Standardization of Pneumococcal Biofilm Release to PncO Expression, a Predictive Measurement of Virulence.

A critical component in clinical trials for vaccines against pneumococcal disease is the establishment of robust preclinical models and clinical correlates of protection, which, in the case of the causative bacterial agent Streptococcus pneumoniae, include standard sepsis/pneumonia mouse models and opsonophagocytic activity (OPA), respectively. Despite broad usage, these gold-standard measures are ill equipped to evaluate nontraditional antigens that target virulence factors beyond capsular polysaccharides and/or proteins not associated with colonization or routine growth. These assays are further complicated by observed inconsistencies in the expression of target protein antigens and in the quantity of usable bacteria provided from respective growth processes. In an effort to overcome these issues, we performed an extensive optimization study of the critical steps in a bacterial biofilm dispersion model (termed "the biofilm model") to identify conditions that yield the greatest quantity of released pneumococci displaying a consistent virulence phenotype. Using this knowledge, we developed a secondary immune absorbance assay to provide immediate insight into the phenotypic state of bacteria conditioned using the biofilm model. Specifically, positive correlations between the expression of PncO (a key virulence-associated protein antigen) and immune absorbance (R2 = 0.96), capsule shedding, and OPA assay titers were translated into a predictive readout of virulence in sepsis and pneumonia challenge models. These results present a methodology for generating consistent lots of virulent bacteria to standardize inputs in preclinical and clinical models for testing vaccines against biofilm-associated bacteria.

Patient experience and overall satisfaction after emergency abdominal surgery.

BACKGROUND: There is a growing recognition of the importance of patient experience in healthcare, however little is known in the context of emergency abdominal surgery. This study sought to quantify the association between patient experience and overall satisfaction. METHODS: Patient demographics, operation details and 30-day clinical outcome data of consecutive patients undergoing emergency abdominal surgery were collected. Data was collected using validated Patient Reported Experience Measures (PREMs) questionnaires. Categorical data were tested using Mann Whitney U test. Multivariable regression was used to determine independent factors associated with satisfaction. RESULTS: In a well-fitting multivariable analysis (R2 = 0.71), variables significantly associated with a higher global satisfaction score were "sufficient information given about treatment" (ß = 0.86, 95% CI 0.01-1.70, p = 0.047), "sufficient explanation of risks and benefits of surgery" (ß = 1.26, 95% CI 0.18-2.34, p = 0.020), "absence of night-time noise" (ß = 1.35, 95% CI 0.56-2.14, p = 0.001) and "confidence and trust in nurses" (ß = 1.51, 95% CI 0.54-2.49, p = 0.003). CONCLUSIONS: Overall patient satisfaction was strongly associated with perceptions of good communication and transfer of information. Confidence and trust in the clinical team is an important determinant of patient experience. Improving the ward environment by reducing noise at night may also improve the overall experience and satisfaction in emergency surgery.

Variations in strength and slip rate along the san andreas fault system.

Convergence across the San Andreas fault (SAF) system is partitioned between strike-slip motion on the vertical SAF and oblique-slip motion on parallel dip-slip faults, as illustrated by the recent magnitude M(s) = 6.0 Palm Springs, M(s) = 6.7 Coalinga, and M(s) = 7.1 Loma Prieta earthquakes. If the partitioning of slip minimizes the work done against friction, the direction of slip during these recent earthquakes depends primarily on fault dip and indicates that the normal stress coefficient and frictional coefficient (micro) vary among the faults. Additionally, accounting for the active dip-slip faults reduces estimates of fault slip rates along the vertical trace of the SAF by about 50 percent in the Loma Prieta and 100 percent in the North Palm Springs segments.

Prevention of mucosal Escherichia coli infection by FimH-adhesin-based systemic vaccination.

Virtually all uropathogenic strains of Escherichia coli, the primary cause of cystitis, assemble adhesive surface organelles called type 1 pili that contain the FimH adhesin. Sera from animals vaccinated with candidate FimH vaccines inhibited uropathogenic E. coli from binding to human bladder cells in vitro. Immunization with FimH reduced in vivo colonization of the bladder mucosa by more than 99 percent in a murine cystitis model, and immunoglobulin G to FimH was detected in urinary samples from protected mice. Furthermore, passive systemic administration of immune sera to FimH also resulted in reduced bladder colonization by uropathogenic E. coli. This approach may represent a means of preventing recurrent and acute infections of the urogenital mucosa.

Thyrotropin receptor-adenylate cyclase function in human thyroid neoplasms.

The action of thyrotropin (TSH) on plasma membranes was studied to elucidate the mechanism of hormonal regulation of malignant versus normal human thyroid tissue. Thyroid plasma membranes of six specimens of papillary or follicular carcinoma and six of adenoma, as well as adjacent normal tissue obtained from these patients, were evaluated with respect to binding of 125I-labeled TSH and stimulation of adenylate cyclase. Scatchard analysis of TSH binding revealed the presence of two species of binding sites in normal thyroid of different affinities and capacities. In 11 of 12 tumors studied, the high-affinity binding site remained intact; however, the total number of low-affinity sites was markedly lower than normal tissue. Other parameters of binding were not altered in neoplastic thyroid. In each of these tissues, the hormone responsiveness and kinetics of adenylate cyclase activation were essentially identical to those observed in normal tissue, although basal activity was typically greater in the neoplasm. One carcinoma was totally deficient in both 125I-labeled TSH binding and TSH-stimulatable adenylate cyclase, although basal activity was detected. Furthermore, adenylate cyclase of this specimen was not activated by prostaglandin, in contrast to normal thyroid and other thyroid tumors. These results suggest that: (a) clinical behavior of thyroid carcinomas may not be reflected by TSH receptor-adenylate cyclase function; (b) lack of clinical response as manifest by tumor regression cannot be ascribed to the absence of functional TSH receptors or adenylate cyclase; and (c) decreased low-affinity binding present in tumors is not correlated with altered hormone responsiveness of adenylate cyclase but may reflect more general cancer-induced changes in membrane structure or composition.

PapD and superfamily of periplasmic immunoglobulin-like pilus chaperones.

The formation of a P pilus requires a molecular chaperone in the periplasm and a molecular usher in the outer membrane. Each pilus is composed of six different types of proteins that are assembled into a composite fiber in a defined order. The correct folding of subunits into domains that can serve as assembly modules requires an association with the periplasmic chaperone. PapD is the prototype member of the family of bacterial pilus chaperones that have a three-dimensional structure consistent with an immunoglobulin fold. In general, proteins with an immunoglobulin fold structure have molecular recognition functions in eukaryotic cells that are often integrated with effector functions. PapD has also a recognition function, binding nascently translocated pilus subunits and maintaining them in assembly-competent conformations. The association of the chaperone with the subunit triggers the targeting of the latter to an outer membrane usher. The usher serves as a molecular gatekeeper, allowing the ordered incorporation of the pilus subunits into the pilus structure from the periplasmic chaperone complexes. The two immunoglobulin-like domains of PapD are oriented to form a cleft that contains the subunit binding site. This is a different binding paradigm from that used by either antibodies or the growth hormone receptor. The blend of genetics, biochemistry, X-ray crystallography, and carbohydrate chemistry in the study of pili biogenesis will continue to give insight into some of the most basic intellectual challenges in molecular biology concerning how proteins fold into domains that serve as modules for the formation of larger assemblies, and relating these processes to microbial pathogenesis.

Fatal haemorrhage following a low-energy fracture of the pubic ramus.

We report a case of fatal haemorrhage following a low-energy fracture of the pubic ramus in an 85-year-old woman.

Regulation of thyroid adenylate cyclase: guanyl nucleotide modulation of thyrotropin receptor-adenylate cyclase function.

The role of guanyl nucleotides in regulating the hormonal responsiveness of adenylate cyclase was studied in thyroid plasma membranes. Guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] alone stimulated the enzyme. At a low ATP concentration (0.2 mM), TSH alone had little or no effect, but when added with Gpp(NH)p, it resulted in a dose-dependent increase of enzyme activity. Kinetic studies revealed that Gpp(NH)p alone stimulated adenylate cyclase activity only after a 10-min lag. TSH abolished the lag, resulting in an apparent increase in activity and a lowering of the activation constant for Gpp(NH)p. GTP caused an initial increase in activity at 2 min, followed by a gradual decline below basal levels. This inhibition was not prevented by TSH. Further examination revealed that GDP caused inhibition of Gpp(NH)p-stimulated activity in a competitive manner, suggesting that conversion of GTP to GDP may be responsible for the time-dependent decay seen with GTP. To study the effects of guanyl nucleotides on coupling of the TSH receptor to adenylate cyclase, plasma membranes were preactivated with saturating amounts of Gpp(NH)p and washed extensively to remove unbound Gpp(NH)p. Incubation of preactivated membranes with either Gpp(NH)p or TSH gave no further stimulation of adenylate cyclase. However, Gpp(NH)p plus TSH produced 30% more stimulation. In contrast, the addition of TSH plus GDP to preactivated membranes led to a dose-dependent decrease in activity. Furthermore, promotion of further stimulation by TSH plus Gpp(NH)p was competitively inhibited by GDP, in the same manner as untreated membranes. This suggested that the dual action of TSH, i.e, stimulation and inhibition, is governed and mediated by specific guanyl nucleotides. Analysis of guanyl nucleotides effects on the binding of [125I[iodo-TSH to membranes revealed that although some inhibition of binding exists, this effect is 1) unrelated to the concentration effect of nucleotides on adenylate cyclase, and 2) not specific for guanosine phosphates. Scatchard analysis of TSH binding in the presence of guanyl nucleotides demonstrated that even at high concentrations, GTP had no effect on the high affinity, low capacity receptor for TSH. These results suggest that GDP or Gpp(NH)p plays no role in modulating TSH-receptor interaction. Thus, guanyl nucleotide regulation of TSH action appears to be limited to adenylate cyclase.

Genetic suppression analysis of sigma E interaction with three promoters in sporulating Bacillus subtilis.

Genetic evidence suggests that the sigma (sigma) subunit of RNA polymerase determines the specificity of promoter utilization, by making sequence-specific contacts with DNA. We examined the effects of two single amino acid(aa) substitutions in sigma E on the utilization of mutated derivatives of three different promoters in sporulating Bacillus subtilis. We found allele-specific suppression of mutations in all three promoters by each aa substitution in sigma E. These results provide strong evidence that sigma E interacts with each of these promoters in vivo. Moreover, the specificity of suppression of the mutations by the aa substitutions in sigma E lead us to speculate that the Met124 of sigma E closely contacts two adjacent bp in the -10 region of the promoters.

Ondansetron therapy for uremic pruritus in hemodialysis patients.

Pruritus is a distressing symptom affecting up to 90% of dialysis patients. Conventional treatment with antihistamines is often ineffective and may have unacceptable side effects. Serotonin (5-hydroxytryptamine type 3 [5-HT(3)]) is known to enhance pain perception and pruritic symptoms through receptors on sensory nerve endings. Antagonism of 5-HT(3) receptors may be of use in treating uremic pruritus. We randomly assigned 16 hemodialysis patients with persistent pruritus to treatment with the 5-HT(3)-receptor antagonist, ondansetron (8 mg), or placebo three times daily for 2 weeks each in a prospective, placebo-controlled, double-blind crossover study. Patients scored their intensity of pruritus daily on a 0-to-10 visual analogue scale (0 = no pruritus, 10 = maximal pruritus), and daily use of antihistamines as escape medication was recorded. The median daily pruritus score did not change significantly during active or placebo treatment (preondansetron, 5. 3; interquartile range [IQR], 3.4 to 6.3; during ondansetron, 3.9; IQR, 2.7 to 5.0; P = not significant; preplacebo, 3.7; IQR, 3.0 to 4. 6; during placebo, 3.6; IQR, 2.4 to 4.8; P = not significant). The median daily percentage of escape medication use decreased from 21% (IQR, 9 to 61) to 9% (IQR, 0 to 33) with ondansetron (P = not significant) and from 53% (IQR, 0 to 88) to 5% (IQR, 0 to 31) with placebo (P = not significant). There was no difference in predialysis biochemistry test results or dialysis efficacy during treatment phases. Ondansetron does not improve pruritus in hemodialysis patients. Use of antihistamines decreased with both ondansetron and placebo.

Dissociation kinetics of the thyrotropin-receptor complex. Characterization of a slowly dissociable component.

The kinetics of the dissociation of thyrotropin (TSH) from human thyroid plasma membranes were studied in an attempt to further understand the molecular dynamics of the TSH--receptor interaction. Dissociation of bound [125I]TSH from thyroid plasma membranes was a biphasic process consisting of rapidly and slowly dissociable components, RDC and SDC, respectively. The dilution induced dissociation of bound [125I]TSH was enhanced by the addition of excess TSH (DEC). DEC was proportional to the dose of unlabeled TSH and its magnitude increased linearly with temperature. These results are in contrast to those reported for the kinetics of [125I]insulin dissociation. The functional significance of DEC remains largely unexplained. It was found that the fraction of SDC was dependent upon time of association in a temperature-dependent and apparently saturable process. It could not be attributed to alterations in the electrophoretic, immunologic or binding properties of [125I]TSH. Furthermore, no correlation was observed between generation of SDC and change in the Scatchard profile of TSH binding, in contrast to studies on growth hormone. These data suggest that, like some other polypeptide hormones, binding of TSH to its receptor does not proceed according to laws describing simple, rapidly reversible, bimolecular reactions. Furthermore, bound TSH undergoes a receptor-mediated conversion from a rapidly to a slowly dissociable state with time of incubation.

Resolution of radioiodinated thyrotropin into receptor active and inactive components by column chromatography.

Following radioiodination by the lactoperoxidase method and subsequent purification on Sephadex G100, it was found that [125I]TSH exhibited varying degrees of binding activities to the thyrotropin receptor. In order to further purify the radiolabeled hormone, the [125I]TSH preparation was chromatographed on Sepharose 6B. Two peaks of radioactive material (Peaks I and II) were recovered, containing approx. 60% of the applied radioactivity. Upon elution with Mg2+, the remainder of the radiolabeled material was recovered as a single peak (Peak III). Characterization of these 3 peaks by radioimmunoassay demonstrated that all 3 were immunocompetent, although Peaks I and III were 3-4-fold more immunoreactive than Peak II. Analysis by radioreceptor assay indicated that Peak III showed an increase in receptor-binding capacity (in comparison with the [125I]TSH preparation purified by Sephadex G100 alone), while both Peaks I and II exhibited significantly reduced binding activity. In contrast, human TSH (NIH) chromatographed mainly as a receptor inactive peak, although it was fully immunocompetent. Scatchard analysis of receptor binding to bovine [125I]TSH from Peak III yielded a curvilinear plot with affinities similar to those we have previously reported for [125I]TSH purified by Sephadex G100 chromatography. The total number of binding sites, however, increased proportionally with the active fraction of the [125I]TSH preparation. Since the mass of bound hormone is calculated from the percent bound of total radioactivity and only a fraction of the measured total participates in the binding, it is therefore necessary to correct for the inactive fraction when calculating the total receptor number.

Linguistic analogies and behavior: the finite-state behavioral grammar of food-hoarding in hamsters.

Rodger and Rosebrugh (1979) developed a method for identifying sets of rules by which discrete responses are combined to form functional sequences of behavior. In the present study, this so-called finite-state behavioral grammar method was used to analyze the food-hoarding behavior of Syrian golden hamsters (Mesocricetus auratus). Specifically, it was used to analyze 127 videotaped food-hoarding sequences that were observed in 14 male, adult hamsters in an apparatus that comprised a home compartment, a foraging area, and a connecting tunnel. An integral part of grammar derivation is the development of hypotheses about the function of the observed response patterns to guide the grammar derivation. Two such hypotheses guided the present analysis: first, we hypothesized that behaviors observed in the foraging area after the hamsters' cheek pouches were filled served an exploratory function; second, we hypothesized that behaviors observed in the home compartment prior to the hamsters emptying their cheeck pouches served to assess potential food deposition sites. In addition to generating these two hypotheses and the grammars for the hoarding sequences, this study demonstrates how the finite-state grammar method can be used as a productive means of gaining insights into the organization of consummatory response sequences.

Quantitative measurement of small-angle gamma ray scattering from water, nylon, and Lucite.

The small-angle (2 degrees-10 degrees) total-differential-scattering cross sections of water, methyl methacrylate, and nylon 6 have been measured at a photon energy of 59.54 keV. An annular target geometry has been used with an acceptance of +/- 0.5 degrees and an experimental uncertainty of approximately 4% achieved. The experimental methods and data corrections necessary to obtain this accuracy are discussed in detail. The results demonstrate clearly the contribution of intermolecular effects to the scattering process and allow critical comparison with calculations using molecular form factors for water obtained from interpolated x-ray diffractometer data; agreement is obtained within the experimental uncertainties. They also support earlier Monte Carlo predictions of scattering in water at angles of the order of 2 degrees which are in disagreement with certain published experimental data.

HDR microSelectron quality-assurance studies using a well-type ion chamber.

A modified Standard Imaging HDR 1000 re-entrant ion chamber has been used with an NE 2570/1 electrometer as part of a quality-assurance programme for checking 192Ir source strengths and for source localization studies. The chamber measurements correlate well with in-air calibration measurements made over a three-month source-decay period; the maximum discrepancy between the in-air measurement and the re-entrant chamber measurement was 0.9%. The source position may be determined with an uncertainty of lower than 0.5 mm. The reproducibility of measurements made with each of four electrometers used with the reentrant ion chamber was typically 0.1% (1 sigma).

Theoretical aspects and practical applications of moiré topography.

When a surface is observed at a distance through a parallel line grating which is illuminated by a source of light laterally displaced to the viewing axis, the grating is superimposed on its shadow and this gives rise to a moiré fringe pattern. The fringes represent cross-sections of the surface parallel to the grating plane. The principle of the formation of the contour map has been studied assuming a grating represented by a Fourier series, a source of light represented by a 3D intensity distribution and a viewing aperture of finite dimensions. The experimental system used for illumination and observation of the moiré fringes consists of an equispaced parallel-lined glass grating and two small light sources positioned symmetrically about the imaging device. The arrangement constitutes a 3D imaging system with an accuracy of the order of 1 mm. The technique has been applied in radiotherapy problems such as the design of contour compensators and the measurement of body contours. Moiré patterns have been analysed with the aid of a PDP-8 computer, and have been used to calculate surface areas and volumes such as in breast studies and superficial tumour measurements.

Evaluation of a moiré imaging system.

Moiré fringe techniques can be used to measure surface topography. The factors that affect the formation of the fringes have been assessed theoretically by developing models of gratings and light sources of finite dimensions. In this way the effect of parameters such as the intensity distribution of the light source, the viewing aperture function, the lateral displacement of the source relative to the viewing axis, the viewing distance, the grating spacing and grating ratio have been evaluated. Other factors, related to the physical properties and geometry of the reflecting surface, are also investigated. Suggestions are made for the design of an optimum moiré imaging system.

Measurement of small-angle photon scattering for some breast tissues and tissue substitute materials.

For photon energies encountered in diagnostic radiology the shape of the scattering distributions for low-atomic-number media exhibits peaks in intensity close to the forward direction that are not predicted by conventional theoretical models. The positions and shapes of the peaks depend upon the interatomic and intermolecular configurations of the scatterers. The phenomenon is of particular interest because of its relevance to the understanding and modelling of x-ray imaging processes and the possibility that the peaking may be characteristic of tissue type. In the present study, peaks in the forward scattering distributions have been demonstrated for 19 samples of breast tissue and three tissue substitute materials using a position-sensitive photon detector and a 60 kVp x-ray source. Prominent features were observed for all samples investigated. Large differences were found in the shapes of the distributions between adipose and fibroglandular tissues and only small differences were found between carcinomas and fibroglandular tissues.

Litter accumulation in woodlands contaminated by Pb, Zn, Cd and Cu.

Close to a primary lead-zinc-cadmium smelter the standing crop of litter in woodlands was found to be elevated relative to more distant sites. The total litter accumulation is similar to that from contaminated sites reported by other authors but in this case the concentrations of heavy metals are considerably lower than those reported for other sites. Evidence is provided to support the hypothesis that within the woodlands studied, litter accumulation is not closely pH dependent, but is clearly related to both cadmium and zinc concentrations in litter. Litter accumulation occurs in certain particle size ranges and fractionation shows that the weight of accumulated litter in these size ranges is highly correlated to cadmium concentrations. These results are discussed in relation to the reported possible long term effects of metal contamination on decomposition processes and the possibility of adaptation to these adverse effects.

Contingent tolerance to the anticonvulsant effects of carbamazepine, diazepam, and sodium valproate in kindled rats.

The effect of convulsive stimulation during periods of drug exposure on the development of tolerance to the anticonvulsant effects of carbamazepine (CBZ), diazepam (DZP), or sodium valproate (VPA) was studied in three similar experiments. In each experiment, amygdala-kindled rats were assigned to one of three groups: one group received a drug injection (CBZ, 70 mg/kg, IP; DZP, 2 mg/kg, IP; VPA, 250 mg/kg, gavage) 1 h before each of a series of 10 bidaily (one every 48 h) convulsive stimulations, a second group received the same dose of the drug 1 h after each of the 10 stimulations, and a third group served as a vehicle control. The drug tolerance test occurred in each experiment 48 h after the 10th tolerance-development trial; every rat received the appropriate dose of CBZ, DZP, or VPA 1 h before being stimulated. In each experiment, only the rats from the drug-before-stimulation group displayed a significant amount of tolerance to the drug's anticonvulsant effect. Thus the development of tolerance to the anticonvulsant effects of CBZ, DZP, and VPA was not an inevitable consequence of drug exposure; the development of tolerance was contingent upon the occurrence of convulsive stimulation during the periods of drug exposure. These results support the idea that functional drug tolerance is an adaptation to a drug's effects on ongoing patterns of neural activity, rather than to drug exposure per se.