatment plant to inactivate Cryptosporidium oocysts during two seasonal temperatures.

We investigated the efficiency of residual ozone from an advanced water treatment plant with an applied dose of 2.5 mg l(-1) to inactivate viable Cryptosporidium oocysts during summer (i.e. 24 degrees C) and winter (i.e. 18.9 degrees C) in Queensland, Australia. Containers for sample collection were inoculated with 1,000 oocysts l(-1) and filled with ozonated water. Ozone residual concentrations were measured at 0, 5 and 10 min intervals. Viability was determined by excystation. Non-ozonated water from the plant, trip and laboratory controls were also analysed. The applied ozone dose of 2.5 mg l(-1) produced an immediate residual concentration of 1.25 mg 1(-1) at 24 degrees C and 1.34 mg 1(-1) at 18.9 degrees C in unseeded samples. The initial ozone residual in seeded containers was 1.22+/-0.03 mg 1(-1) at 24 degrees C and 1.37+/-0.04 mg 1(-1) at 18.9 degrees C. There was a gradual increase in inactivation of oocysts, with 49% of oocysts inactivated at 0 min to 92% after 10 min at 24 degrees C and 57% at 0 min to 92.8% at 10 min at 18.9 degrees C.

Characterization of hns genes from Erwinia amylovora.

The small basic histone-like protein H-NS is known for bacteria to attenuate virulence of several animal pathogens. An hns homologue from E. amylovora was identified by complementing an E. coli hns-mutant strain with a cosmid library from E. amylovora. A 1.6 kb EcoRI-fragment complemented the mucoid phenotype and repressed the ss-glucosidase activity of E. coli PD32. The open reading frame encoding an H-NS-like protein of 134 amino acid was later shown to be located on plasmid pEA29 (McGhee and Jones 2000). A chromosomal hns gene was amplified with PCR consensus primers and localized near galU of E. amylovora. E. amylovora mutants were created by insertion of a resistance cassette, and the intact gene was inserted into a high copy number plasmid for constitutive expression. Purified chromosomal H-NS protein preferentially bound to a DNA fragment from the lsc region and bending was predicted for an adjacent fragment with the rlsB-promoter. Levan production was significantly increased by hns mutations. Synthesis of the capsular exopolysaccharide amylovoran and of levan were reduced, when hns from the E. amylovora plasmid was overexpressed. A mutation in chromosomal hns of E. amylovora increased amylovoran synthesis, and both mutations retarded symptom formation on immature pears.

The FtsH protease is involved in development, stress response and heat shock control in Caulobacter crescentus.

The ftsH gene of Caulobacter crescentus has been isolated and identified as a component of the general stress response of this organism. In C. crescentus, ftsH expression is transiently induced after temperature upshift and in stationary phase. Consistent with this, mutants deprived of the FtsH protease are viable at normal growth conditions, but are highly sensitive to elevated temperature, increased salt concentration or the presence of antibiotics. Overexpression of ftsH resulted in an increased salt but not thermotolerance, emphasizing the importance of the FtsH protease in stress response. Mutants lacking FtsH were unable to undergo morphological and physiological adaptation in stationary phase and, upon starvation, experienced a more pronounced loss of viability than cells containing FtsH. In addition, cells lacking FtsH had an increased cellular concentration of the heat shock sigma factor sigma32, indicating that, as in Escherichia coli, the FtsH protease is involved in the control of the C. crescentus heat shock response. In agreement with this, transcription of the heat-induced sigma32-dependent gene dnaK was derepressed at normal temperature when FtsH was absent. In contrast, the groEL gene, which is controlled in response to heat stress by both sigma32 and a HcrA/CIRCE mechanism, was not derepressed in an ftsH mutant. Finally, FtsH is involved in C. crescentus development and cell cycle control. ftsH mutants were unable to synthesize stalks efficiently and had a severe cell division phenotype. In the absence of FtsH, swarmer cells differentiated into stalked cells faster than when FtsH was present, even though the entire cell cycle was longer under these conditions. Thus, directly or indirectly, the FtsH protease is involved in the inherent biological clock mechanism, which controls the timing of cell differentiation in C. crescentus.

How and when are substrates selected for type III secretion?

Many Gram-negative bacteria use type III secretion systems to secrete virulence factors as well as the structural components of the flagellum. Some bacterial secretion systems use a secretion signal contained in the amino acid sequence of the secreted substrate. However, substrates of type III systems lack a single, defined secretion signal. There is evidence for the existence of three independent secretion signals - the 5' region of the mRNA, the amino terminus of the substrate and the ability of a secretion chaperone to bind the substrate before secretion - that direct substrates for secretion through the type III pathways. One or more of these signals might be used for a given substrate. A recent study of flagellar assembly presented evidence for a role of translation in the type III secretion mechanism. We present a unifying model for type III secretion that can be applied to flagellar assembly, needle assembly and the secretion of virulence factors. The potential role of translation in regulating the timing of substrate secretion is also discussed.

Cell cycle-dependent degradation of a flagellar motor component requires a novel-type response regulator.

The poles of each Caulobacter crescentus cell undergo morphological development as a function of the cell cycle. A single flagellum assembled at one pole during the asymmetric cell division is later ejected and replaced by a newly synthesized stalk when the motile swarmer progeny differentiates into a sessile stalked cell. The removal of the flagellum during the swarmer-to-stalked cell transition coincides with the degradation of the FliF flagellar anchor protein. We report here that the cell cycle-dependent turnover of FliF does not require the structural components of the flagellum itself, arguing that it is the initial event leading to the ejection of the flagellum. Analysis of a polar development mutant, pleD, revealed that the pleD gene was required for efficient removal of FliF and for ejection of the flagellar structure during the swarmer-to-stalked cell transition. The PleD requirement for FliF degradation was also not dependent on the presence of any part of the flagellar structure. In addition, only 25% of the cells were able to synthesize a stalk during cell differentiation when PleD was absent. The pleD gene codes for a member of the response regulator family with a novel C-terminal regulatory domain. Mutational analysis confirmed that a highly conserved motif in the PleD C-terminal domain is essential to promote both FliF degradation and stalk biogenesis during cell differentiation. Signalling through the C-terminal domain of PleD is thus required for C. crescentus polar development. A second gene, fliL, was shown to be required for efficient turnover of FliF, but not for stalk biogenesis. The possible roles of PleD and FliL in C. crescentus polar development are discussed.

Characterization of a gene locus from Erwinia amylovora with regulatory functions in exopolysaccharide synthesis of Erwinia spp.

In a genomic library of Erwinia amylovora, a locus has been identified that can suppress an Erwinia stewartii rcsA mutant. In addition, the locus induced a mucoid sticky phenotype of colonies in a wild-type strain of Erwinia stewartii and increased exopolysaccharide synthesis in several species of bacteria belonging to the genus Erwinia. An open reading frame was identified at this locus encoding a 225 amino acid protein that contained a helix-turn-helix motif typical of transcriptional regulators. The corresponding gene was subsequently named rcsV (regulator of capsular synthesis affecting viscosity). A mutant of rcsV in wild-type Erwinia amylovora had no detectable phenotype and produced typical levels of amylovoran under laboratory conditions. The rcsV gene on a high copy number plasmid under the control of its own promoter did not alter amylovoran production, in contrast to in-frame fusions of the structural gene in expression vectors. Since even the lac promoter was inert in the expression of rcsV, a DNA-binding protein could inhibit transcription of the gene in Erwinia amylovora. On the other hand, an Erwinia amylovora rcsA mutant was suppressed by rcsV when its promoter was replaced and the structural gene fused in-frame with lacZ' or malE. Northern blots, with total RNA from Erwinia amylovora, or promoter analysis using the GUS reporter gene did not show expression of rcsV in Erwinia amylovora, although primer extension analysis did. RcsV could be a component involved in the regulation of amylovoran synthesis, and gene expression may require an unknown external signal during the life cycle or pathogenesis of Erwinia amylovora.

Formant transition duration and speech recognition in normal and hearing-impaired listeners.

Listeners with sensorineural hearing loss often have difficulty discriminating stop consonants even when the speech signals are presented at high levels. One possible explanation for this deficit is that hearing-impaired listeners cannot use the information contained in the rapid formant transitions as well as normal-hearing listeners. If this is the case, then perhaps slowing the rate of frequency change in formant transitions might assist their ability to perceive these speech sounds. In the present study, sets of consonant plus vowel (CV) syllables were synthesized corresponding to /ba, da, ga/ with formant transitions for each set ranging from 5 to 160 ms in duration. The listener's task was to identify the consonant in a three-alternative, closed-set response task. The results for normal-hearing listeners showed nearly perfect performance for transitions of 20 ms and longer, whereas the shortest transitions yielded poorer performance. A group of eight hearing-impaired listeners pure-tone averages (PTAs) ranging from 30 to 62 dB HL) was also tested. The hearing-impaired listeners tended to show poorer performance than the normals for transitions of all durations; however, the performance of a few hearing-impaired subjects was equal to that of normals for the shortest-duration transitions. A strong inverse relation was observed between degree of hearing loss and improvement in score as a function of transition duration. These results suggest that increasing the duration of formant transitions for listeners with more severe hearing losses may not provide a helpful solution to their speech recognition difficulties.

Genetics of sorbitol metabolism in Erwinia amylovora and its influence on bacterial virulence.

A chromosomal DNA fragment from Erwinia amylovora was identified that complemented a deletion mutant in the gut(srl) operon of Escherichia coli. The E. amylovora srl operon on the cloned fragment was localized by transposon mutagenesis. A DNA fragment including the srl genes of E. amylovora was sequenced and found to contain six open reading frames (ORFs). These ORFs were highly homologous to genes of the gut operon of E. coli. No large gene was found that encoded a protein equivalent to GutA of E. coli; instead two ORFs with extensive similarity to GutA were identified in the E. amylovora srl operon. All transposon insertions were mapped by PCR analysis, and several insertions in a plasmid bearing the srl operon were unable to complement a mutation in the E. coli gutD gene. All E. amylovora srl mutants could be complemented by introducing the sorbitol operon from E. coli. The direction of transcription was confirmed by analysis of lacZ fusions. Expression of the srl operon in E. amylovora was high in the presence of sorbitol in the medium and was repressed by glucose. Mutants with a sorbitol deficiency were still virulent on slices of immature pears, but were unable to cause significant fire blight symptoms on apple shoots. Since sorbitol is used for carbohydrate transport in host plants of E. amylovora, this sugar alcohol may be an important factor in determining host specificity for the fire blight pathogen.

Automated system for the on-line monitoring of powder blending processes using near-infrared spectroscopy. Part I. System development and control.

An automated system for the on-line monitoring of powder blending processes is described. The system employs near-infrared (NIR) spectroscopy using fibre-optics and a graphical user interface (GUI) developed in the LabVIEW environment. The complete supervisory control and data analysis (SCADA) software controls blender and spectrophotometer operation and performs statistical spectral data analysis in real time. A data analysis routine using standard deviation is described to demonstrate an approach to the real-time determination of blend homogeneity.

On-line monitoring of powder blend homogeneity by near-infrared spectroscopy.

Near-infrared spectroscopy is evaluated as an on-line technique for monitoring the homogeneity of a pharmaceutical blend during the blending process. Blends containing 10% sodium benzoate (model active), which provided an aromatic functionality typical of many pharmaceutical compounds, 39% microcrystalline cellulose (Avicel PH102), 50% lactose, and 1% magnesium stearate were developed to mimic the properties of an actual pharmaceutical blend. A twin-shell V-blender was modified to allow installation of a diffuse reflectance fiber-optic probe at the axis of rotation, and spectra were collected during three experiments using a commercially available near-infrared spectrophotometer. In each experiment, blender control and spectral data collection were controlled by a compilation of software packages. The experiments detected spectral changes which eventually converged to a point of constant variance. Further analysis of the spectral data showed the blend is homogeneous long before a typical blending period is complete. Near-infrared spectroscopy has proven to be a feasible and effective method for the "real time" noninvasive determination of homogeneity in a pharmaceutical blend.

Appearance of discontinuities in spectra transformed by the piecewise direct instrument standardization procedure.

Several years ago, we noted that spectra transformed by the piecewise direct standardization (PDS) method may contain discontinuities. Having noticed that the problem was a recurring one, we studied it and recently diagnosed its source. Our investigations suggest that this problem also occurs in applications of window factor analysis, evolving factor analysis, and any other procedure that uses piecewise principal component models. In this work, we report the source of the problem and illustrate it with one example. A procedure is presented for eliminating the problem that is effective in PDS pattern recognition applications. Further work is needed to develop modified algorithms suitable for calibration applications.

A robotic dissolution system with on-line fiber-optic UV analysis.

An automated, robotic system has been developed with on-line UV fiber optics for real time dissolution analysis of solid dosage forms. The system is comprised of "off-the-shelf" hardware including a UV-Vis diode array spectrophotometer, fiber optic coupler, immersion probe, robot, and dissolution apparatus. The software system is modular with the functionalities of control, data acquisition, and spectral analysis separated into three Windows applications with communications performed via Dynamic Data Exchange (DDE). The fiber optic spectrophotometer collects a full spectrum over the range of 190-810 nm. Single wavelength UV analysis is performed on dosage forms in six dissolution vessels. The robotic system automates all facets of the analyses: measuring, degassing, and dispensing of the media; thermostating the media to physiologic temperature; dropping the dosage forms into the vessels; immersing the fiber optic probe at the appropriate time intervals; initiating the data acquisition, analyses, and reporting; and emptying and washing of the vessels prior to the next automated run. As a representative dosage form, 10 mg active tablets were selected and analyzed by this method. This fiber optic system has significantly improved the throughput of the robotic systems by eliminating the need for time consuming off-line HPLC or UV analyses. In addition, with the exception of system calibration, it is no longer necessary for laboratory personnel to come in contact with samples.

Observation of individual DNA molecules undergoing gel electrophoresis.

Individual DNA molecules undergoing agarose gel electrophoresis were viewed with the aid of a fluorescence microscope. Molecular shape and orientation were studied in both steady and pulsed electric fields. It was observed that (i) DNA macromolecules advanced lengthwise through the gel in an extended configuration, (ii) the molecules alternately contracted and lengthened as they moved, (iii) the molecules often became hooked around obstacles in a U-shape for extended periods, and (iv) the molecules displayed elasticity as they extended from both ends at once. A computer model has been developed that simulates the migration of the molecules in a rotating-field gel electrophoresis experiment.

Use of the indirect fluorescent antibody test in the detection of bovine respiratory syncytial virus antibodies in bovine serum.

The indirect fluorescent antibody test was adapted for identifying bovine respiratory syncytial virus and its specific antibody, using goat turbinate (GTU) cells. The virus caused maximal cytopathic effects in GTU cells 4 to 8 days postinfection, but fluorescence was not readily detected during this period. Fluorescence was maximal in infected GTU cells at 24 to 36 hours postinfection, but could be detected 48 hours postinfection. Bovine serums (331) which had been submitted to the Oklahoma Animal Disease Diagnostic Laboratory were tested for antibodies to this virus, and 73.6% were found to be positive.

Frequency of occurrence of viruses associated with respiratory tract disease of cattle in Oklahoma: serologic survey for bovine herpesvirus DN599.

Serum samples from 351 Oklahoma cattle were tested for antibodies to DN599 virus by the indirect fluorescent antibody test. Seven cattle (approximately 2%) were seropositive for this virus.