Endpoint Determination Research Articles

Present Status and Problems on Molecular Targeted Therapy of Cancer

Numerous clinical trials of molecular targeted drugs for cancer have been conducted, with remarkable results for certain drugs and accumulation of "negative data" causing a hitch in the development plan for some other compounds. Five recent issues and problems of molecular targeted therapies were discussed critically. Drug discovery and effects against driver mutations (activating mutations) and problems: possibility for circumventing inherent and acquired resistance with the aim of achieving radical cure. Synthetic lethality: reasonable patient selection in individualized treatment strategy. Response rate and progression-free survival improvement with or without overall survival benefit and enhancement of toxicity in bevacizumab therapy: best endpoints for the evaluation of effect of antiangiogenic therapy. Negative data on small-molecule targeted therapy, primarily vascular endothelial growth factor tyrosine kinase inhibitors: loose GO or NO-GO decision criteria for further development of new compounds in early clinical trials. Effect of immunotherapy: difficulty to verify by proof of principle study. We are faced to many questions for the development of efficient personalized therapy. Accumulation of scientific global preclinical and clinical evidences is essential to use these new therapeutic modalities for the improvement of oncologic health care.

Robust Real-Time-Constrained Estimation of Respiratory Motion for Interventional MRI on Mobile Organs

Real-time magnetic resonance imaging is a promising tool for image-guided interventions. For applications such as thermotherapy on moving organs, a precise image-based compensation of motion is required in real time to allow quantitative analysis, retrocontrol of the interventional device, or determination of the therapy endpoint. Reduced field-of-view imaging represents a promising way to improve spatial and/or temporal resolution. However, it introduces new challenges for target motion estimation, since structures near the target may appear transiently due to the respiratory motion and the limited spatial coverage. In this paper, a new image-based motion estimation method is proposed combining a global motion estimation with a novel optical flow approach extending the initial Horn and Schunck (H&S) method by an additional regularization term. This term integrates the displacement of physiological landmarks into the variational formulation of the optical flow problem. This allowed for a better control of the optical flow in presence of transient structures. The method was compared to the same registration pipeline employing the H&S approach on a synthetic dataset and in vivo image sequences. Compared to the H&S approach, a significant improvement (p<0.05) of the Dice's similarity criterion computed between the reference and the registered organ positions was achieved.

Two-Phase Titration Method for Cationic Surface Active Agents Determination with Use of 5-(3-Bromo-4-hydroxy-5-methoxyphenyl)-7,7-dimethyl-7H-indolo[1,2-a]quinolinium Perchlorate Dye

Abstract The direct two-phase titration method with use of solvatochromic dye, i. e. 5-(3-bromo-4-hydroxy-5-methoxyphenyl)-7,7-dimethyl-7H-indolo[1,2-a]quinolinium perchlorate (1) for cationic surfactants determination is presented which is the modification of an earlier described procedure for anionic surface active substances detection. The method was tested on standard solution of benzethonium chloride (cationic detergent) which enables to estimate its accuracy as well as on few commercially available products to verify its utility. The influence of pH value of the probe on the titration endpoint determination was discussed and a simple solution of this problem has been proposed.

Critical evaluation of methods for end-point determination in pharmaceutical blending processes

This paper compares existing approaches to the monitoring of pharmaceutical blending processes and the detection of their end-point by use of near infrared spectroscopy (NIRS). To this end, we examined homogenization in a pharmaceutical mixture containing a relatively low concentration (<4%) of an API that exhibits low absorption in the NIR region and is difficult to blend with excipients. The end-point of the process (viz. where a uniform distribution of the API in the mixture was reached) was determined by using various qualitative and quantitative spectral treatments for comparison. The results revealed the capabilities of different spectral treatments for establishing blend homogeneity, which can be especially useful with a view to optimizing their industrial use. API distribution uniformity in the final blend was confirmed by near infrared chemical imaging (NIR-CI) analysis.

근적외분광분석법과 라만분광분석법을 이용한 트리메부틴말레인산 서방정의 혼합 과정 모니터링

본 연구에서는 위장관에 작용하는 트리메부틴말레인산염을 주성분으로 하는 서방정의 제조 과정 중 혼합과정의 혼합 진행 정도의 확인에 근적외분광분석법과 라만분광분석법을 적용하였다. 서방정의 제조의 혼합 과정에서 소량의 부형제로 콜로이달실리콘디옥사이드, 탈크, 스테아르산, 스페타르산마그네슘을 넣어 혼합하는 과정에서, 각 혼합 시간 별, 혼합기 내에서 구역 별 혼합물 시료를 채취한 후 주성분(active ingredient)인 트리메부틴말레인산염의 양을 HPLC로 분석하였고, 동시에 채취한 시료의 근적외스펙트럼과 라만스펙트럼을 측정하여 PCA (Principal Component Analysis)를 수행하였다. 혼합기로는 U자형 혼합기를 사용하였고, 상, 중, 하와 좌, 우 각각 6개의 영역에서 시료를 채취하여 주성분과 부형제의 분포에 따른 혼합물의 균일도를 확인하고자 하였다. HPLC법으로는 소량의 부형제의 혼합을 주성분을 분석하는 것으로 시간에 따른 혼합도를 확인할 수 없는 반면, 근적외스펙트럼과 라만 스펙트럼에서는 주성분과 각각의 부형제의 특징적인 피크들을 확인할 수 있었고, 이러한 특징적인 흡광도를 가진 영역을 이용하여 전체 혼합물에서의 부형제 영향에 의한 변화를 PCA를 수행하여 혼합의 진행 정도를 성공적으로 확인하였다. 산란의 영향에 의한 바탕선의 변이를 제거하기 위해 전처리 방법으로는 미분을 사용하였고, 근적외스펙트럼에서는 5000-7500 <TEX>$cm^{-1}$</TEX>를 사용하였고, 라만스펙트럼에서는 1000-1500 <TEX>$cm^{-1}$</TEX>를 이용하여, PCA를 수행하였을 때 효과적으로 혼합의 진행정도를 확인할 수 있었다. Chemometrics using near-infrared (NIR) and Raman spectroscopy have found significant uses in a variety quantitative and qualitative analyses of pharmaceutical products in complex matrixes. Most of the pharmaceutical can be measured directly with little or no sample preparation using these spectroscopic methods. During pharmaceutical manufacturing process, analytical techniques with no or less sample preparation are very critical to confirm the quality. This study showed NIR and Raman spectroscopy with principal component analysis (PCA) was very effective for the blending processing control. It is of utmost importance to evaluate critical parameters related to quality of products during pharmaceutical processing. The blending is confirmed by off-line determination of active pharmaceutical ingredient (API) by a conventional method such as high performance liquid chromatography (HPLC) and UV spectroscopy. These analytical methods are time-consuming and ineffective for real time control. This study showed the possibility for the determination of blend uniformity end-point of CR tablets with the use of both NIR and Raman spectroscopy. The samples were acquired from six positions during blending processing with U-type blender from 0 to 30 min. Using both collected NIR and Raman spectral data, principal component analysis (PCA) was used to follow the uniformity of blending and finally determine the end-point. The variation of homogeneity of six samples during blending was clearly found and blend uniformity end-point was successfully confirmed in the domains of principal component (PC) scores.

To determine the end point of wet granulation by measuring powder energies and thermal properties

Wet granulation has been widely used in pharmaceutical industry as a tablet manufacturing process. However, end-point determination of wet granulation process has always remained a challenge. Many traditional methods are available for end-point determination, yet accuracy and reproducibility still remain a challenge. Microcrystalline cellulose, widely used as an excipient in pharmaceutical industry, was granulated using water. Wet mass was passed through sieve # 12 and dried till constant percentage loss on drying was obtained and dried granules were obtained. Wet and dried granules collected were subjected to basic flow energy, specific energy, bulk density, pressure drop, differential scanning calorimetry and effusivity measurements. Analysis of data revealed various stages of granule growth from initial seed formation by adding 200–400 g of water, granule growth was observed by adding 600–800 g of water and over wetting was observed at 1155 g of water. In this work, we have justified our work to properly identify and utilize this technique for practical purpose to correctly identify the end-point determination of microcrystalline cellulose and explain various principles underlying energies associated with powder and thermal measurements.

A novel microplate‐based HPLC‐fluorescence assay for determination of NADPH‐cytochrome P450 reductase activity

A 96-well microplate-based HPLC endpoint assay is described for the determination of NADPH-cytochrome P450 reductase (CPR) activity. Novel sampling of NADPH into microplates was optimized. Separation was performed on a Zorbax Eclipse XDB-C₁₈ analytical 4.6 × 150 mm, 5 µm column. To validate the method, recombinant human NADPH-P450 reductase and microsomes with cytochrome P450 CYP1A1 were used. The mobile phase consisted of 80% acetonitrile and 20% water at a flow-rate of 0.8 mL/min. The CPR activity was quantified using NADPH fluorescence at λ(Ex) = 340 nm and λ(Em) = 450 nm. Enzymatic activity was directly proportional to the decrease in NADPH fluorescence. This analytical process enables a highly sensitive endpoint determination for reductase activity in vitro and monitoring of the consumption of NADPH in enzymatic reactions. The method avoids the use of substrates and of organic solvents that may affect CPR and cytochrome P450 activity. In the reaction, molecular oxygen served as a proton source. The method can substitute spectrophotometric detection methods for its accuracy, high reproducibility (~100%) and sensitivity. The lower limit of detection, shown using the Agilent 1200 aparatus, is in the 250 nmol range. In addition, using this method it is possible to set up reactions in a high-throughput format.

Findings of Feasibility Assessment From Multicenter Clinical Trials of Antithrombotic Therapy in Pediatric Venous Thromboembolism

Abstract 4323BACKGROUND:Two major multicenter pediatric randomized controlled clinical trials (RCTs) in pediatric venous thromboembolism (VTE) have been conducted in the last decade, and each was closed early due to poor accrual. With the recent trend in reduced federal grant support for clinical trials, an emphasis on demonstration of feasibility is critical.OBJECTIVE: In order to inform the design and execution of future RCTs in pediatric VTE, we sought to report findings of feasibility assessment from two ongoing and one proposed clinical trials in the field. METHODS: In the UNBLOCK study (ongoing phase 1 trial of bivalirudin in acute DVT; clinicaltrials.gov registration #NCT00812370), feasibility was assessed informally in 2008/9 during grant development teleconferences and early investigator meetings. In the Kids-DOTT trial (ongoing phase 3 RCT of shortened vs. conventional duration of therapy in acute VTE associated with a transient/reversible clinical risk factor; NCT00687882), feasibility was assessed in 2005 and updated in 2011 using standardized questionnaires, with response data summarized via descriptive statistics. The latter approached was also used in 2011 for the PHLO trial (proposed phase II/III RCT of catheter-directed thrombolysis followed by therapeutic anticoagulation vs. anticoagulation alone in completely veno-occlusive acute DVT of the proximal leg). RESULTS: Response rate (Kids-DOTT and PHLO questionnaires) was 100%. Number of investigative sites/principal investigators assessed and geographical distribution, by trial, were as follows: UNBLOCK: n=6 (all USA); Kids-DOTT: n=26 (25 USA, 1 Canada); PHLO: n=7 (all USA). Of the 26 site PIs in Kids-DOTT, 84% reported past experience with 5 or more trials, with a median experience of 20 per PI. Across all 3 trial feasibility assessments, all but one site used electronic health records in clinical care, and all but one investigative team had trial experience in using electronic data capture systems. With regard to size of the screening population for pediatric VTE trials, Figure 1 shows the reported average annual volume of new pediatric VTE cases managed by each Kids-DOTT investigator/site, using data from the prior 2 years. All sites reported use of inpatient/consult lists in screening efforts. CONCLUSIONS: These findings indicate appropriate site experience and capacity to execute multicenter clinical trials in pediatric VTE. Therefore, feasibility of ongoing and future trials in the field is more likely related to such issues as: suitability of funding and number of sites to targeted enrollment; experience/capabilities of the clinical coordinating and data coordinating centers; and pragmatic protocol design, particularly eligibility criteria, enrollment/randomization windows, and frequency, timing, and invasiveness of follow-up and endpoint determinations. Disclosures:No relevant conflicts of interest to declare. [Display omitted]

Phenotype and Genotype of 22 Subjects with Heritable Dysfibrinogenaemia

Abstract 1212▪▪This icon denotes a clinically relevant abstractThe heritable dysfibrinogenaemias (HD) are a heterogenous group of qualitative disorders of fibrinogen in which there is abnormal fibrin clot formation. Affected individuals usually display prolonged thrombin (TCT) and reptilase clotting times (RCT) and reduced plasma fibrinogen activity. The plasma fibrinogen concentration is normal or is reduced less than the fibrinogen activity. HD is associated with pathogenic mutations in FGA, FGB or FGG genes that encode the fibrinogen Aα, Bβ and g chains respectively. However, most clinical descriptions of HD comprise single case reports and the range of phenotype and genotype in the wider population is difficult to estimate. In order to improve understanding of this disorder, we now report a cross-sectional survey of 22 subjects with HD identified at 2 UK regional Haemostasis centres from 2001–11.We recruited index cases from our Haemostasis laboratory referral practice who satisfied all the following criteria- 1) prolonged TCT; 2) prolonged TCT with protamine (TCTP) and/or prolonged RCT; 3) reduced Clauss fibrinogen and 4) normal fibrinogen dry clot weight. Demographic and clinical characteristics were recorded using a standardised case report form. Genetic analysis was performed by Sanger sequencing of FGA exon 2, FGB exon 2 and FGG exon 8 which we identified as mutation hotspots from the fibrinogen mutation database (http://www.geht.org/databaseang/fibrinogen/). Phenotype and genotype was also determined in available first degree family members.We identified 16 index cases and 6 family members with HD (median age 41, range 12–78; 9 males). HD was identified in plasma submitted to our Haemostasis laboratories for the investigation of bleeding in 8/16 (50%) index cases, for stroke in 1/16 (6%) and for ‘routine coagulation screening' in 7/16 (44%). Mild mucocutaneous bleeding or menorrhagia was documented in the clinical records of 13/22 (59%) subjects and thrombotic stroke in 1/22 (5%). 8/22 (36%) subjects were asymptomatic.The prothrombin time (PT) was prolonged in 21/22 (95%) subjects (median 15.3 s; range 11.4–16.7; reference interval (RI) 9.6–11.6) but the activated partial thromboplastin time (aPTT) was prolonged in only 1/22 (5%; median 23.8 s; range 22.8–37.0, RI 24.0–32.0). The median TCT was 28.6 s (range 20.6–45.7; RI 13–19) and TCTP 31.9 s (n=11; range 20.0–35.5; RI 13–19). The median RCT was 37s (n=12; range 19–68; RI 15–19), Clauss fibrinogen 0.49 g/dL (range 0.29–1.3; RI 1.8–3.6) and dry clot weight 2.3 g/dL (range 1.9–4.2; RI 1.8–3.6). Heterozygous missense nucleotide variations were identified in the candidate exons of the fibrinogen genes in all 16/16 (100%) index cases and segregated with the HD phenotype in all available family members. These variations predicted aminoacid substitutions at the fibrinopeptide A cleavage site in the fibrinogen Aa chain in 10/16 (63%) index cases (FGA p.R35H (n=4); p.R35S (n=3); p.R35P (n=2) and p.G36S (n=1); numbered from RefSeq NP_000499.1) or at nearby residues (FGA p.V39D (n=1) and p.F27C (n=1)). FGG variations were identified in 3 index cases that predicted aminoacid substitutions in the fibrinogen g chain region necessary for D:D domain interactions during fibrin monomer assembly (FGG p.R301H (n=1), p.D342G (n=1) and p.A367T (n=1); RefSeq NP_000499.1). One subject harboured a FGB variation that predicted a p.R44C substitution (Refseq NP_005132.2) at the fibrinopeptide B cleavage site in the fibrinogen Bb chain. The FGA p.R35S, p.R35H, FGB p.R44C and FGG p.R301H and p.A367T substitutions have been associated previously with HD.We show that the HD laboratory phenotype is rare and was frequently identified in subjects with mild bleeding. However, HD was also commonly asymptomatic and did not segregate with bleeding symptoms within affected families suggesting that the HD variants identified in this study do not confer significant bleeding risk. The prevalence of mild bleeding in some study subjects may reflect recruitment bias. We also show that HD is strongly associated with nucleotide variations in the FGA, FGB or FGG genes. All the observed nucleotide variations have been previously associated with HD and/or predict aminoacid substitutions in critical regions for normal fibrin polymerization. Defective fibrin polymerization may affect endpoint determination in some automated clotting assays and is likely to account for the consistent finding of prolonged PT in the study subjects. Disclosures:No relevant conflicts of interest to declare.

A spectrophotometric assay for the detection of fungal peroxygenases

Rapid and simple spectrophotometric methods are required for the unambiguous detection of recently discovered fungal peroxygenases in vivo and in vitro. This paper describes a peroxygenase-specific assay using 5-nitro-1,3-benzodioxole as substrate. The product, 4-nitrocatechol, produces a yellow color at pH 7, which can be followed over time at 425 nm ( ε 425 = 9,700 M −1 cm −1), and a red color when adjusted to pH >12, which can be measured in form of an end-point determination at 514 nm ( ε 514 = 11,400 M −1 cm −1). The assay is suitable for detecting peroxygenase activities in complex growth media and environmental samples as well as for high-throughput screenings.

A Bayesian approach to integrate temporal data into probabilistic risk analysis of monitored NAPL remediation

Upon their release into the subsurface, non-aqueous phase liquids (NAPLs) dissolve slowly in groundwater and/or volatilize in the vadose zone threatening the environment and public health over extended periods of time. The failure of a treatment technology at any given site is often due to the unnoticed presence of dissolved NAPL trapped in low permeability areas and/or the remaining presence of substantial amounts of pure phase NAPL after remediation efforts. The design of remediation strategies and the determination of remediation endpoints are traditionally carried out without quantifying risks associated with the failure of such efforts. We conduct a probabilistic risk analysis (PRA) to estimate the likelihood of failure of an on-site NAPL treatment technology. The PRA integrates all aspects of the problem (causes, pathways, and receptors) without resorting to extensive modeling. It accounts for a combination of multiple mechanisms of failure of a monitoring system, such as bypassing, insufficient sampling frequency and malfunctioning of the observation wells. We use a Bayesian framework to update the likelihood of failure of the treatment technology with observed measurements of concentrations at nearby monitoring wells.

Autonomous subpixel satellite track end point determination for space-based images

An algorithm for determining satellite track end points with subpixel resolution in spaced-based images is presented. The algorithm allows for significant curvature in the imaged track due to rotation of the spacecraft capturing the image. The motivation behind the subpixel end point determination is first presented, followed by a description of the methodology used. Results from running the algorithm on real ground-based and simulated spaced-based images are shown to highlight its effectiveness.

Treatment end point determinants for pulmonary tuberculosis: Human resistin as a surrogate biomarker

Treatment of tuberculosis (TB), which takes one human life every 15s, globally, requires a prolonged (>6 months) antitubercular treatment (ATT) which, is known to have hepatotoxic side effects. This study was designed to explore the utility of human resistin, a proinflammatory hormone, as a sensitive biomarker to determine TB treatment end points. Patients for pulmonary tuberculosis enrolled under the directly observed treatment, short-course (DOTS) program were followed-up for six months and were monitored by sputum analysis, body weight and ELISA-based serum resistin and C-reactive protein (CRP) levels at 0, 2, 4 and 6 months, along with close family contacts of TB patients and healthy controls. The mean circulating resistin levels were found to be significantly higher (P<0.001) in patients (n=48, 25.74±9.45ng/ml) reporting for the first time for treatment (T0) as compared to healthy subjects (n=45, 7.18±2.40ng/ml). Resistin levels in contacts (n=48, 19.61±7.88ng/ml) also were found to be significantly (P<0.001) elevated as compared to healthy controls. Significant increase in body weight after four months (P=0.006) and at 6 months (P<0.001) of treatment inversely correlated with resistin levels. Our data suggest resistin could be a surrogate marker for TB treatment in addition to its utility as an early prognostic biomarker for monitoring TB disease onset.

Procalcitonin for Triage of Patients with Respiratory Tract Symptoms: A Case Study in the Trial Design Process for Approval of a New Diagnostic Test for Lower Respiratory Tract Infections

Symptoms of cough, fever, chest pain, and shortness of breath are common reasons that patients seek medical care, and they can be due to a variety of medical conditions, including lower respiratory tract infection (LRTI). Only a small proportion of these patients will actually have a bacterial etiology, but many will receive antibiotic treatment because physicians cannot readily determine the etiology at the time of presentation. Current diagnostic methodologies are not sensitive or specific enough to reliably distinguish bacterial from viral or noninfectious etiologies. Procalcitonin (PCT) is a marker of host response. PCT serum levels are elevated in patients with bacterial infection, compared with levels in those with viral infections or other inflammatory pulmonary conditions. Studies have suggested that the determination of PCT levels can identify a subset of patients with LRTI symptoms who can safely avoid antibiotic treatment. As with any new test, clinical trials are necessary to demonstrate the safety and efficacy of the test to obtain U.S. Food and Drug Administration clearance. However, in the absence of standard reference methods for comparison that are reliably sensitive and specific, meeting the regulatory requirements for proof of safety and efficacy is a major challenge. Additional challenges include the choice of study design, the definition and determination of end points, and the justification of statistical analysis.

5-(3-Bromo-4-hydroxy-5-methoxyphenyl)-7,7-dimethyl-7H-indolo[1,2-a]quinolinium Perchlorate as a New Indicator for Anionic Surface Active Agents Determination

Abstract The work refers to the already published modification of widely used method for anionic active agents determination which concerns the replacement of the mixed indicator, consisting of Acid Blue 1 and carcinogenic Dimidium Bromide, applied in the International Standard ISO 2271:1989 with solvatochromic dye i.e. 5-(3-bromo-4-hydroxy-5-methoxyphenyl)-7,7-dimethyl-7H-indolo[1,2-a]quinolinium perchlorate (1). The value of pKa for examined dye (1) amounts to 6.59 thus the influence of different pH values of the probes on the analysis results was studied i.e. precision of determination of titration endpoint and the same the result of the quantitative analysis. The change of pH for aqueous and apparent pH for organic phase were determined during the titration process. It was shown that there is no effect of the pH change in its range varying from 3.5 to 5 on the results. The modification of the method for the probes which pH do not fit to the range mentioned above is presented.

Blend uniformity end-point determination using near-infrared spectroscopy and multivariate calibration

A multivariate calibration approach using near-infrared (NIR) spectroscopy for determining blend uniformity end-point of a pharmaceutical solid dosage form containing 29.4% (w/w) drug load with three major excipients (crospovidone, lactose, and microcrystalline cellulose) is presented. A set of 21 off-line, static calibration samples were used to develop a multivariate partial least-squares (PLS) calibration model for on-line predictions of the API content during the blending process. The concentrations of the API and the three major excipients were varied randomly to minimize correlations between the components. A micro-electrical-mechanical-system (MEMS) based NIR spectrometer was used for this study. To minimize spectral differences between the static and dynamic measurement modes, the acquired NIR spectra were preprocessed using standard normal variate (SNV) followed by second derivative Savitsky-Golay using 21 points. The performance of the off-line PLS calibration model were evaluated in real-time on 67 production scale (750 L bin size) blend experiments conducted over 3 years. The real-time API-NIR (%) predictions of all batches ranged from 93.7% to 104.8% with standard deviation ranging from 0.5% to 1.8%. These results showed the attainment of blend homogeneity and were confirmed with content uniformity by HPLC of respective manufactured tablets values ranging from 95.4% to 101.3% with standard deviation ranging from 0.5% to 2.1%. Furthermore, the performance of the PLS calibration model was evaluated against off-target batches manufactured with high and low amounts of water during the granulation phase of production. This approach affects the particle size and hence blending. All the off-target batches exhibited API-NIR (%) predictions of 94.6% to 103.5% with standard deviation ranging from 0.7% to 1.9%. Using off-target data, a systematic approach was developed to determine blend uniformity end-point. This was confirmed with 3 production scale batches whereby the blend uniformity end-point was determined using the PLS calibration model. Subsequently, the uniformity was also ascertained with conventional thief sampling followed by HPLC analysis and content uniformity by HPLC of the manufactured tablets.

Simultaneous determination of bisphenol A and bisphenol B in beverages and powdered infant formula by dispersive liquid–liquid micro-extraction and heart-cutting multidimensional gas chromatography-mass spectrometry

The purpose of this study was to establish a reliable, cost-effective, fast and simple method to quantify simultaneously both bisphenol A (BPA) and bisphenol B (BPB) in liquid food matrixes such as canned beverages (soft drinks and beers) and powdered infant formula using dispersive liquid–liquid micro-extraction (DLLME) with in-situ derivatisation coupled with heart-cutting gas chromatography-mass spectrometry (GC-MS). For the optimisation of the DLLME procedure different amounts of various extractive and dispersive solvents as well as different amounts of the derivative reagent were compared for their effects on extraction efficiency and yields. The optimised procedure consisted of the injection of a mixture containing tetrachloroethylene (extractant), acetonitrile (dispersant) and acetic anhydride (derivatising reagent) directly into an aliquot of beverage samples or into an aqueous extract of powdered milk samples obtained after a pretreatment of the samples. Given the compatibility of the solvents used, and the low volumes involved, the procedure was easily associated with GC-MS end-point determination, which was accomplished by means of an accurate GC dual column (heart-cutting) technique. Careful optimisation of heart-cutting GC-MS conditions, namely pressure of front and auxiliary inlets, have resulted in a good analytical performance. The linearity of the matrix-matched calibration curves was acceptable, with coefficients of determination (r2) always higher than 0.99. Average recoveries of the BPA and BPB spiked at two concentration levels into beverages and powdered infant formula ranged from 68% to 114% and the relative standard deviation (RSD) was <15%. The limits of detection (LOD) in canned beverages were 5.0 and 2.0 ng l–1 for BPA and BPB, respectively, whereas LOD in powdered infant formula were 60.0 and 30.0 ng l–1, respectively. The limits of quantification (LOQ) in canned beverages were 10.0 and 7.0 ng l–1 for BPA and BPB, respectively, whereas LOQ in powdered infant formula were 200.0 and 100.0 ng l–1, respectively. BPA was detected in 21 of 30 canned beverages (ranging from 0.03 to 4.70 µg l–1) and in two of seven powdered infant formula samples (0.23 and 0.40 µg l–1) collected in Portugal. BPB was only detected in canned beverages being positive in 15 of 30 samples analysed (ranging from 0.06 to 0.17 µg l–1). This is the first report about the presence of BPA and BPB in canned beverages and powdered infant formula in the Portuguese market.

Process Analysis of Variables for Standardization of Antifungal Susceptibility Testing of Nonfermentative Yeasts

Nonfermentative yeasts, such as Cryptococcus spp., have emerged as fungal pathogens during the last few years. However, standard methods to measure their antifungal susceptibility (antifungal susceptibility testing [AST]) are not completely reliable due to the impaired growth of these yeasts in standard media. In this work, we have compared the growth kinetics and the antifungal susceptibilities of representative species of nonfermentative yeasts such as Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus albidus, Rhodotorula spp., Yarrowia lipolytica, Geotrichum spp., and Trichosporon spp. The effect of the growth medium (RPMI medium versus yeast nitrogen base [YNB]), glucose concentration (0.2% versus 2%), nitrogen source (ammonium sulfate), temperature (30°C versus 35°C), shaking, and inoculum size (10(3), 10(4), and 10(5) cells) were analyzed. The growth rate, lag phase, and maximum optical density were obtained from each growth experiment, and after multivariate analysis, YNB-based media demonstrated a significant improvement in the growth of yeasts. Shaking, an inoculum size of 10(5) CFU/ml, and incubation at 30°C also improved the growth kinetics of organisms. Supplementation with ammonium sulfate and with 2% glucose did not have any effect on growth. We also tested the antifungal susceptibilities of all the isolates by the reference methods of the CLSI and EUCAST, the EUCAST method with shaking, YNB under static conditions, and YNB with shaking. MIC values obtained under different conditions showed high percentages of agreement and significant correlation coefficient values between them. MIC value determinations according to CLSI and EUCAST standards were rather complicated, since more than half of isolates tested showed a limited growth index, hampering endpoint determinations. We conclude that AST conditions including YNB as an assay medium, agitation of the plates, reading after 48 h of incubation, an inoculum size of 10(5) CFU/ml, and incubation at 30°C made MIC determinations easier without an overestimation of MIC values.

Randomized clinical trials and the treatment of pediatric cardiomyopathy

Randomized clinical trials are essential for the development of an evidence base for treating virtually all medical conditions. Opportunities for randomized clinical trials in pediatric cardiomyopathy exist in many areas, including determination of the utility of biomarkers in diagnosis and management, preventative strategies for children at risk for cardiomyopathies, pharmacogenomic studies, and drug or device treatment studies. Because of the rare nature of pediatric cardiomyopathies, many obstacles exist to the implementation of clinical trials in this entity. Obstacles include realistic enrollment targets, endpoint determinations, and funding of these very expensive kinds of trials. This review discusses each of these topics and provides some examples and some options for performing randomized clinical trials in children with cardiomyopathy.

Practical pathology of aging mice

Old mice will have a subset of lesions as part of the progressive decline in organ function that defines aging. External and palpable lesions will be noted by the research, husbandry, or veterinary staff during testing, cage changing, or physical exams. While these readily observable lesions may cause alarm, not all cause undue distress or are life-threatening. In aging research, mice are maintained until near end of life that, depending on strain and genetic manipulation, can be upwards of 33 months. Aging research has unique welfare issues related to age-related decline, debilitation, fragility, and associated pain of chronic diseases. An effective aging research program includes the collaboration and education of the research, husbandry, and veterinary staff, and of the members of the institution animal care and use committee. This collaborative effort is critical to humanely maintaining older mice and preventing excessive censorship due to non-lethal diseases. Part of the educational process is becoming familiar with how old mice appear clinically, at necropsy and histopathologically. This baseline knowledge is important in making the determination of humane end points, defining health span, contributing causes of death and effects of interventions. The goal of this paper is to introduce investigators to age-associated diseases and lesion patterns in mice from clinical presentation to pathologic assessment. To do so, we present and illustrate the common clinical appearances, necropsy and histopathological lesions seen in subsets of the aging colonies maintained at the University of Washington.