Core Outcome Set for IgE-mediated food allergy clinical trials and observational studies of interventions: International Delphi consensus study 'COMFA'.

BACKGROUND: IgE-mediated food allergy (FA) is a global health concern with substantial individual and societal implications. While diverse intervention strategies have been researched, inconsistencies in reported outcomes limit evaluations of FA treatments. To streamline evaluations and promote consistent reporting, the Core Outcome Measures for Food Allergy (COMFA) initiative aimed to establish a Core Outcome Set (COS) for FA clinical trials and observational studies of interventions. METHODS: The project involved a review of published clinical trials, trial protocols and qualitative literature. Outcomes found as a result of review were categorized and classified, informing a two-round online-modified Delphi process followed by hybrid consensus meeting to finalize the COS. RESULTS: The literature review, taxonomy mapping and iterative discussions with diverse COMFA group yielded an initial list of 39 outcomes. The iterative online and in-person meetings reduced the list to 13 outcomes for voting in the formal Delphi process. One more outcome was added based on participant suggestions after the first Delphi round. A total of 778 participants from 52 countries participated, with 442 participating in both Delphi rounds. No outcome met a priori criteria for inclusion, and one was excluded as a result of the Delphi. Thirteen outcomes were brought to the hybrid consensus meeting as a result of Delphi and two outcomes, 'allergic symptoms' and 'quality of life' achieved consensus for inclusion as 'core' outcomes. CONCLUSION: In addition to the mandatory reporting of adverse events for FA clinical trials or observational studies of interventions, allergic symptoms and quality of life should be measured as core outcomes. Future work by COMFA will define how best to measure these core outcomes.

[Multidisciplinary meetings in pelvic floor disease in women: A national survey by the CUROPF among French urologists]. / Réunion de concertation pluridisciplinaire en pelvipérinéologie en France : résultats d'une enquête nationale du CUROPF auprès des urologues français.

INTRODUCTION: Multidisciplinary team meetings (MTMs) in the field of pelvic floor diseases in women tend to generalize, as they are required as mandatory before mid-urethral sling implantation or sacrocolpopexy by recent decrees published by the French health authorities. However, access to these meetings is variable in the French territory. The goal of the present study was to describe the existence and the settings of these kinds of meetings in France. MATERIEL AND METHODS: An on-line survey was conducted between June and July 2020 (stage 1) then between November 2021 and January 2022 (stage 2). A 15-item questionnaire was sent to all members of the Association française d'urologie (AFU). A descriptive analysis was conducted. RESULTS: Three hundred and twenty-two completed questionnaires were sent back during stage 1 and 158 during stage 2. Early 2022, 61.3% of respondents had access to a pelviperineology MTM, with important difference according to geographical areas. Main activity of MTMs was case discussion of complex situations (68% of meetings). At the end of 2021, 22% of the respondents declared willing to stop partially or totally their pelviperineology activity, given the new regulations set in place by the authorities. CONCLUSION: Despite being absolutely mandatory in current clinical practice, MTMs in pelvic floor disease have spread slowly. MTMs implementation was still insufficient in 2022, and variable on the French territory. Some urologists declare having no access to such resources and about 1 out of 5 were considering to voluntary stop of decrease significantly their activity in this difficult context.

Magnetic susceptibility to measure total protein concentration from NMR metabolite spectra: Demonstration on blood plasma.

PURPOSE: Accurate metabolite and protein quantification in blood plasma and other body fluids from one single NMR measurement, allowing for improved quantitative metabolic profiling and better assessment of metabolite-protein interactions. THEORY AND METHODS: The total protein concentration is derived from the common chemical-shift changes-caused by protein-induced bulk magnetic susceptibility (BMS)-measured on well-accessible and exchange-free metabolite resonances. These BMS shifts are simply obtained by external referencing with respect to 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt in a coaxial insert. RESULTS: Based on blood-plasma data from five volunteers, the estimated accuracy of the BMS method is ≤ 5% with respect and comparable to the 3.8% error of the standard colorimetric, Biuret, method. Valine, alanine, glucose, leucine, and lactate display no exchange-induced shift changes. Their well-accessible signals act as reliable probes for pure protein-induced BMS. The slopes and intercepts of their chemical-shift change versus protein concentration were derived from metabolite mixtures with (fatted) human and bovine albumin acting as blood-plasma mimics. CONCLUSION: The BMS method, demonstrated on blood plasma, can also be used on other samples containing sufficient protein (> 10 g/L). Also, it allows measurement of the presence and sign of exchange-induced chemical-shift changes.

Prolonged urinary leakage after partial nephrectomy: a novel management pathway.

INTRODUCTION: To describe an original method for managing prolonged urinary leakage after partial nephrectomy. We placed a Malecot catheter in the ureter to improve the urinary drainage and therefore avoid the renal percutaneous treatment of the fistula or potential open surgery. TECHNICAL CONSIDERATIONS: We performed ureteral stenting using a 16 F Malecot catheter in 3 patients who had a prolonged urinary fistula after partial nephrectomy in which the placement of a ureteral stent could not resolve the urine leak. Drainage using 2 ureteral catheters was performed, which proved to be insufficient for the urinary fistula to resolve. We subsequently placed in the dilated ureter a 16 F Malecot catheter into the renal pelvis using a cystoscopic approach and a bladder catheter to complete the drainage. In all cases, the urine leak stopped after stenting with the Malecot catheter. At 1 month after the stenting, computed tomography and magnetic resonance imaging showed complete healing of the fistula. No infection or secondary ureteral stricture was reported. CONCLUSION: This technique with a low complication profile can be used as an additional endoscopic step, before more invasive procedures.

Does the use of a barbed polyglyconate absorbable suture have an impact on urethral anastomosis time, urethral stenosis rates, and cost effectiveness during robot-assisted radical prostatectomy?

OBJECTIVE: To evaluate the use of a single needle driver with the V-Loc (Covidien, Dublin, Ireland) running suture and compare this with the use of 2 needle drivers with polyglactin interrupted sutures (IS) in dividing the dorsal venous complex (DVC) and forming the urethrovesical anastomosis (UVA) during robot-assisted radical prostatectomy (RARP). MATERIALS AND METHODS: A prospective cohort study was performed to compare V-Loc (n = 40) with polyglactin (n = 40) sutures. Division of the dorsal venous complex and formation of the UVA during robot-assisted radical prostatectomy using V-Loc or polyglactin sutures were studied. Preoperative, intraoperative, and postoperative parameters were measured. RESULTS: V-Loc sutures were associated with a statistically significant reduction in mean dorsal vein suture time (3.15 minutes V-Loc vs 3.75 minutes IS, P = .02) and UVA anastomosis time (8.5 minutes V-Loc vs 11.5 minutes IS, P = .001). No significant difference was noted between operative time (121 minutes V-Loc vs 130 minutes IS, P = .199), delayed healing rates (5% V-Loc vs 7.5% IS, P = .238), continence rate at 12 months (97.5% V-Loc vs 95% IS, P = .368), and urethral stenosis rates (2.5% V-Loc vs 2.5% IS, P = .347) in both groups. CONCLUSION: The use of a V-Loc running suture with a single needle driver is a feasible, reproducible, and economic technique with no significant difference in continence rates and urethral stenosis rates, compared with the use of a traditional interrupted suture.

Feasibility and oncological outcomes of laparoscopic treatment for local relapse of renal cell carcinoma.

UNLABELLED: What's known on the subject? and what does the study add?: Local relapse of renal cell carcinoma following radical nephrectomy is rare, and surgical removal provides the only opportunity for cure. Open surgery has been established as the usual approach for these tumours. It is, however, associated with significant morbidity. Our study describes the largest series of laparoscopic treatment of local relapse of renal cell carcinoma with the longest follow-up. We show that the laparoscopic approach is feasible in expert centres. It provides faster recovery and fewer complications with satisfactory oncological outcomes in selected patients. OBJECTIVE: To assess the feasibility and oncological outcomes of laparoscopic treatment for local relapse of renal cell carcinoma. PATIENTS AND METHODS: Nine patients were treated by a pure laparoscopic approach for local recurrence of a renal tumour between 2005 and 2011 by a single surgeon (HB), following an initial open radical nephrectomy for the primary tumour. Clinical and histopathological data were collected prospectively and analysed retrospectively. Seven patients were treated by a transperitoneal approach and two patients had a retroperitoneal approach. RESULTS: Relapse occurred within a mean time of 83 months (7-168) following nephrectomy. Recurrent tumour size varied from 2.5 to 4.5 cm. All surgeries were performed laparoscopically without need for conversion. Mean operative duration was 144 min (40-240), mean estimated blood loss was 430 mL (50-1300) and mean hospital stay was 4.5 days (3-6). Three patients had Clavien grade I intraoperative complications. Late complications were noted in two patients (Clavien I and IIIb). Pathology confirmed clear cell carcinoma in all patients with an absence of sarcomatoid features and negative surgical margins. Three patients had neoadjuvant treatment and two patients had adjuvant treatment. In all, 67% of patients were disease free with a mean follow-up period of 3 years. CONCLUSIONS: Surgical removal of isolated local recurrence remains the only possibility of cure in patients with renal cell carcinoma. We demonstrated that the laparoscopic approach is a safe and feasible alternative treatment option for selected cases with low morbidity and satisfactory oncological outcomes.

NMR identification of endogenous metabolites interacting with fatted and non-fatted human serum albumin in blood plasma: Fatty acids influence the HSA-metabolite interaction.

Metabolites and their concentrations are direct reporters on body biochemistry. Thanks to technical developments metabolic profiling of body fluids, such as blood plasma, by for instance NMR has in the past decade become increasingly accurate enabling successful clinical diagnostics. Human Serum Albumin (HSA) is the main plasma protein (∼60% of all plasma protein) and responsible for the transport of endogenous (e.g. fatty acids) and exogenous metabolites, which it achieves thanks to its multiple binding sites and its flexibility. HSA has been extensively studied with regard to its binding of drugs (exogenous metabolites), but only to a lesser extent with regard to its binding of endogenous (non-fatty acid) metabolites. To obtain correct NMR measured metabolic profiles of blood plasma and/or potentially extract information on HSA and fatty acids content, it is necessary to characterize these endogenous metabolite/plasma protein interactions. Here, we investigate these metabolite-HSA interactions in blood plasma and blood plasma mimics. The latter contain the roughly twenty metabolites routinely detected by NMR (also most abundant) in normal relative concentrations with fatted or non-fatted HSA added or not. First, we find that chemical shift changes are small and seen only for a few of the metabolites. In contrast, a significant number of the metabolites display reduced resonance integrals and reduced free concentrations in the presence of HSA or fatted HSA. For slow-exchange (or strong) interactions, NMR resonance integrals report the free metabolite concentration, while for fast exchange (weak binding) the chemical shift reports on the binding. Hence, these metabolites bind strongly to HSA and/or fatted HSA, but to a limited degree because for most metabolites their concentration is smaller than the HSA concentration. Most interestingly, fatty acids decrease the metabolite-HSA binding quite significantly for most of the interacting metabolites. We further find that competition between the metabolites for binding is absent for most of these metabolites. These mappings in plasma mimics may thus open new opportunities for improved metabolic profiling of blood plasma. For instance, correct metabolite concentrations can be determined for the non-interacting metabolites and/or concentration corrections made for interacting metabolites. Secondly, the interacting metabolites could be used to act as reporters on HSA and fatty acid concentration in plasma, and thus potentially act as biomarker in diagnostic studies of trauma or cardiovascular diseases. Finally, we find in the blood plasma mimics that after ultrafiltration, commonly used to remove the protein from plasma, the measured concentration equals the total metabolite concentration, except for the strongest binding metabolite citrate.

Reference dosimetry using radiochromic film.

The objectives of this study are to identify and quantify factors that influence radiochromic film dose response and to determine whether such films are suitable for reference dosimetry. The influence of several parameters that may introduce systematic dose errors when performing reference dose measurements were investigated. The effect of the film storage temperature was determined by comparing the performance of three lots of GAFCHROMIC EBT2 films stored at either 4ºC or room temperature. The effect of high (> 80%) or low (< 20%) relative humidity was also determined. Doses measured in optimal conditions with EBT and EBT2 films were then compared with an A12 ionization chamber measurement. Intensity-modulated radiation therapy quality controls using EBT2 films were also performed in reference dose. The results obtained using reference dose measurements were compared with those obtained using relative dose measurements. Storing the film at 4ºC improves the stability of the film over time, but does not eliminate the noncatalytic film development, seen as a rise in optical density over time in the absence of radiation. Relative humidity variations ranging from 80% to 20% have a strong impact on the optical density and could introduce dose errors of up to 15% if the humidity were not controlled during the film storage period. During the scanning procedure, the film temperature influences the optical density that is measured. When controlling for these three parameters, the dose differences between EBT or EBT2 and the A12 chamber are found to be within ± 4% (2σ level) over a dose range of 20-350 cGy. Our results also demonstrate the limitation of the Anisotropic Analytical Algorithm for dose calculation of highly modulated treatment plans.

Simultaneous analysis of plasma and CSF by NMR and hierarchical models fusion.

Because cerebrospinal fluid (CSF) is the biofluid which interacts most closely with the central nervous system, it holds promise as a reporter of neurological disease, for example multiple sclerosis (MScl). To characterize the metabolomics profile of neuroinflammatory aspects of this disease we studied an animal model of MScl-experimental autoimmune/allergic encephalomyelitis (EAE). Because CSF also exchanges metabolites with blood via the blood-brain barrier, malfunctions occurring in the CNS may be reflected in the biochemical composition of blood plasma. The combination of blood plasma and CSF provides more complete information about the disease. Both biofluids can be studied by use of NMR spectroscopy. It is then necessary to perform combined analysis of the two different datasets. Mid-level data fusion was therefore applied to blood plasma and CSF datasets. First, relevant information was extracted from each biofluid dataset by use of linear support vector machine recursive feature elimination. The selected variables from each dataset were concatenated for joint analysis by partial least squares discriminant analysis (PLS-DA). The combined metabolomics information from plasma and CSF enables more efficient and reliable discrimination of the onset of EAE. Second, we introduced hierarchical models fusion, in which previously developed PLS-DA models are hierarchically combined. We show that this approach enables neuroinflamed rats (even on the day of onset) to be distinguished from either healthy or peripherally inflamed rats. Moreover, progression of EAE can be investigated because the model separates the onset and peak of the disease.

Protoflavonoids from ferns impair centrosomal integrity of tumor cells.

Six protoflavonoids, including two new compounds, were isolated during a large scale screening of fern extracts for original interaction with mitosis. The new compounds isolated from PHEGOPTERIS decursive-pinnata and EQUISETUM fluviatile were 2',3'-dihydroprotogenkwanone (1) and 2',3'-dihydro-2'-hydroxyprotoapigenone (2). Known compounds were: protoapigenone, protogenkwanone, protoapigenin, and 4'- O- ß-D-glucopyranosyl protoapigenin. They showed a cytotoxic activity against HeLa cells at a micromolar level. IC50 values were 2 µM for compound 1 > 10 µM for compound 2, and respectively 2.4, 0.6, > 10 µM for the known compounds. Their cytotoxic effects were associated with phenotypic changes never observed before and characterized by the loss of centrosomal γ-tubulin labelling in both mitotic and interphasic cells.

NIF unconverted light and its influence on DANTE measurements.

NIF laser facility produces 1053 nm light and a fundamental requirement for NIF is to give up to 1.8 MJ of 351 nm light for target physics experiments. The 351 nm light is provided by frequency tripling the 1053 nm light in nonlinear crystals in the final optics assembly, just before the laser light enters the target chamber. Since this tripling process is not 100% efficient, unconverted light from the conversion process also enters the chamber. This unconverted light does not directly hit the target but it can strike target support structures at average intensities of few TW/cm2 where it can generate unwanted, background soft x-rays that are measured by the soft x-ray diagnostic DANTE installed on the NIF target chamber. This diagnostic quantifies the x-radiation intensity inside the hohlraum by measuring the x-ray flux coming from the target's laser entrance hole. Due to its centimeter wide field of view, it integrates x-ray emission from both the flux exiting a hohlraum laser entrance hole and from the target support structure irradiated by residual 1omega and 2omega unconverted light. This work gives quantitative evaluations of the unconverted light for the first time and the effects on DANTE measurements for the future NIF tuning experiment called "Shock timing." Emission spectra are significantly modified leading to an overestimation of radiative temperature during the foot of the laser pulse since background x-rays are predominant in first two DANTE channel measurements. Mitigations of these effects by coating silicon paddle with plastic, using a smaller collimator to reduce DANTE field of view or eliminating DANTE channels in the analysis have been investigated.

Preparation of selective and segmentally labeled single-stranded DNA for NMR by self-primed PCR and asymmetrical endonuclease double digestion.

We demonstrate a new, efficient and easy-to-use method for enzymatic synthesis of (stereo-)specific and segmental (13)C/(15)N/(2)H isotope-labeled single-stranded DNA in amounts sufficient for NMR, based on the highly efficient self-primed PCR. To achieve this, new approaches are introduced and combined. (i) Asymmetric endonuclease double digestion of tandem-repeated PCR product. (ii) T4 DNA ligase mediated ligation of two ssDNA segments. (iii) In vitro dNTP synthesis, consisting of in vitro rNTP synthesis followed by enzymatic stereo-selective reduction of the C2' of the rNTP, and a one-pot add-up synthesis of dTTP from dUTP. The method is demonstrated on two ssDNAs: (i) a 36-nt three-way junction, selectively (13)C(9)/(15)N(3)/(2)H((1',2'',3',4',5',5''))-dC labeled and (ii) a 39-nt triple-repeat three-way junction, selectively (13)C(9)/(15)N(3)/(2)H((1',2'',3',4',5',5''))-dC and (13)C(9)/(15)N(2)/(2)H((1',2'',3',4',5',5''))-dT labeled in segment C20-C39. Their NMR spectra show the spectral simplification, while the stereo-selective (2)H-labeling in the deoxyribose of the dC-residues, straightforwardly provided assignment of their C1'-H2' and C2'-H2' resonances. The labeling protocols can be extended to larger ssDNA molecules and to more than two segments.

Mutations in domain I interhelical loops affect the rate of pore formation by the Bacillus thuringiensis Cry1Aa toxin in insect midgut brush border membrane vesicles.

Pore formation in the apical membrane of the midgut epithelial cells of susceptible insects constitutes a key step in the mode of action of Bacillus thuringiensis insecticidal toxins. In order to study the mechanism of toxin insertion into the membrane, at least one residue in each of the pore-forming-domain (domain I) interhelical loops of Cry1Aa was replaced individually by cysteine, an amino acid which is normally absent from the activated Cry1Aa toxin, using site-directed mutagenesis. The toxicity of most mutants to Manduca sexta neonate larvae was comparable to that of Cry1Aa. The ability of each of the activated mutant toxins to permeabilize M. sexta midgut brush border membrane vesicles was examined with an osmotic swelling assay. Following a 1-h preincubation, all mutants except the V150C mutant were able to form pores at pH 7.5, although the W182C mutant had a weaker activity than the other toxins. Increasing the pH to 10.5, a procedure which introduces a negative charge on the thiol group of the cysteine residues, caused a significant reduction in the pore-forming abilities of most mutants without affecting those of Cry1Aa or the I88C, T122C, Y153C, or S252C mutant. The rate of pore formation was significantly lower for the F50C, Q151C, Y153C, W182C, and S252C mutants than for Cry1Aa at pH 7.5. At the higher pH, all mutants formed pores significantly more slowly than Cry1Aa, except the I88C mutant, which formed pores significantly faster, and the T122C mutant. These results indicate that domain I interhelical loop residues play an important role in the conformational changes leading to toxin insertion and pore formation.

Helix alpha 4 of the Bacillus thuringiensis Cry1Aa toxin plays a critical role in the postbinding steps of pore formation.

Helix alpha 4 of Bacillus thuringiensis Cry toxins is thought to play a critical role in the toxins' mode of action. Accordingly, single-site substitutions of many Cry1Aa helix alpha 4 amino acid residues have previously been shown to cause substantial reductions in the protein's pore-forming activity. Changes in protein structure and formation of intermolecular disulfide bonds were investigated as possible factors responsible for the inactivity of these mutants. Incubation of each mutant with trypsin and chymotrypsin for 12 h did not reveal overt structural differences with Cry1Aa, although circular dichroism was slightly decreased in the 190- to 210-nm region for the I132C, S139C, and V150C mutants. The addition of dithiothreitol stimulated pore formation by the E128C, I132C, S139C, T142C, I145C, P146C, and V150C mutants. However, in the presence of these mutants, the membrane permeability never reached that measured for Cry1Aa, indicating that the formation of disulfide bridges could only partially explain their loss of activity. The ability of a number of inactive mutants to compete with wild-type Cry1Aa for pore formation in brush border membrane vesicles isolated from Manduca sexta was also investigated with an osmotic swelling assay. With the exception of the L147C mutant, all mutants tested could inhibit the formation of pores by Cry1Aa, indicating that they retained receptor binding ability. These results strongly suggest that helix alpha 4 is involved mainly in the postbinding steps of pore formation.

Chemical modification of Bacillus thuringiensis Cry1Aa toxin single-cysteine mutants reveals the importance of domain I structural elements in the mechanism of pore formation.

Bacillus thuringiensis Cry toxins form pores in the apical membrane of insect larval midgut cells. To investigate their mechanism of membrane insertion, mutants in which cysteine replaced individual amino acids located within the pore-forming domain of Cry1Aa were chemically modified with sulfhydryl-specific reagents. The thiol group of cysteine was highly susceptible to oxidation and its reactivity was significantly increased when the toxins were purified under reducing conditions. Addition of a biotin group to the cysteine had little effect on the ability of the toxins to permeabilize Manduca sexta brush border membrane vesicles except for a slight reduction in activity for S252C and a large increase in activity for Y153C. The activity of Y153C was also significantly increased after modification by reagents that added an aromatic or a charged group to the cysteine. When permeability assays were performed in the presence of streptavidin, a large biotin-binding protein, the pore-forming activity of several mutants, including Y153C, where the altered residue is located within the hairpin comprising helices alpha4 and alpha5, or in adjacent loops, was significantly reduced. These results support the umbrella model of toxin insertion.

Multiple segmental and selective isotope labeling of large RNA for NMR structural studies.

Multiple segmental and selective isotope labeling of RNA with three segments has been demonstrated by introducing an RNA segment, selectively labeled with (13)C(9)/(15)N(2)/(2)H((1', 3', 4', 5', 5''))-labeled uridine residues, into the central position of the 20 kDa epsilon-RNA of Duck Hepatitis B Virus. The RNA molecules were produced via two efficient protocols: a two-step protocol, which uses T4 DNA ligase and T4 RNA ligase 1, and a one-pot protocol, which uses T4 RNA ligase 1 alone. With T4 RNA ligase 1 all not-to-be-ligated termini are usually protected to prevent formation of side products. We show that such labor-intensive protection of termini is not required, provided segmentation sites can be chosen such that the segments fold into the target structure or target-like structures and thus are not trapped into stable alternate structures. These sites can be reliably predicted via DINAMelt. The simplified NMR spectrum provided evidence for the presence of a U28 H(3)-imino resonance, previously obscured in the fully labeled sample, and thus of the non-canonical base pair U28:C37. The demonstrated multiple segmental labeling protocols are generally applicable to large RNA molecules and can be extended to more than three segments.

Cysteine scanning mutagenesis of alpha4, a putative pore-lining helix of the Bacillus thuringiensis insecticidal toxin Cry1Aa.

Helix alpha4 of Bacillus thuringiensis Cry toxins is thought to line the lumen of the pores they form in the midgut epithelial cells of susceptible insect larvae. To define its functional role in pore formation, most of the alpha4 amino acid residues were replaced individually by a cysteine in the Cry1Aa toxin. The toxicities and pore-forming abilities of the mutated toxins were examined, respectively, by bioassays using neonate Manduca sexta larvae and by a light-scattering assay using midgut brush border membrane vesicles isolated from M. sexta. A majority of these mutants had considerably reduced toxicities and pore-forming abilities. Most mutations causing substantial or complete loss of activity map on the hydrophilic face of the helix, while most of those having little or only relatively minor effects map on its hydrophobic face. The properties of the pores formed by mutants that retain significant activity appear similar to those of the pores formed by the wild-type toxin, suggesting that mutations resulting in a loss of activity interfere mainly with pore formation.

Cost-effectiveness of various diagnostic approaches for occupational asthma.

BACKGROUND: Diagnosis of occupational asthma (OA) by specific inhalation challenge (SIC) can be costly and is not always available. The use of sputum testing to avoid this in some patients may be a more cost-effective alternative. OBJECTIVES: To compare the cost-effectiveness of SIC with serial measurements of sputum cell counts (sputum testing) and peak expiratory flow (PEF) monitoring. METHODS: Clinical data and testing costs for OA in 49 patients were collected during a previously published trial, modelled and compared using TreeAge Pro. Clinical outcome was the percentage of accurately diagnosed patients, using SIC as the gold standard. The PEF approach used the most accurate assessment of five experts who were blinded to SIC results. Differences in the proportion of eosinophils during periods on and off work were used for the sputum testing approach and in PEF/sputum for the combined approach. Unit costs were estimated from charges in Canadian hospitals. Data were analyzed by one-way and two-way analyses, and by probabilistic sensitivity analysis using a Monte Carlo simulation technique. RESULTS: The PEF approach had an estimated accuracy of 52% and cost $365 per patient tested. Compared with PEF monitoring, sputum testing was more accurate and cost an estimated $255 for each additional OA patient correctly diagnosed. SIC costs per additional correct diagnosis were $11,032 compared with sputum testing and $6,458 compared with PEF monitoring. The combined PEF/sputum testing approach was not cost-effective in the base case analysis, but cannot be excluded according to probabilistic sensitivity analyses. CONCLUSIONS: Although SIC remains the reference test to diagnose OA, when this test is not available, sputum testing is a cost-effective alternative to PEF for diagnosis of OA.

Thermodynamics and NMR studies on Duck, Heron and Human HBV encapsidation signals.

Hepatitis B virus (HBV) replication is initiated by binding of its reverse transcriptase (P) to the apical stem-loop (AL) and primer loop (PL) of epsilon, a highly conserved RNA element at the 5'-end of the RNA pregenome. Mutation studies on duck/heron and human in vitro systems have shown similarities but also differences between their P-epsilon interaction. Here, NMR and UV thermodynamic data on AL (and PL) from these three species are presented. The stabilities of the duck and heron ALs were found to be similar, and much lower than that of human. NMR data show that this low stability stems from an 11-nt internal bulge destabilizing the stem of heron AL. In duck, although structured at low temperature, this region also forms a weak point as its imino resonances broaden to disappearance between 30 and 35 degrees C well below the overall AL melting temperature. Surprisingly, the duck- and heron ALs were both found to be capped by a stable well-structured UGUU tetraloop. All avian ALs are expected to adhere to this because of their conserved sequence. Duck PL is stable and structured and, in view of sequence similarities, the same is expected for heron - and human PL.

Solution structure of the apical stem-loop of the human hepatitis B virus encapsidation signal.

Hepatitis B virus (HBV) replication is initiated by HBV RT binding to the highly conserved encapsidation signal, epsilon, at the 5' end of the RNA pregenome. Epsilon contains an apical stem-loop, whose residues are either totally conserved or show rare non-disruptive mutations. Here we present the structure of the apical stem-loop based on NOE, RDC and (1)H chemical shift NMR data. The (1)H chemical shifts proved to be crucial to define the loop conformation. The loop sequence 5'-CUGUGC-3' folds into a UGU triloop with a CG closing base pair and a bulged out C and hence forms a pseudo-triloop, a proposed protein recognition motif. In the UGU loop conformations most consistent with experimental data, the guanine nucleobase is located on the minor groove face and the two uracil bases on the major groove face. The underlying helix is disrupted by a conserved non-paired U bulge. This U bulge adopts multiple conformations, with the nucleobase being located either in the major groove or partially intercalated in the helix from the minor groove side, and bends the helical stem. The pseudo-triloop motif, together with the U bulge, may represent important anchor points for the initial recognition of epsilon by the viral RT.