Target Quantity Research Articles

Gait Modification to Treat Knee Osteoarthritis

By changing pathological joint mechanics, gait modification may have disease modifying potential for medial compartment knee OA. The most effective gait modification methods may well be a combination of an assisted modification and a learned modification taught via a real-time feedback system—for example, variable stiffness shoes with medial thrust gait. Addition of other holistic elements such as strength or flexibility training may be valuable as well. Determination of the best target quantity will be critical for gait modification to make a positive clinical impact.

Toolbox approach for fast generation of stable CHO production cell lines from different hosts

Background Protein production in CHO cell lines is a highly dynamic process, where the yield limiting step can shift among transcription, translation, and secretion under different cell growth stages and culture conditions. In addition, the choice of CHO cell line has been shown to affect target protein quality and quantity. Further advancements in titer and specific productivity require elimination of cellular/process bottlenecks along the generation of stable production cell lines including vector design, clone selection, genetic engineering, and media and feed optimization. To overcome these limitations, we developed a toolbox for all process phases from transfection to production. This toolbox is based on a chemically defined media platform and includes pre-adapted CHO-K1 or CHO-DG44 cell lines, optimized vectors for single and multi-chain proteins as well as fine-tuned protocols. In addition, it enables the production of antibodies with specialized glycan structures (GlymaxX [1]) as well as clone engineering with regard to the expression and secretion machinery (CDC42).

Rapid monitoring of Escherichia coli and Enterococcus spp. in bathing water using Reverse Transcription-quantitative PCR

For accompanying actions to pollution crisis management of recreational waters, rapid methods providing a quantified faecal indicator which can be completed in less than half a day, are currently needed. Two sensitive rRNA-targeted Reverse Transcription-quantitative PCR (RT-qPCR) methods were developed for quantifying Escherichia coli and Enterococcus spp. cells in marine bathing waters. Threshold cycle (Ct) values were found to be linear with a target quantity over a 4log dynamic range (from 10(5) to 100 cell equivalents per 100ml). In order to confirm the scope of application of such alternative methods, 80 and 85 seawater samples from the Atlantic Ocean and Mediterranean sea were analysed by both RT-qPCR and ISO culture-based methods for E. coli and Enterococcus spp. enumerations. This study demonstrates that by giving reliable results in 3h, the RT-qPCR method has high potential as a rapid test for recreational water quality monitoring. In natural waters, significant linear log-log relations between the RT-qPCR and culture method measurements for E. coli and Enterococcus spp. assays were shown (correlation coefficient (r) values of 0.814 and 0.715, p<0.0001, respectively). The sensitivities (defined as the probability of a sample testing positive if the criterion number is exceeded) of RT-qPCR for E. coli and Enterococcus spp. were respectively 94.4% and 83.9%. By contrast, specificities (defined as the probability of a test being negative if a contamination is truly absent) could be considered as lower (65.9% and 50.0% for E. coli and Enterococcus spp.). This reflects that RT-qPCR assays detect all viable cells (culturable and non-culturable) whereas culture methods only detect cells which are viable and culturable.

A two-stage hybrid procedure for estimating an inverse regression function

We consider a two-stage procedure (TSP) for estimating an inverse regression function at a given point, where isotonic regression is used at stage one to obtain an initial estimate and a local linear approximation in the vicinity of this estimate is used at stage two. We establish that the convergence rate of the second-stage estimate can attain the parametric n1/2 rate. Furthermore, a bootstrapped variant of TSP (BTSP) is introduced and its consistency properties studied. This variant manages to overcome the slow speed of the convergence in distribution and the estimation of the derivative of the regression function at the unknown target quantity. Finally, the finite sample performance of BTSP is studied through simulations and the method is illustrated on a data set.

A Java Program for LRE-Based Real-Time qPCR that Enables Large-Scale Absolute Quantification

BackgroundLinear regression of efficiency (LRE) introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis.FindingsUtilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification.ConclusionsThe LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples.

Akaike’s information criterion correction for the least-squares autoregressive spectral estimator

In this article we propose a new correction for the penalty term of the Akaike’s information criterion (AIC), when it is used in the context of order selection for an autoregressive fit of the spectral density of a stationary time series. The classical AIC penalty term may be viewed as an approximation of an appropriate target quantity. Simulations show that the quality of this approximation strongly depends on the type of autoregressive estimator used, as well as on the discrepancy used. Therefore here we consider the least squares autoregressive estimator and the Whittle discrepancy only. In this context we propose a closed formula correction of the AIC penalty term. We also develop asymptotic theory which justifies this proposal: an asymptotically valid second-order expansion of a stochastic approximation of the target quantity. This expansion assumes a non-parametric framework: it does not assume gaussianity of the process and only requires its spectral density to be smooth enough. Simulations show that, as compared to previously introduced corrections, this new correction performs similarly to finite sample information criterion, while they both outperform AIC corrected and AIC.

Optimal stockpile voxel identification based on reclaimer minimum movement for target grade

The supply of iron ore closest to the target grade with lowest operation cost is the main concern for the ore producer in mining industries. An automatic optimal reclaiming system to reclaim iron ore from stockpiles using the bucket wheel reclaimer (BWR) is proposed. However, this paper only presents a study on identifying optimal stockpile voxels for the target quantity and quality grade. Stockpiles here are represented by combinations of voxels, each possessing certain quality composition. The main focus of this paper is to identify the combination of voxels from available stockpile voxels that will fulfil quality and quantity demand of iron ore while maintaining BWR minimum movement on reclaiming.

Assessing the Performance Capabilities of LRE-Based Assays for Absolute Quantitative Real-Time PCR

BackgroundLinear regression of efficiency or LRE introduced a new paradigm for conducting absolute quantification, which does not require standard curves, can generate absolute accuracies of ±25% and has single molecule sensitivity. Derived from adapting the classic Boltzmann sigmoidal function to PCR, target quantity is calculated directly from the fluorescence readings within the central region of an amplification profile, generating 4–8 determinations from each amplification reaction.FindingsBased on generating a linear representation of PCR amplification, the highly visual nature of LRE analysis is illustrated by varying reaction volume and amplification efficiency, which also demonstrates how LRE can be used to model PCR. Examining the dynamic range of LRE further demonstrates that quantitative accuracy can be maintained down to a single target molecule, and that target quantification below ten molecules conforms to that predicted by Poisson distribution. Essential to the universality of optical calibration, the fluorescence intensity generated by SYBR Green I (FU/bp) is shown to be independent of GC content and amplicon size, further verifying that absolute scale can be established using a single quantitative standard. Two high-performance lambda amplicons are also introduced that in addition to producing highly precise optical calibrations, can be used as benchmarks for performance testing. The utility of limiting dilution assay for conducting platform-independent absolute quantification is also discussed, along with the utility of defining assay performance in terms of absolute accuracy.ConclusionsFounded on the ability to exploit lambda gDNA as a universal quantitative standard, LRE provides the ability to conduct absolute quantification using few resources beyond those needed for sample preparation and amplification. Combined with the quantitative and quality control capabilities of LRE, this kinetic-based approach has the potential to fundamentally transform how real-time qPCR is conducted.

Bias in the C q value observed with hydrolysis probe based quantitative PCR can be corrected with the estimated PCR efficiency value

For real-time monitoring of PCR amplification of DNA, quantitative PCR (qPCR) assays use various fluorescent reporters. DNA binding molecules and hybridization reporters (primers and probes) only fluoresce when bound to DNA and result in the non-cumulative increase in observed fluorescence. Hydrolysis reporters (TaqMan® probes and QZyme™ primers) become fluorescent during DNA elongation and the released fluorophore remains fluorescent during further cycles; this results in a cumulative increase in observed fluorescence. Although the quantification threshold is reached at a lower number of cycles when fluorescence accumulates, in qPCR analysis no distinction is made between the two types of data sets. Mathematical modeling shows that ignoring the cumulative nature of the data leaves the estimated PCR efficiency practically unaffected but will lead to at least one cycle underestimation of the quantification cycle ( C q value), corresponding to a 2-fold overestimation of target quantity. The effect on the target–reference ratio depends on the PCR efficiency of the target and reference amplicons. The leftward shift of the C q value is dependent on the PCR efficiency and with sufficiently large C q values, this shift is constant. This allows the C q to be corrected and unbiased target quantities to be obtained.

Permanent sample plots for natural tropical forests: A rationale with special emphasis on Central Africa

Permanent sample plots (PSP), where trees are individually and permanently marked, have received increased interest in Central Africa as a tool to monitor vegetation changes. Although techniques for mounting PSP in tropical forests are well known, their planning still deserves attention. This study aims at defining a rationale for determining the size and number of replicates for setting up PSP in mixed tropical forests. It considers PSP as a sampling plan to estimate a target quantity with its associated margin of error. The target quantity considered here is the stock recovery rate, which is a key parameter for forest management in Central Africa. It is computed separately for each commercial species. The number of trees to monitor for each species defines the margin of error on the stock recovery rate. The size and number of replicated plots is obtained as the solution of an optimization problem that consists in minimizing the margin of error for every species while ensuring that the mounting cost remains below a given threshold. This rationale was applied using the data from the M'Baïki experimental site in the Central African Republic. It showed that the stock recovery rate is a highly variable quantity, and that the typical cost that forest managers are prone to devote to PSP leads to high margins of error. It also showed that the size and number of replicated plots is related to the spatial pattern of trees: clustered or spatially heterogeneous patterns favor many small plots, whereas regular or spatially homogeneous patterns favor few large plots.

Real-time PCR microfluidic devices with concurrent electrochemical detection

Electrochemistry-based detection methods hold great potential towards development of hand-held nucleic-acid analyses instruments. In this work, we demonstrate the implementation of in situ electrochemical (EC) detection method in a microfluidic flow-through EC-qPCR (FTEC-qPCR) device, where both the amplification of the target nucleic-acid sequence and subsequent EC detection of the PCR amplicon are realized simultaneously at selected PCR cycles in the same device. The FTEC-qPCR device utilizes methylene blue (MB), an electroactive DNA intercalator, for electrochemical signal measurements in the presence of PCR reagent components. Our EC detection method is advantageous, when compared to other existing EC methods for PCR amplicon analysis, since FTEC-qPCR does not require probe-modified electrodes, or asymmetric PCR, or solid-phase PCR. Key technical issues related to surface passivation, electrochemical measurement, PCR inhibition by metal electrode, bubble-free PCR, were investigated. By controlling the concentration of MB and the exposure of PCR mixture to the bare metal electrode, we successfully demonstrated electrochemical measurement of MB in solution-phase, symmetric PCR by amplifying a fragment of lambda phage DNA. The threshold cycle (Ct) values for both the electrochemical and fluorescence-based assays decreased linearly with the increase of the input target quantity. The sensitivity of EC-based detection of PCR products is comparable to the sensitivity of an optical fluorescence detection system.

Statistical Grounds for Favoring the Characteristic Magnitude Model: A Case Study

Abstract In the frame of probabilistic seismic hazard analysis, the characteristic magnitude model is often favored on the basis of general geological, physical, and mechanical considerations. However, it is generally recognized that the seismic record in most seismic zones is too short for a statistical validation of the characteristic hypothesis: different and simpler models can explain available catalogs as successfully as a characteristic magnitude model, even if they lead (all other things being equal) to important differences in the final results of the seismic hazard analysis. In other words, when two competing magnitude models are proposed, it is difficult to justify (on the basis of statistical tests) favoring one or the other model for the interpretation of the available catalog. We observe that most of the problems of seismic hazard analysis actually raise the question: which one of the alternative models is more reliable for the estimation of a specific hazard quantity at a given site? In this article, we describe a method that can give a statistically based answer to this question. The answer depends, obviously, on the mathematical structure of the two models, as well as on the nature of the site and on the target quantity. Changing the site or the target quantity leads in general to different outcomes of the comparison between the models. What is interesting to observe is that, with the proposed method, given the site and the target quantity, the comparison is independent of the magnitude values contained in the available catalog. We describe in detail the comparison between a characteristic magnitude model and the doubly truncated exponential model, alternatively applied to the evaluation of the peak ground acceleration (PGA) with a 500-yr return period at a specific site. We show that, for the two test sites considered in our study, a set of appropriate statistical tests clearly indicate the characteristic model as more reliable than the exponential model. A simple nonparametric procedure (free from modeling errors) is also presented and compared with the mathematical models.

A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR

BackgroundBased upon defining a common reference point, current real-time quantitative PCR technologies compare relative differences in amplification profile position. As such, absolute quantification requires construction of target-specific standard curves that are highly resource intensive and prone to introducing quantitative errors. Sigmoidal modeling using nonlinear regression has previously demonstrated that absolute quantification can be accomplished without standard curves; however, quantitative errors caused by distortions within the plateau phase have impeded effective implementation of this alternative approach.ResultsRecognition that amplification rate is linearly correlated to amplicon quantity led to the derivation of two sigmoid functions that allow target quantification via linear regression analysis. In addition to circumventing quantitative errors produced by plateau distortions, this approach allows the amplification efficiency within individual amplification reactions to be determined. Absolute quantification is accomplished by first converting individual fluorescence readings into target quantity expressed in fluorescence units, followed by conversion into the number of target molecules via optical calibration. Founded upon expressing reaction fluorescence in relation to amplicon DNA mass, a seminal element of this study was to implement optical calibration using lambda gDNA as a universal quantitative standard. Not only does this eliminate the need to prepare target-specific quantitative standards, it relegates establishment of quantitative scale to a single, highly defined entity. The quantitative competency of this approach was assessed by exploiting "limiting dilution assay" for absolute quantification, which provided an independent gold standard from which to verify quantitative accuracy. This yielded substantive corroborating evidence that absolute accuracies of ± 25% can be routinely achieved. Comparison with the LinReg and Miner automated qPCR data processing packages further demonstrated the superior performance of this kinetic-based methodology.ConclusionCalled "linear regression of efficiency" or LRE, this novel kinetic approach confers the ability to conduct high-capacity absolute quantification with unprecedented quality control capabilities. The computational simplicity and recursive nature of LRE quantification also makes it amenable to software implementation, as demonstrated by a prototypic Java program that automates data analysis. This in turn introduces the prospect of conducting absolute quantification with little additional effort beyond that required for the preparation of the amplification reactions.

Gene Expression of HIF-1α and XRCC4 Measured in Human Samples by Real-Time RT-PCR Using the Sigmoidal Curve-Fitting Method

Quantitative reverse transcription PCR (RT-PCR) has become an important tool for studying functional gene expression. However the most often used cycle threshold (CT)-based method, primarily related to the required amplification efficiency determination via serial dilution, can call into question the level of quantitative reliability and accuracy that can be achieved, in addition to the impracticalities inherent to CT-based methodologies. In this study, an alternative method, named the sigmoidal curve-fitting (SCF) method, was compared with the classic CT method for two target genes (XRCC4 and HIF-1alpha) and a reference gene (HPRT). The PCR conditions were optimized for each gene on a LightCycler apparatus. Fluorescence data were fitted to a four-parametric sigmoidal function, and the initial messenger RNA (mRNA) copy number was determined by a theoretical fluorescence (F0) value calculated from each fitting curve. The relative expression of the target gene versus that of the reference gene was calculated using an equation based upon these F0 values. The results show that the F0 value had a good linearity with the initial number of target genes between 10(7) and 10(1) copies. The reproducibility tests showed that the variations of initial target quantity were well reflected by F0 values. Relative expression of target gene calculated by the SCF method and by the CT method showed similar results. In our hands, the SCF method gave reliable results and a more precise error description of quantitative RT-PCR.

P-422: Granulosa cell gene expression of IGF-II and IGFBP-3 in PCOS patients as a predictor of IVF outcome

IGF-II is the predominant intraovarian IGF and its synthesis is hormonally-sensitive (Proc Nat Acad Sci, 1987). IGFBP-3 is the primary intraovarian binding protein and is present in dominant follicles (JCEM 1993,1994). Our objective was to examine the association between granulosa cell gene expression of IGF-II and IGFBP-3 and oocyte maturity in a cohort of PCOS patients undergoing IVF. Analysis of granulosa cell mRNA from PCOS and male factor infertility (control) patients undergoing IVF/ICSI with OCP/GnRH agonist dual suppression followed by low dose stimulation (<2amps/day). Nine PCOS patients and 9 normally ovulating controls were recruited with IRB approval. Follicular fluid aspirates were obtained following removal of oocyte-cumulus complexes. Samples were centrifuged, and granulosa cells were prepared and isolated with 50% Percoll. For each patient, half of the cells were cultured for 5 days in uniform conditions (1:1 DMEM:F12 with 10% fetal calf serum and 0.1% Gentamicin)(“cultured”), and half of the cells underwent immediate RNA extraction (“fresh”). RNA extraction was performed with Trizol reagent. Following cDNA synthesis, real-time quantitative PCR was performed with IGF-II and IGFBP-3 primers and probes (Applied Biosystems). Samples were run in duplicate. For PCR we report Ct values which decrease linearly with increasing target quantity and reflect input sample concentration. PCOS patients had a higher mean BMI, but stimulation parameters and outcome were comparable (Table 1). Mean expression of IGF-II and IGFBP-3 was comparable between PCOS and control patients (Figure 1). Adjustment for BMI did not affect this finding. When individual patients’ cultured and fresh cells’ gene expression was compared, cultured cells demonstrated significantly increased expression. Ct levels of IGF-II for freshly cultured cells correlated negatively with peak E2 levels in controls (R= -0.99, p=0.05) and PCOS (R= -0.84, p=0.07), suggesting that higher granulosa cell IGF-II production correlated with higher peak E2 levels. IGF-II and IGFBP-3 expression did not correlate with number of oocytes retrieved, oocyte maturity, or ampules of gonadotropins. Gene expression did not differ between pregnant and nonpregnant patients.Tabled 1View Large Image Figure ViewerDownload (PPT) Although no significant differences were found between PCOS and controls’ expression of IGF-I and IGFBP-3, real-time quantitative PCR offers the potential to detect differences between PCOS and control granulosa cells both in vivo and in vitro. Study of a greater number of patients will hopefully allow further insight into the PCOS granulosa cell gene expression.

Carboxymethylcellulose as a new carrier substance for intravitreal injection of reproducible amounts of triamcinolone

Intravitreal application of triamcinolone acetonide has become increasingly popular for the treatment of various retinal disorders. However, dosage, mode of preparation and application differ worldwide. The aim of this study was to find a safe vehicle that would allow intravitreal injection of an exact amount of triamcinolone acetonide without potentially retinotoxic preservatives. Solutions of triamcinolone acetonide with a theoretical concentration of 4 mg/0.2 ml were prepared following one sedimentation (A) and two filtration (B, C) methods. In addition, a filtration method using carboxymethylcellulose 2% as a carrier (D) was established. During processing and after injection into an eye model, the crystals were quantified by weight and high-performance liquid chromatography (HPLC), and, hence, the rate of crystal loss during this process was determined. The initial preparation contained 93-106% of the calculated quantity. Method A, containing the entire vehicle, delivered 45%+/-7.3% of the target quantity to the eye model, whereas the vehicle-free methods B and C delivered 15%+/-6.9% and 11%+/-3.2%, respectively. Using carboxymethylcellulose 2% as a preservative-free vehicle, we found 93%+/-3.7% of the calculated amount in the eye model. The missing crystals were mainly sticking to the walls of the syringes and needles used for transfer. Common methods for preparing triamcinolone acetonide vary in the amount of drug actually injected intravitreally. Carboxymethylcellulose is an ideal carrier substance for intravitreal application of an exact dose of triamcinolone acetonide without preservatives.

Is supplementation with elemental diet feasible in patients undergoing pelvic radiotherapy?

Acute gastrointestinal upset occurs in approximately 80% of patients undergoing radiotherapy for pelvic cancers. Underlying changes relate to denudation of the mucosal layer which renders the small intestine vulnerable to additional damage from proteolytic enzymes and bile acids. Severe acute bowel symptoms may predispose to progressive fibrotic and ischaemic changes. Elemental diet given during treatment may reduce acute and chronic bowel symptoms induced by pelvic radiotherapy. This study aimed to assess compliance with elemental diet during pelvic radiotherapy. Patients with gynaecological, urological or rectal malignancy undergoing radical or adjuvant pelvic radiotherapy were randomised to one of five groups. Each group was assigned a target quantity of three different elemental sip feeds (Group 1, 20%, Group 2, 50%, Group 3, 75% of calorie requirements taken as E028 extra liquid; Group 4, 50% of calorie requirements taken as E028 extra powder; Group 5, 50% of calorie requirements taken as Emsogen powder). Compliance was assessed using a diary card and weekly assessment. Statistical analyses were conducted on an intention to treat basis. Fifty patients (44 female, six male) mean age 58 yr (95% CI 55-61), were recruited to the study (24 endometrial, 17 cervical, 7 rectal, 1 vulval and 1 bladder carcinoma). Mean weight at baseline was 74.5 kg (95% CI 69-80) and this did not change during treatment. Full compliance was achieved in three patients, all from Group 1. By week 5, only 46% of patients were consuming elemental diet. Post hoc analysis of variance with repeated measures indicated that there were no significant differences in volume of elemental diet consumed between groups (P = 0.937). There was a significant inverse linear relationship between intake of elemental diet and time (P < 0.001). Different formulations of elemental diet do not influence compliance. Patients are unlikely to be able to consume more than one-third of their calorie requirements in the form of an elemental sip feed. Further investigation is warranted to determine if intervention with this volume of elemental diet is beneficial.

Using Real-Time PCR to Study Hormonal Regulation of Implantation-Related Gene Expression

Endometrial cytokines and adhesion molecules involved in implantation may be hormonally regulated. The purpose of our study was to use real-time PCR to quantify steroid influence on endometrial gene expression. Mouse endometrial tissue was cultured with hormonal media. Gene expression patterns were studied by real time PCR. Endometrial cells were cultured from 3-week old BDF1 mice (Charles River Co.) The enzymatically digested glands and stroma (2x105 cell density) were seeded in 4 groups and incubated for 10 days with different hormonal media: Control, Estradiol (E2, 1x10-8 M), Progesterone (P, 1x10-8M) and E2+P. RNA was extracted from endometrial cells on day 10 of incubation using an Rneasy Mini Kit (Qiagen®) and quantitated. Reverse transcription was performed. Relative quantitation of mRNA was performed with the ABI Prism 7700 Sequence Detection System (Applied Biosystems®) using TaqMan real-time technology. Cytokine primers and probes were designed using Applied Biosystems inventoried probes. Quantitation was normalized to endogenous control mouse β-actin. Decidualization was confirmed by measuring decidual/trophoblastic prolactin-related protein (d/tPRP) mRNA in P and E2+P treated cells (Gynecol Endocrinol 2001;15: 426-432). Multiplex PCR was performed with 6-FAM as the reporter dye for each protein and VIC for β-actin. 30mcl of total RNA was obtained from each group: Control concentration, 240.4mcg/mL; E2, 296.4mcg/mL; P, 460.9 mcg/mL; E2+P, 432.5 mcg/mL. All samples were detected twice, and mean results are reported. The threshold cycle (Ct) was calculated by determining the point at which fluorescence emission by degradation of the hybridization probe exceeded a threshold limit (Genome Research 1996;6: 986-994). Ct values decrease linearly with increasing target quantity and reflect input sample concentration. The Ct for blank sample cycles was 40, and protein mRNA levels were stratified by hormonal treatment. Hormonal regulation of the studied genes’ expression was observed. TIMP-1 mRNA was highly expressed in all cells, consistent with the strong signal detected in cell media in our protein chip analysis (submitted ASRM 2005). Hormonal influence on the expression of IGFBP-3 and -6 mRNA paralleled their protein secretion, as both were significantly upregulated by P treatment (mRNA, p<.001). We found significantly increased TIMP-1 mRNA expression in P treated cells (p<.001), contrasting with the finding of relatively decreased secretion of TIMP-1 by P cells compared to control. We also found significantly increased expression of VCAM-1 mRNA in E2 treated cells (p<.001), whereas secretion of VCAM-1 by all cell groups with hormonal treatment was relatively lower than that of control cells. These discrepancies between mRNA expression and hormonal secretion of TIMP-1 and VCAM-1 suggest that they may have post-transcription control mechanisms. The utilization of quantitative PCR to study hormonal influence on gene expression in cultured endometrial cells shows promise as a method to better understand the regulation of genes that prepare the endometrium for implantation.

EVALUATION AND SIGNIFICANCE OF TETRACYCLINE STABILITY IN RABIES VACCINE BAITS

Tetracycline is widely used as a biomarker for bait consumption by wildlife; tetracycline is incorporated into bones and teeth and can be detected by fluorescence microscopy several weeks postconsumption. During 2003, the United States Department of Agriculture distributed more than 10 million tetracycline-containing rabies-vaccine baits to control the spread of wildlife vectored rabies to humans, pets, and livestock. To estimate the percentage of target species consuming the baits, raccoons and skunks were collected in baited areas and teeth were analyzed for the presence of the biomarker. Several incidents of low biomarker detection rates prompted an investigation of the stability of the biomarker in the baits. Baits were collected at several points along the manufacturing and distribution chain. Baits were analyzed for free and polymer-bound tetracycline and the less active isomer epitetracycline. Results indicated that a portion of the tetracycline was converted to epitetracycline. Additionally, significant quantities of both compounds were trapped in the polymer, which is homogeneously distributed throughout the bait. The results of this study suggest that approximately 40% of the target quantity of tetracycline was unavailable for absorption. This situation could contribute to low biomarker detection rates and suggests that formulation modification should be considered.

Surface-Integral QSPR Models: Local Energy Properties

Surface-integral models based on AM1 semiempirical molecular orbital calculations are presented for the free energies of solvation in water, n-octanol, and chloroform and for the enthalpy of solvation in water. A parametrized function of four local properties calculated at the isodensity surface (the molecular electrostatic potential, local ionization energy, electron affinity, and polarizability) is integrated over the triangulated surface area to obtain the target quantity. The resulting models give results only slightly less accurate than those reported for parametrized generalized Born/polar surface area models despite relying only on gas-phase calculations. The water and octanol free-energy models were validated by calculating the water-octanol partition coefficient for a test set of organic compounds with moderate success. The models lead to a local solvation energy, which can be projected onto the molecular isodensity surface and provides insight into "hot" areas for solvation in water or the other solvents.