Mapping manifestations of parametric uncertainty in projected pelagic oxygen concentrations back to contemporary local model fidelity.

Pelagic biogeochemical models (BGCMs) have matured into generic components of Earth System Models. BGCMs mimic the effects of marine biota on oceanic nutrient, carbon and oxygen cycles. They rely on parameters that are adjusted to match observed conditions. Such parameters are key to determining the models' responses to changing environmental conditions. However, many of these parameters are difficult to constrain and constitute a major source of uncertainty in BGCM projections. Here we use, for the first time, variance-based sensitivity analyses to map BGCM parameter uncertainties onto their respective local manifestation in model entities (such as oceanic oxygen concentrations) for both contemporary climate and climate projections. The mapping effectively relates local uncertainties of projections to the uncertainty of specific parameters. Further, it identifies contemporary benchmarking regions, where the uncertainties of specific parameters manifest themselves, thereby facilitating an effective parameter refinement and a reduction of the associated uncertainty. Our results demonstrate that the parameters that are linked to uncertainties in projections may differ from those parameters that facilitate model conformity with present-day observations. In summary, we present a practical approach to the general question of where present-day model fidelity may be indicative for reliable projections.

Inter-joint coordination and the flexion-relaxation phenomenon among adults with low back pain during bending.

BACKGROUND: Altered inter-joint coordination and reduced flexion-relaxation at end-range trunk flexion are common in people with low back pain. Inconsistencies in these behaviors, however, make assessment and treatment challenging for this population. RESEARCH QUESTION: The study objective was to investigate patterns of regional lumbo-pelvic coordination and flexion-relaxation in adults with and without low back pain, during a bending task. METHODS: Adults with low back pain (n = 16) and a healthy group (n = 21) performed three trials of a bending task. Motion capture and surface electromyography systems measured joint kinematics (hip, lower and upper lumbar spine) and muscle activity (erector spinae longissimus, iliocostalis, and multifidus). Continuous relative phase analysis determined inter-joint coordination of the hip/lower lumbar and lower lumbar/upper lumbar joint pairs, during flexion and extension periods. Flexion-relaxation ratios using normalized surface electromyography data determined the extent of flexion-relaxation for each muscle, during each period. For inter-joint coordination, two-way repeated measure mixed ANOVAs calculated the effects of group (healthy/low back pain), period, and their interactions. Separate hierarchical linear models were constructed and tested relationships between flexion-relaxation ratios and our independent variables, group and muscle, while controlling for patient characteristics. RESULTS: The low back pain group had more out-of-phase coordination of the hip/lower lumbar joint pair compared to the healthy group (mean difference = 24.7°; 95 % confidence interval = 3.93-45.4), independent of movement period. No significant between group differences in lower lumbar/upper lumbar coordination were observed. The low back pain group demonstrated reduced flexion-relaxation of all muscles during full flexion (21.7 % reduction on average), with multifidus showing the least relaxation. SIGNIFICANCE: Regional differences in the lumbar spine and the possibility of subgroups with distinct movement pattern should be considered when analyzing coordination in people with low back pain. Multifidus showed the largest changes in flexion-relaxation and should be included when measuring this construct.

Correction to: The effect of beta blocker withdrawal on adenosine myocardial perfusion imaging.

Regrettably the original version of the above article contained errors in Table 2 and wrong values in the text. The corrected table is presented here and the values which have been corrected now appear in bold text. Page 1223 abstract Global MBF showed an increase from 180.2 ± 59.9 to 193.6 ± 60.8 mL minute/100 g (P = .002) after beta blocker withdrawal. Page 1225 Mean systolic and mean diastolic blood pressure during adenosine were nearly identical (P = .77 and P = .79) with and without beta blocker. Mean heart rate and mean RPP during adenosine significantly increased after beta blocker withdrawal by 15.2% ± 17% (P = .001) and 16.2% ± 23% (P = .004), respectively. Page 1226 The data are listed in Table 2, lower third. Global MBF showed a significant increase by 7.4% ± 10% (P = .002) after beta blocker withdrawal. The individual data are depicted in Figure 1. All but three patients had a lower global MBF without beta blocker than with. The segmental MBF values (Figure 2) demonstrated a strong correlation over the entire range of perfusion values. The average effect was a slight perfusion shift of about 1015 mL minute-1/100 g in the range of 100-300 mL minute-1/100 g. The mCR under adenosine declined by 8.1% ± 11% (P = .038) and the normalized RPP by 16.2% ± 21% (P = .004) after betablocker discontinuation. Table 2 Hemodynamic response under adenosine, perfusion, and left-ventricular function.

The role of FDG-PET-CT in pediatric cardiac patients and patients with congenital heart defects.

AIM: Medical imaging by using FDG-PET/CT (PET-CT) can detect, confirm or eliminate with high sensitivity areas of suspected infections in case of persistent fever of unknown origin in combination with other bacteriological examinations. The aim of this study was to assess the potential role of PET-CT in detecting or excluding infections or other inflammatory processes in patients with congenital heart defects (CHD). In addition we wanted to evaluate the practical impact of PET-CT on the subsequent clinical management. METHODS: In this retrospective study we analyzed the data of all CHD patients who underwent PET-CT over a 5year period in our institution. The results were then evaluated with regard to the potential impact on clinical decision making. RESULTS: Between 2010 and 2015 PET-CT was performed in 30 patients. The mean age was 26years (SD 15years, range 1 to 66years). The diagnoses covered a large field of CHD. 11 patients (4/11 with assist device) were assessed before heart transplantation; suspected malignancies or infections were excluded and transplant listing was possible. In another 5/6 patients suspected assist device infection could be confirmed with PET/CT. Endocarditis was suspected in 13 patients, 2 of whom underwent previous MRI without confirmation and ECHO was inconclusive. Endocarditis was finally excluded in 5/13 patients but confirmed in 8/13 patients by PET-CT. CONCLUSION: In this study we could show a high sensitivity of PET-CT for specific localization of infections and with high impact on subsequent therapy. Based on this results clinical management could be targeted and adapted. We could demonstrate that PET-CT has a high impact on the subsequent clinical therapy.

The effect of beta blocker withdrawal on myocardial SPECT modeled from adenosine 13N-ammonia PET.

AIM: The effect of beta blockers (BB) on myocardial imaging has been studied in several SPECT and PET studies with divergent results concerning perfusion and impact on diagnostic accuracy. The present study evaluated the effect of BB withdrawal on virtual SPECT studies modeled from quantitative PET perfusion scans. PATIENTS, METHODS: Data from 20 CAD patients scheduled for adenosine 13N-ammonia imaging with and without BB were considered. Modeling the uptake characteristics of 99mTc-MIBI, all parametric stress PET polarmaps were transferred to virtual 20-segment SPECT polarmaps. The SPECT studies were categorized with a 5-point score and read to assess the effect of the BB withdrawal on scan result and interpretation. RESULTS: The SPECT analysis revealed a mean score of 6.0 ± 4.7 with, and of 5.9 ± 4.5 without BB (p = 0.84). In 260 (74.9%) segments the scores were equal in both conditions. Without BB a downstaging was recorded in 44 segments (12.7%), an upstaging in 43 segments (12.4%). An essentially different interpretation (shift from medical therapy recommendation to angiography) was recorded in one patient. In six cases the interpretation differed mildly. CONCLUSION: In the majority of patients studied, scan results and interpretation remain unchanged after discontinuation of the BB. Nevertheless, the segmental scan results are not uniformly affected. The recommendation to stop BBs prior to stress testing in order to ensure the highest MBF remains advisable. If temporary BB withdrawal is unfeasible due to contraindications, a tight clinical schedule, or because a patient forgot to withhold the BB, it is appropriate to perform adenosine stress testing according to the results of this study.

The influence of the strength of bone on the deformation of acetabular shells: a laboratory experiment in cadavers.

Concerns have been raised that deformation of acetabular shells may disrupt the assembly process of modular prostheses. In this study we aimed to examine the effect that the strength of bone has on the amount of deformation of the acetabular shell. The hypothesis was that stronger bone would result in greater deformation. A total of 17 acetabular shells were inserted into the acetabula of eight cadavers, and deformation was measured using an optical measuring system. Cores of bone from the femoral head were taken from each cadaver and compressed using a materials testing machine. The highest peak modulus and yield stress for each cadaver were used to represent the strength of the bone and compared with the values for the deformation and the surgeon's subjective assessment of the hardness of the bone. The mean deformation of the shell was 129 µm (3 to 340). No correlation was found between deformation and either the maximum peak modulus (r² = 0.011, t = 0.426, p = 0.676) or the yield stress (r² = 0.024, t = 0.614, p = 0.549) of the bone. Although no correlation was found between the strength of the bone and deformation, the values for the deformation observed could be sufficient to disrupt the assembly process of modular acetabular components.

The French-Canadian validation of a disease-specific, patient-reported outcome measure for lupus.

OBJECTIVES: The objective of this paper is to perform the cross-cultural validation of the French version of the LupusPRO, a disease-targeted patient-reported outcome measure, among systemic lupus erythematosus (SLE) patients in Canada. METHODS: The French version of the LupusPRO and the MOS SF-36 were administered; demographic, clinical and serological characteristics were obtained. Disease activity (SELENA-SLEDAI and the Lupus Foundation of America definition of flare) and damage (SLICC/ACR SDI) were assessed. Physician disease activity and damage assessments were ascertained using visual analog scales. Internal consistency reliability (ICR), test-retest reliability (TRT), convergent and discriminant validity (against corresponding domains of the SF-36), criterion validity (against disease activity, damage or health status) and known group validity were tested. RESULTS: A total of 99 French-Canadian SLE patients participated (97% women, mean (SD) age 45.2 (14.5) years). The median (IQR) SELENA-SLEDAI and SDI were 3.5 (6.0) and 1.0 (2.0), respectively. The ICR of the LupusPRO domains ranged from 0.81 to 0.93 (except for lupus symptoms, procreation and coping), while TRT ranged from 0.72 to 0.95. Convergent and discriminant validity, criterion validity and known group validity against disease activity, damage and health status measures were observed. Confirmatory factor analysis showed a good fit. CONCLUSION: The LupusPRO has fair psychometric properties among French-Canadian patients with SLE.

The effect of beta blocker withdrawal on adenosine myocardial perfusion imaging.

BACKGROUND: The effect of beta blockers on myocardial blood flow (MBF) under vasodilators has been studied in several SPECT and PET myocardial perfusion imaging (MPI) studies with divergent results. The present study evaluated the effect of a beta blocker withdrawal on quantitative adenosine MBF and on MPI results. METHODS: Twenty patients with beta blockers and CAD history were studied with quantitative adenosine N-13 ammonia PET. The first study was performed under complete medication and the second after beta blocker withdrawal. The PET studies were independently read with respect to MPI result and clinical decision making. RESULTS: Global MBF showed an increase from 180.2 ± 59.9 to 193.6 ± 60.8 mL·minute(-1)/100 g (P = .02) after beta blocker withdrawal. The segmental perfusion values were closely correlated (R(2) = 0.82) over the entire range of perfusion values. An essentially different interpretation after beta blocker discontinuation was found in two cases (10%). CONCLUSION: A beta blocker withdrawal induces an increase in adenosine MBF. In the majority of cases, MPI interpretation and decision making are independent of beta blocker intake. If a temporary beta blocker withdrawal before MPI is not possible or was not realized by the patient, it is appropriate to perform adenosine stress testing without loss of the essential MPI result.

Cross-cultural validation of a disease-specific patient-reported outcome measure for lupus in Philippines.

OBJECTIVE: LupusPRO is a disease-targeted patient-reported outcome measure that was developed and validated among US patients with systemic lupus erythematosus (SLE). We report the cross-cultural validation results of the LupusPRO English-language version among Filipino SLE patients. METHOD: The 43-item LupusPRO was pretested in 15 SLE individuals, then administered to 106 SLE patients, along with short-form SF36 and the EQ5D visual analogue scale. A mail/drop-back LupusPRO and change in health status item survey were returned within two to three days. Demographics, clinical and serological characteristics, disease activity and damage measured by PGA, SELENA-SLEDAI, LFA Flare, and SLICC-ACR SLE damage index (SDI) were collected. Internal consistency reliability (ICR), test-retest reliability (TRT), convergent validity (corresponding SF36 domains) and criterion validity (against general health and disease activity measures) were tested. Reported p values are two tailed. RESULTS: A total of 121 Filipino SLE subjects (95% women, median age 31.0 ± 16 years) with at least a high school level of English instruction participated. Median (IQR) PGA, SLEDAI and SDI were 0.0 (1.0), 2.0 (10) and 0 (1), respectively. ICR exceeded 0.7 for all domains except the lupus symptoms domain. TRT was greater than 0.85 for all LupusPRO domains. Convergent and criterion validity were observed against corresponding SF36 domains and disease activity measures. The tool was well received by patients. Confirmatory factor analysis showed good fit. CONCLUSION: English LupusPRO has fair psychometric properties among SLE patients in the Philippines, and is now available for inclusion in clinical trials and longitudinal studies to test responsiveness to change.

Evidence against nitric oxide-quenching effects of chemically defined Maillard reaction products.

Direct interaction between Maillard reaction products (MRPs) and nitric oxide (NO) has been suggested as a pathophysiological mechanism involved in enhanced diabetic arteriosclerosis. Only MRPs without structural characterization have been studied to date. Using chemically synthesized and analytically well defined individual MRPs, we investigated whether the native nitric oxide concentration is directly affected by the Amadori compound N-epsilon-fructosyllysine or the advanced glycation end product N-epsilon-carboxymethyllysine. MRPs were incubated with nitric oxide solution or NO donors (SNAP, spermine-NONOate). Changes in the nitrite (oxidative metabolite of NO) concentration served as indicator of NO availability. MRPs, either as free amino acids or covalently bound to bovine serum albumin (BSA), had no influence on nitrite concentration when using NO solution. In contrast, incubation of the respective NO donors with several covalently protein-bound MRPs as well as native BSA significantly reduced nitrite concentration. If SNAP was co-incubated with EDTA or with Fe (2+) ions, nitrite concentration was decreased or increased, respectively, suggesting a metal ion-dependent alteration of the NO liberation rate. Native NO concentration was not affected by the MRPs tested. Substitution of native NO by NO-releasing substances may be inadequate as a model of NO-MRP interaction, as metal ions or chelators present in compound preparations may affect the NO-liberating mechanism of the donor.

3-Hydroxybenzo[a]pyrene in the urine of workers with occupational exposure to polycyclic aromatic hydrocarbons in different industries.

OBJECTIVES: This study was conducted to assess external and internal exposure of workers to polycyclic aromatic hydrocarbons (PAHs). In this context, the analytical and diagnostical reliability of 3-hydroxybenzo[a]pyrene (3OH-BaP) as a biomarker of internal exposure to PAHs was established. METHODS: Ambient and biological monitoring was carried out of 225 PAH-exposed employees of different industries. External exposure was determined by personal air sampling and analysis of 16 EPA-PAH. Internal exposure was examined by the urinary metabolites 3OH-BaP, 1-hydroxypyrene (OH-Pyr) and monohydroxylated phenanthrenes (OH-Phens). RESULTS: Benzo[a]pyrene (BaP) was detected at all workplaces. Concentrations in the breathing zone of the workers ranged from below the limit of detection up to 44.3 mug/m(3). In biological monitoring, urinary 3OH-BaP was found in median concentrations of 0.8 ng/g creatinine (crea) and the 95th percentile of 6.7 ng/g crea. The results ranged from the limit of detection up to 19.5 ng/g crea. Only 1% of the analysed samples showed concentrations below the limit of detection (0.05 ng/l). Regarding median concentrations, workers in coking plants showed lower 3OH-BaP concentrations (0.5 ng/g crea) than those employed in the production of fireproof material in refractories (1.1 ng/g crea), converter infeed (1.2 ng/g crea) and graphite electrode production (1.3 ng/g crea). Strong correlations of 3OH-BaP with OH-Pyr and the sum of OH-Phens were found for the workplaces converter infeed, coking plants and graphite electrode production (r(Pearson) ranging from 0.618 to 0.867, p<0.001). The poor correlation of BaP in the air and 3OH-BaP in urine is most probably caused by routes of uptake other than via air-for example, dermal uptake. CONCLUSION: 3OH-BaP as a metabolite of the carcinogenic BaP could be shown to be a diagnostically specific and sensitive biomarker for determining the internal exposure of workers in different industries. Using this method, the estimation of health risks for workers can be fundamentally improved, because the 3OH-BaP represents the group of carcinogenic PAHs. The procedure for analysing 3OH-BaP is complex, but it is robust and produces reliable results.

Energy-confinement scaling for high-beta plasmas in the W7-AS stellarator.

High-beta energy-confinement data are subjected to comparisons of scaling invariant, first-principles physical models. The models differ in the inclusion of basic equations indicating the nature of transport. The result for high-beta data of the W7-AS stellarator is that global transport is described best with a collisional high-beta model, which is different from previous outcomes for low-beta data. Model predictive calculations indicate the validation of energy-confinement prediction with respect to plasma beta and collisionality nu*. The finding of different transport behaviors in distinct beta regimes is important for the development of fusion energy based on magnetic confinement and for the assessment of different confinement concepts.

Di(2-ethylhexyl)phthalate (DEHP): human metabolism and internal exposure-- an update and latest results.

Di(2-ethylhexyl)phthalate (DEHP) is a reproductive and developmental toxicant in animals and a suspected endocrine modulator in humans. There is widespread exposure to DEHP in the general population. Patients can be additionally exposed through DEHP-containing medical devices. Toxicokinetic and metabolic knowledge on DEHP in humans is vital not only for the toxicological evaluation of DEHP but also for exposure assessments based on human biomonitoring data. Secondary oxidized DEHP metabolites like mono-(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono-(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP), mono-(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP) and mono-[2-(carboxymethyl)hexyl]phthalate (2cx-MMHP) are most valuable biomarkers of DEHP exposure. They represent the major share of DEHP metabolites excreted in urine (about 70% for these four oxidized metabolites vs. about 6% for MEHP); they are immune to external contamination and possibly the ultimate developmental toxicants. Long half-times of elimination make 5cx-MEPP and 2cx-MMHP excellent parameters to measure the time-weighted body burden to DEHP. 5OH-MEHP and 5oxo-MEHP more reflect the short-term exposure. We calculated the daily DEHP intake for the general population (n = 85) and for children (n = 254). Children were significantly higher exposed to DEHP than adults. Exposures at the 95th percentile (21 and 25 microg/kg/day, respectively) scooped out limit values like the Reference Dose (RfD, 20 microg/kg/day) and the Tolerable Daily Intake (TDI, 20-48 microg/kg/day) to a considerable degree. Up to 20-fold oversteppings for some children give cause for concern. We also detected significant DEHP exposures for voluntary platelet donors (n = 12, 38 microg/kg/apheresis, dual-needle technique). Premature neonates (n = 45) were exposed to DEHP up to 100 times above the limit values depending on the intensity of medical care (median: 42 microg/kg/day; 95th percentile: 1,780 microg/kg/day).

Genotoxic risk assessment in white blood cells of occupationally exposed workers before and after alteration of the polycyclic aromatic hydrocarbon (PAH) profile in the production material: comparison with PAH air and urinary metabolite levels.

OBJECTIVE: Workers in various industries can be exposed to polycyclic aromatic hydrocarbons (PAHs). The relationship between biomarkers of genotoxic risk, PAH compounds in air (ambient monitoring) and PAH metabolites in urine (internal exposure) were studied in 17 workers exposed to PAHs in a fireproof-material producing plant before and 3 months after the PAH profile was altered in the binding pitch. METHODS: Two biomarkers of exposure, specific DNA adducts of (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) and non-specific DNA adduct of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were determined in white blood cells (WBCs). In addition, DNA strand breaks were analysed in lymphocytes by single-cell gel electrophoresis in a genotoxic risk assessment. Sixteen PAH compounds in air were determined by personal air sampling, and hydroxylated metabolites of phenanthrene, pyrene and naphthalene were determined in urine. RESULTS: After substitution of the binding pitch the concentrations of benzo[a]pyrene in air decreased (P<0.01). No changes could be observed for pyrene, while levels of phenanthrene (P=0.0013) and naphthalene (P=0.0346) in air increased. Consequently, median DNA adduct rates of anti-BPDE decreased after alteration of the production material (from 0.9 to <0.5 adducts/10(8) nucleotides). No changes in the excretion of 1-hydroxypyrene in urine could be determined, whereas increased levels of 1-, 2+9-, 3- and 4-hydroxyphenanthrene (P<0.0001) and 1-naphthol and 2-naphthol (P=0.0072) were found in urine. In addition, a statistically significant increase in DNA strand break frequencies (P<0.01) and elevated 8-oxodGuo adduct levels (P=0.7819, not statistically significant) were found in the WBCs of exposed workers 3 months after the PAH profile in the binding pitch had been altered. CONCLUSION: The results presented here show that the increased concentration of naphthalene and/or phenanthrene in the air at the work place could induce the formation of DNA strand breaks and alkali-labile sites in WBCs of exposed workers.

A simple method to estimate force plate inertial components in a moving surface.

Support surface perturbations are a common paradigm for the study of balance and postural control. Forces and moments acquired from force plates mounted on, or within, the moving surface will contain components resulting from the inertia of the force plate itself. These force plate inertial components must be removed in order to accurately estimate forces resulting from contact with the force plate. This is particularly important when these contact forces are to be used in further calculations, such as an inverse dynamics analysis of joint kinetics. An estimate of the FPIC can be derived using the kinematics of the moving surface and the inertial properties of the force plate. This technique allowed for a reduction of up to 85% of the peak and integrated FPIC acquired from AMTI (OR6-7) force plates during translations of 0.1m, and surface rotations of 10 degrees, using a ramp stimulus of 150 ms duration.

Decomposition of multicomponent mass spectra using Bayesian probability theory.

We present a method for the decomposition of the mass spectra of mixed gases using Bayesian probability theory. The method works without any calibration measurement and therefore applies also to the analysis of spectra containing unstable species. For the example of mixtures of three different hydrocarbon gases the algorithm provides concentrations and cracking coefficients of each mixture component and also their confidence intervals. The amount of information needed to obtain reliable results and its relation to the accuracy of our analysis are discussed.

Analysis of multicomponent mass spectra applying Bayesian probability theory.

In this paper we develop a method for the decomposition of mass spectra of gas mixtures, together with the relevant calibration measurements. The method is based on Bayesian probability theory. Given a set of spectra, the algorithm returns the relative concentrations and the associated margin of confidence for each component of the mixture. In addition to the concentrations, such a data set enables the derivation of improved values of the cracking coefficients of all contributing species, even for those components for which the set does not contain a calibration measurement. This latter feature also allows one to analyze mixtures that contain radicals in addition to stable molecules. As an example, we analyze and discuss the mass spectra obtained from the pyrolysis of azomethane, which contain the radical CH3 apart from nitrogen and C1- and C2-hydrocarbons.

Design and synthesis of inhibitors of adenosine and AMP deaminases.

Nucleosides and nucleotides which are able to undergo covalent hydration in the aglycone ring system are potential inhibitors of the enzymes adenosine deaminase (ADA) and AMP deaminase, respectively. Calculations of the enthalpy of covalent hydration and of enzyme binding energy have been used to design new inhibitors of ADA. The ribosyl triazolotriazine 16, which was synthesized as a result of these calculations, exists predominantly as the covalent hydrate 18 in water and is a potent inhibitor of mammalian ADA (IC(50) 50 nM).

Probing RNA-protein interactions using pyrene-labeled oligodeoxynucleotides: Qbeta replicase efficiently binds small RNAs by recognizing pyrimidine residues.

Binding of small RNAs by the RNA-dependent RNA polymerase of coliphage Qbeta was studied utilizing a fluorometric assay. A DNA oligonucleotide probe of sequence 5'-d(TTTTTCC) was 5'-end-labeled with pyrene. In this construct, the proximal thymine residues efficiently quench the fluorophore emission in solution. Upon stoichiometric binding of one probe per polymerase molecule, the pyrene steady-state fluorescence increases by two orders of magnitude, the fluorescence anisotropy increases, and a long fluorescence lifetime component of 140 ns appears. With addition of replicable RNA, steady-state fluorescence decreases in a concentration dependent manner and the long lifetime component is lost. This observation most likely reflects displacement of the pyrene-labeled probe from the proposed nucleic acid binding site II of Qbeta replicase. The effect was utilized to access binding affinities of different RNAs to this site in a reverse titration assay format. In 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, at 16 degrees C, equilibrium dissociation constants for different template midi- and minivariant RNAs were calculated to be in the nanomolar range. In general, the minus and plus strands, concomitantly synthesized by Qbeta replicase during replication, exhibited discriminative affinities, while their hybrid bound less efficiently than either of the single strands. Different non-replicable tRNAs also bound to the polymerase with comparable dissociation constants. By titration with DNA homo-oligonucleotides it was shown that the probed site on Qbeta replicase does not require a 2' hydroxyl group for binding nucleic acids, but recognizes pyrimidine residues. Its interaction with thymine is lost in an A.T base-pair, while that with cytosine is retained after Watson-Crick base-pairing. These findings can explain the affinities of RNA-Qbeta replicase interactions reported here and in earlier investigations. The sensitivity of the described fluorometric assay allows detection of RNA amplification by Qbeta replicase in real-time.