Exact Quantification Research Articles

Abstract 4844: High throughput detection of PRKD1 mutations in salivary gland tumors using in situ mutation detection in tissue microarrays

Abstract Recently, unique activating mutations in PRKD1 were observed in salivary gland polymorphous low-grade adenocarcinoma (PLGA). PRKD1 is a serine-threonine kinase that participates in important cell functions such as survival, adhesion, and migration. PLGA is a salivary gland tumor that usually shows indolent clinical presentation. Due to morphological similarities within different subtypes of salivary gland neoplasms, differential diagnosis may be puzzling. Although salivary gland cancer is a rare disease (around 3,000 new cases per year in the USA), the gravity of the disease should not be undermined. Currently, the salivary gland cancer biology is poorly understood. Comprehending the molecular biology of this cancer is instrumental in improving the diagnosis and prognostic prediction of this disease. Our goal is to assess the incidence of c.2130A>T and c.2130A>C PRKD1 mutations in different salivary gland tumors by a high throughput mutation detection technique. We first assessed the c.2130 PRKD1 point mutations in 8 PLGA cases using conventional Sanger sequencing. Hotspot mutations were present in 75% (6/8) of the samples. Subsequently, we built tissue microarrays slides that included the following: 5 PLGA, 54 adenoid cystic carcinomas, 30 mucoepidermoid carcinomas, 14 acinic cell carcinomas, 6 adenocarcinomas not otherwise specified, 34 pleomorphic adenocarcinomas, 3 mammary analogue secretory carcinomas, and 1 salivary duct carcinoma. Additional sample collection is ongoing. We optimized a recently described in situ mutation detection to directly visualize single mutations with microscopy in fixed cells and tissue sections. The technique is based on in situ proximity ligation assays that utilize sequence specific probes. Ligation of the padlock probe will only occur if there is a perfect match between the probe and the target. The probes are labeled with fluorophores that allow generated signals to be visualized as bright spots with a fluorescence microscope. This technique allows exact quantification of the detected target molecules while identifying each individual signal (mutant or wild type) within the tissue sections. Once the methodology is validated to detect these mutations, it will allow us to quickly analyze a cohort of salivary gland neoplasms which have been already prepared as tumor microarrays. Additionally, this technique will open new avenues to analyze other mutations in different tumor cohorts already prepared as tumor microarray slides. In the context of salivary gland tumors, differentiating distinct primary tumors subtypes and/ or metastasis from primary tumors with other sites of origin would be invaluable for clinical decisions. Citation Format: Piotr T. Wysocki, William H. Westra, Shahnaz Begum, David Sidransky, Mariana Brait. High throughput detection of PRKD1 mutations in salivary gland tumors using in situ mutation detection in tissue microarrays. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4844. doi:10.1158/1538-7445.AM2015-4844

Penetración espermática intracervical e intrauterina en la infertilidad funcional por factor cervical

En 14 mujeres con infertilidad funcional por factor cervical se realizaron 30 pruebas postcoitales en días preovulatorios, con recuentos exactos de espermatozoides en moco cervicaI y contenido intrauterino. Los valores se comparan con los de 19 mujeres senos, multíparas. Una paciente normal y dos infértiles fueron seguidas con determinaciones seriadas a través de un ciclo completo. La cuantificación exacta de la cristalización del moco, realizada mediante un nuevo método, resultó ser un buen criterio de evaluación de la penetrabilidad cervical. A nivel intracervical los resultados del grupo infértil no fueron significativamente diferentes de los controles; una leve disminución de los espermios móviles direccionales carece de valor práctico. Por el contrario, a nivel intrauterino, en el grupo infértil se detectaron espermios sólo en 7 de los 30 casos, con concentraciones muy bajas; en el grupo control fueron 18 de 19 casos. Se insiste en el valor del recuento espermático intrauterino, muy superior a las pruebas intracervicales. La técnica empleada permite obtener resultados cuantitativos más exactos que los reportados por la literatura. El tratamiento consistió principalmente en estrógenos a dosis baja en días 8 o 14 del ciclo. Se consiguió mejoría de la calidad del moco en 11 pacientes y gestación en 8 (57%).

A novel coplanar probe design for fast scanning of edema in human brain tissue via dielectric measurements

As part of every standard forensic autopsy, the examination of the brain includes assessment with respect to possible edema. The quantification of edema is helpful to make a sound diagnosis in presence of multiple affections and multiple possible causes of death. The water content in certain brain regions is furthermore a promising marker to distinguish between causes of death with no visible evidence, such as suffocation, shaking impact syndrome and sudden infant death syndrome. However, in todays’ forensic medicine, no technique is available for the objective and exact quantification of edema. Therefore, the aim of this work is to develop a fast and easy-to-use measuring system for the accurate determination of the water content in human brain tissue that fits into the procedure of a routine autopsy. For our setups, the dependency between relative permittivity and water content is utilized. In former works, we presented measurements of human brain tissue using a coaxial measuring chamber and an open-ended coaxial probe. However, some drawbacks of the used methods emerged. Thus, a novel probe design using a coplanar transmission line has been developed, addressing the drawbacks of the formerly used methods. This new probe is easy to calibrate and allows fast and accurate sequential scanning for edema in human brain tissue.

Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods.

Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEONLA) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEONLA with an additional protein corona formed by bovine serum albumin (SEONLA-BSA) and commercially available Rienso® particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products.

Heme Degradation by Heme Oxygenase Protects Mitochondria but Induces ER Stress via Formed Bilirubin

Heme oxygenase (HO), in conjunction with biliverdin reductase, degrades heme to carbon monoxide, ferrous iron and bilirubin (BR); the latter is a potent antioxidant. The induced isoform HO-1 has evoked intense research interest, especially because it manifests anti-inflammatory and anti-apoptotic effects relieving acute cell stress. The mechanisms by which HO mediates the described effects are not completely clear. However, the degradation of heme, a strong pro-oxidant, and the generation of BR are considered to play key roles. The aim of this study was to determine the effects of BR on vital functions of hepatocytes focusing on mitochondria and the endoplasmic reticulum (ER). The affinity of BR to proteins is a known challenge for its exact quantification. We consider two major consequences of this affinity, namely possible analytical errors in the determination of HO activity, and biological effects of BR due to direct interaction with protein function. In order to overcome analytical bias we applied a polynomial correction accounting for the loss of BR due to its adsorption to proteins. To identify potential intracellular targets of BR we used an in vitro approach involving hepatocytes and isolated mitochondria. After verification that the hepatocytes possess HO activity at a similar level as liver tissue by using our improved post-extraction spectroscopic assay, we elucidated the effects of increased HO activity and the formed BR on mitochondrial function and the ER stress response. Our data show that BR may compromise cellular metabolism and proliferation via induction of ER stress. ER and mitochondria respond differently to elevated levels of BR and HO-activity. Mitochondria are susceptible to hemin, but active HO protects them against hemin-induced toxicity. BR at slightly elevated levels induces a stress response at the ER, resulting in a decreased proliferative and metabolic activity of hepatocytes. However, the proteins that are targeted by BR still have to be identified.

Dual 3'Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells.

BackgroundThe interaction of eukaryotic host and prokaryotic pathogen cells is linked to specific changes in the cellular proteome, and consequently to infection-related gene expression patterns of the involved cells. To simultaneously assess the transcriptomes of both organisms during their interaction we developed dual 3’Seq, a tag-based sequencing protocol that allows for exact quantification of differentially expressed transcripts in interacting pro- and eukaryotic cells without prior fixation or physical disruption of the interaction.ResultsHuman epithelial cells were infected with Salmonella enterica Typhimurium as a model system for invasion of the intestinal epithelium, and the transcriptional response of the infected host cells together with the differential expression of invading and intracellular pathogen cells was determined by dual 3’Seq coupled with the next-generation sequencing-based transcriptome profiling technique deepSuperSAGE (deep Serial Analysis of Gene Expression). Annotation to reference transcriptomes comprising the operon structure of the employed S. enterica Typhimurium strain allowed for in silico separation of the interacting cells including quantification of polycistronic RNAs. Eighty-nine percent of the known loci are found to be transcribed in prokaryotic cells prior or subsequent to infection of the host, while 75% of all protein-coding loci are represented in the polyadenylated transcriptomes of human host cells.ConclusionsDual 3’Seq was alternatively coupled to MACE (Massive Analysis of cDNA ends) to assess the advantages and drawbacks of a library preparation procedure that allows for sequencing of longer fragments. Additionally, the identified expression patterns of both organisms were validated by qRT-PCR using three independent biological replicates, which confirmed that RELB along with NFKB1 and NFKB2 are involved in the initial immune response of epithelial cells after infection with S. enterica Typhimurium.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1489-1) contains supplementary material, which is available to authorized users.

Development of a Silver/Silver Ibuprofenate Potentiometric Sensor for Ibuprofen Quantification in Pharmaceutical Products

Nonsteroidal antiinflamatory drugs (NSAIDs) are the most used drugs due to their analgesic, antipyretic and antiinflamatory properties. Within this group of pharmaceutical compounds, Ibuprofen (acid-α-methyl-4(isobutyl)-phenylacetic is found. It is useful for treating headaches and arthritis. To assure consumer’s health, it is necessary to develop analytical methodologies to allow an accurate and exact quantification of ibuprofen. Potentiometry has demonstrated to be an advantageous technique, standing out the use of second type electrodes, which in accordance with the Nernst equation can detected anions activities. Considering this last, in this work we present the development and analytical characterization of a second-type electrode based on the Silver/Silver Ibuprofenate (Ag/AgIbu) pair for the quantification of Ibuprofen in pharmaceutical drugs.

Chemical and Radiative Size—Related Effects on the Performance of Liquid Rocket Engines fed with Different Propellant Combinations

The effects of rocket engine size on the effective exhaust velocity, for geometrically similar nozzles, are investigated as far as finite-rate chemistry and (gas-phase) radiation are concerned. Propulsion systems burning LOX/LH and LOX/LCH4 combinations are considered. The engine performance is estimated via a CFD code based on a particularly convenient formulation. It is found that finite-rate chemistry alone leads to performance steadily increasing with engine size, whereas introducing the effects of radiation reveals the occurrence of a maximum of effective exhaust velocity as a function of nozzle throat diameter. The exact quantification of radiation effects depends anyway to some extent on the modelling assumptions, as well as on the engine operating cycle.

Digital PCR to assess hematopoietic chimerism after allogeneic stem cell transplantation

Analysis of hematopoietic chimerism after allogeneic stem cell transplantation represents a crucial method to evaluate donor-cell engraftment. Whereas sensitivity of classical approaches for chimerism monitoring is limited to ≥1%, quantitative polymerase chain reaction (qPCR)-based techniques readily detect one patient cell in >1,000 donor cells, thus facilitating application of chimerism assessment as a surrogate for minimal residual disease. However, due to methodologic specificities, qPCR combines its high sensitivity with limited resolution power in the state of mixed chimerism (e.g., >10% patient cells). Our aim was to overcome this limitation by employing a further development of qPCR, namely digital PCR (dPCR), for chimerism analysis. For proof-of-principle, we established more than 10 dPCR assays detecting Indel polymorphisms or Y-chromosome sequences and tested them on artificial cell mixtures and patient samples. Employing artificial cell mixtures, we found that dPCR allows exact quantification of chimerism over several orders of magnitude. Digital PCR results proved to be highly reproducible (deviation<5%), particularly in the "difficult" range of mixed chimerism. Excellent performance of the new assays was confirmed by analysis of multiple retrospective blood samples from patients after allogeneic stem cell transplantation, in comparison with established qPCR (14 patients) and short-tandem repeat PCR (4 patients) techniques. Finally, dPCR is easy to perform, needs only small amounts of DNA for chimerism assessment (65ng corresponds to a sensitivity of approximately 0.03%), and does not require the use of standard curves and replicate analysis. In conclusion, dPCR represents a very promising method for routine chimerism monitoring.

Development of an improved method for quantitative analysis of skin blotting: increasing reliability and applicability for skin assessment.

A novel skin assessment tool named 'skin blotting' has been recently developed, which can easily predict the skin status to avoid its deterioration. The aim of this study was to propose a normalization method for skin blotting to compensate for individual differences that can hamper the quantitative comparisons and clinical applications. To normalize individual differences, we utilized a total protein as a 'normalizer' with calibration curves. For evaluation, we performed a simple simulation experiment, in which the same concentration of a protein of interest [tumour necrosis factor (TNF)-α] was applied at different volumes as a virtual individual difference. Moreover, to demonstrate the applicability of this normalization, male volunteers were recruited for skin blotting followed by the estimation of TNF-α with normalization. We obtained good calibration curves for total protein (R(2) =0.995) and TNF-α (R(2) =0.997), both of which were necessary for an exact quantification. In the simulation experiment, we estimated the exact concentration of TNF-α regardless of the applied volume, demonstrating the applicability of this normalization method in skin blotting. Further, skin blotting on human subjects showed a wide range of variation in the total protein content, although the normalization was thought to reduce such individual variations. This study has proposed total protein normalization for skin blotting with calibration curves. This method may strengthen the quantitative performance of skin blotting, which may expand the applicability of this method as a skin assessment tool in broader fields, such as nursing and cosmetology.

Monitoring recovery from iron deficiency using total hemoglobin mass.

Using hemoglobin concentration ([Hb]) to diagnose borderline iron deficiency and monitor the progress of its treatment is difficult because of the confounding effects of plasma volume. Because hemoglobin mass (Hbmass) is not affected by plasma volume, it may be a more sensitive parameter. The aim of this study was to monitor Hbmass, iron storage, and maximal oxygen consumption (V˙O2max) during and after oral iron therapy in subjects with severe and moderate iron deficiency. Three groups of female recreational athletes were monitored for at least 22 wk, as follows: 1) severe iron deficiency group (SID) (n = 8; ferritin, ≤12 ng·mL), 2) moderate iron deficiency group (MID) (n = 14; ferritin, ≤25 ng·mL), and 3) control group (n = 8; ferritin, >25 ng·mL). Hbmass and iron status were determined before, during, and up to 12 wk after at least 10 wk of oral iron supplementation. In total, five V˙O2max tests were performed before, during, and after the supplementation period. Hbmass increased markedly in the SID group (15.6% ± 11.0%, P < 0.001) and slightly in the MID group (2.2% ± 3.7%, P < 0.05) by the end of the supplementation period and remained at this level for the following 12 wk. [Hb] and Hbmass were similarly affected, but Hbmass was more closely related to mean corpuscular volume and mean corpuscular hemoglobin than [Hb]. The SID group incorporated 534 ± 127 mg of iron into ferritin and hemoglobin, whereas the MID group incorporated 282 ± 68 mg of iron. V˙O2max increased only in the SID group by 0.20 ± 0.18 L·min (P < 0.05) and was closely related to Hbmass (P < 0.01). Hbmass is a sensitive tool for monitoring recovery from iron deficiency anemia and assessing the effectiveness of iron supplementation in individuals with severe or moderate iron deficiency.

Determination of charge on asphaltene nanoaggregates in air using electrostatic force microscopy.

In this paper, we provide measurement of charge of asphaltene nanoaggregates in air using electrostatic force microscopy. We obtain the average surface charge density of the nanoaggregates as 43.7 nC/cm(2). Among the different aspects of asphaltene, one of the least known is its charge and the effect of solvent and compositional variability (of asphaltene) in dictating this charge. For aqueous systems, asphaltene charge demonstrates a strong dependence on the pH and the salt concentration, indicating that a possible ionization of the surface groups leads to this charging. On the contrary, for asphaltene in nonpolar media (e.g., toluene and heptane), it is believed that asphaltene native charge is central in dictating this charging. This native charge is the solvent-independent charge or the asphaltene charge in air. Our measurements, therefore, provide the first direct quantification (i.e., a quantification of charge not from the measurement of the asphaltene mobilities, which in turn requires specification of the nonuniform asphaltene size distribution) of this asphaltene native charge by conducting the measurements in air. Similar measurements in a solvent may introduce a solvent-dependent value, thereby forbidding not only the exact quantification of this native charge but also the understanding of the specific role of the solvent. This measurement, therefore, will provide a useful starting point to quantify the mechanism of asphaltene charging in nonpolar solvents with important ramifications in deciphering the role of asphaltene in transport and handling of crude and heavy oils.

Utility of different glycemic control metrics for optimizing management of diabetes.

The benchmark for assessing quality of long-term glycemic control and adjustment of therapy is currently glycated hemoglobin (HbA1c). Despite its importance as an indicator for the development of diabetic complications, recent studies have revealed that this metric has some limitations; it conveys a rather complex message, which has to be taken into consideration for diabetes screening and treatment. On the basis of recent clinical trials, the relationship between HbA1c and cardiovascular outcomes in long-standing diabetes has been called into question. It becomes obvious that other surrogate and biomarkers are needed to better predict cardiovascular diabetes complications and assess efficiency of therapy. Glycated albumin, fructosamin, and 1,5-anhydroglucitol have received growing interest as alternative markers of glycemic control. In addition to measures of hyperglycemia, advanced glucose monitoring methods became available. An indispensible adjunct to HbA1c in routine diabetes care is self-monitoring of blood glucose. This monitoring method is now widely used, as it provides immediate feedback to patients on short-term changes, involving fasting, preprandial, and postprandial glucose levels. Beyond the traditional metrics, glycemic variability has been identified as a predictor of hypoglycemia, and it might also be implicated in the pathogenesis of vascular diabetes complications. Assessment of glycemic variability is thus important, but exact quantification requires frequently sampled glucose measurements. In order to optimize diabetes treatment, there is a need for both key metrics of glycemic control on a day-to-day basis and for more advanced, user-friendly monitoring methods. In addition to traditional discontinuous glucose testing, continuous glucose sensing has become a useful tool to reveal insufficient glycemic management. This new technology is particularly effective in patients with complicated diabetes and provides the opportunity to characterize glucose dynamics. Several continuous glucose monitoring (CGM) systems, which have shown usefulness in clinical practice, are presently on the market. They can broadly be divided into systems providing retrospective or real-time information on glucose patterns. The widespread clinical application of CGM is still hampered by the lack of generally accepted measures for assessment of glucose profiles and standardized reporting of glucose data. In this article, we will discuss advantages and limitations of various metrics for glycemic control as well as possibilities for evaluation of glucose data with the special focus on glycemic variability and application of CGM to improve individual diabetes management.

Laboratory diagnosis of acquired hemophilia A: limitations, consequences, and challenges.

Acquired hemophilia A (AHA) should be suspected in patients with a new onset of bleeding and an isolated prolongation of activated partial thromboplastin time. About 10% of patients do not bleed at the time of diagnosis, but are at risk of future bleeding, particularly during interventions or surgery. Diagnosis of AHA is confirmed by demonstrating markedly reduced factor VIII activity (FVIII:C) and neutralizing anti-FVIII antibodies, so-called inhibitors. Several limitations and pitfalls exist with the assays used to diagnose AHA. Interference can result from anticoagulants or lupus anticoagulant. The Bethesda assay used to measure inhibitor potency assumes a log-linear relationship between inhibitor concentration and effect on residual FVIII:C activity to allow exact quantification. However, this relationship is not present for the type 2 inhibitors typically seen in AHA. Therefore, this assay only provides a rough estimate of inhibitor potency. These limitations can explain, in part, why laboratory data, such as inhibitor potency, failed to predict bleeding or response to treatment in AHA. This article reviews the diagnostic approach to AHA, discusses assay-specific limitations and addresses some of the challenges for future research.

High-sensitivity cardiac troponin in acute conditions.

We aim to help clinicians to use and interpret high-sensitivity cardiac troponins (cTns) in different acute care settings. This guidance is timely and relevant as high-sensitivity cTns are currently replacing conventional cTn assays in most parts of the world. cTn I and T are structural proteins unique to the heart. Detection of cTn in peripheral blood indicates cardiomyocyte injury. Although acute myocardial infarction is a very common, dangerous, but treatable and therefore clinically important cause of cardiomyocyte injury, multiple other acute conditions are associated with substantial amounts of cardiomyocyte injury and corresponding elevations in cTn. These include acute heart failure, tachyarrhythmias, pulmonary embolism, sepsis, shock, and noncardiac surgery. Recent advances in assay technology have led to more sensitive and precise cTn assays that now allow the detection and exact quantification of cardiomyocyte injury also in many predominately noncardiac acute conditions. In all of these, elevated levels of high-sensitivity cTn are associated with increased mortality risk. In some of these, concepts are evolving as to how the pathophysiological signal of cardiomyocyte injury could be used to alter patient management and potentially improve outcomes.

Open-ended coaxial probe for the quantification of edema in human brain tissue

The major task in most autopsies is the evaluation of circumstances and cause of a person's death. The quantification of edema, primarily in certain brain regions, is a promising marker to distinguish between certain causes of death. This is especially important if there is no visible reason, for example, in the case of distinguishing Sudden Infant Death Syndrome from Shaken Impact Syndrome. However, until now there is no standardized procedure in forensic medicine for the quantification of edema in parenchymatous organs. Therefore, the aim of our work is to develop a fast low-cost measuring system that fits into the procedure of an autopsy. One approach for an exact quantification of edema is to determine the humidity content in the tissue with the so-called oven dry method, which we used as a reference. The approach presented here is the determination of the humidity content via a measurement of the relative permittivity of the tissue. We use two different setups: a transmission and a reflection measurement. Thereby a linear regression between the humidity content and the relative permittivity was found with excellent regression coefficients of R=0.98 for the transmission measurement and R=0.97 for the reflection measurement. We compare the results of the used methods regarding accuracy and measuring time.

Development of a Silver/Silver Ibuprofenate Potentiometric Sensor for Ibuprofen Quantification in Pharmaceutical Products

Nonsteroideal antiinflamatory drugs (NSAIDs) are the most used drugs due to their analgesic, antipyretic and antiinflamatory properties, along with the fact that these are sold without the requirement of a medical prescription. Within this group of pharmaceutical compounds, ibuprofen (acid-α-methyl-4(isobutyl)-phenylacetic) is found. The use of Ibuprofen has increased because it is useful for treating headaches and arthritis. It is considered a safe drug since the human body can degrade it to its main metabolites and then excrete them through urine.Nevertheless, to assure consumer’s health, it is necessary the development of analytical methodologies to allow an accurate and exact quantification of ibuprofen. Potentiometry has demonstrated to be an advantageous technique, where stands out the use of second type electrodes, which in accordance with the Nernst equation can detected anions activities.Considering this last, in this work we present the development and analytical characterization of a second-type electrode based on the Silver/Silver Ibuprofenate (Ag/AgIbu) pair.A pure silver wire was modified using a 0.05 M sodium ibuprofenate solution and 0.1 M of sodium nitrate as supporting electrolyte. Chronoamperometry was used to achieve the modification of the electrode (Ag/AgIbu), the technique parameters were optimized and the registered optimums where: 0.8 V (vs Ag/AgCl) potential for 30 s. Calibration curves were performed to obtain the best analytical parameters: it was registered a sensitivity of -41.69 ± 1.61 mV/log (ibuprofenate molar activity), a linear range of 1.22x10-4 - 8.98 x 10-3 M, and a detection limit of 8.11x10-5 M. Repeatability interpreted as the relative error percentage, was 2.46%. The pH effect on the system response was studied, it was found that the best working pH is 7.0. The electrode’s time response was 3.3 min with a drift of -0.01 mV s-1. With the Matched Potential Method it was possible to determine that chloride and carbonate are the most important interferents.Ibuprofen was measured in commercial drugs with no significant differences found between the manufacture’s reported value and the one obtained with the proposed sensor.

Aged human mesenchymal stem cells: the duration of bone morphogenetic protein-2 stimulation determines induction or inhibition of osteogenic differentiation.

Bone morphogenetic protein 2 (BMP-2) is a potent osteoinductive cytokine and a growing number of in vitro studies analyze its effects on human mesenchymal stem cells (hMSC) derived from aged or osteoporotic donors. In these studies the exact quantification of osteogenic differentiation capacity is of fundamental interest. Nevertheless, the experimental conditions for osteogenic differentiation of aged hMSC have not been evaluated systematically and vary to a considerable extend. Aim of the study was to assess the influence of cell density, osteogenic differentiation media (ODM) change intervals and duration of BMP-2 stimulation on osteoinduction. Furthermore, time series were carried out for osteogenic differentiation and BMP-2 concentration in ODM/BMP-2 cell culture supernatants. The experiments were performed using hMSC isolated from femoral heads of aged patients undergoing hip joint replacement. ODM change intervals of 96 hours resulted in significantly higher calcium deposition compared to shorter intervals. A cell density of 80% prior to stimulation led to stronger osteoinduction compared to higher cell densities. In ODM, aged hMSC showed a significant induction of calcium deposition after 9 days. Added to ODM, BMP-2 showed a stable concentration in the cell culture supernatants for at least 96 hours. Addition of BMP-2 to ODM for the initial 4 days led to a significantly higher induction of osteogenic differentiation compared to ODM alone. On the other hand, addition of BMP-2 for 21 days almost abrogated the osteoinductive effect of ODM. We could demonstrate that the factors investigated have a substantial impact on the extent of osteogenic differentiation of aged hMSC. Consequently, it is of upmost importance to standardize the experimental conditions in order to enable comparability between different studies. We here define standard conditions for osteogenic differentiation in regard to the specific features of aged hMSC. The finding that BMP-2 induces or inhibits osteogenic differentiation in a time dependent manner indicates an age related alteration in signal transduction of hMSC and requires further investigation.

Three-dimensional facial analysis in acromegaly: a novel tool to quantify craniofacial characteristics after long-term remission

The exact quantification of craniofacial characteristics in patients with acromegaly is important because it provides insight in the pathophysiology of the disease and offers a tool to evaluate the effects of treatment on tissue specific endpoints. However, until recently this was not feasible due to limitations of available cephalometric methods. The new technique of three-dimensional (3D) cephalometry enables the accurate quantification of facial anatomical characteristics of both soft tissue and bone. This is the first study that uses 3D cephalometry to analyze craniofacial disproportions in patients in long-term remission of acromegaly. Sixteen patients in remission of acromegaly for over 24 months (50% male, mean age 56.0 ± 10.7 years, mean body mass index 29.3 ± 5.5 kg/m(2)) were compared to 16 matched control subjects. A 3D cone beam computed tomography scan and 3D stereophotograph of each individual were acquired and analyzed using 3D cephalometry. In addition to an accurate quantification of the classical craniofacial characteristics, 3D cephalometry, shows that many typical soft tissue deformities persist, even after long-term remission. Furthermore, we found that, compared to controls, the patients in remission of acromegaly have a wider face at the level of the zygoma and longer maxilla (p < 0.05). 3D cephalometry is an attractive novel imaging modality to accurately investigate craniofacial disproportions of both soft tissue and bony parts of the face in patients with acromegaly, which makes it a promising technique for future research purposes and clinical practice.

Mesoscale and macroscale kinetic energy fluxes from granular fabric evolution.

Recent advances in high-resolution measurements means it is now possible to identify and track the local "fabric" or contact topology of individual grains in a deforming sand throughout loading history. These provide compelling impetus to the development of methods for inferring changes in the contact forces and energies at multiple spatiotemporal scales, using information on grain contacts alone. Here we develop a surrogate measure of the fluctuating kinetic energy based on changes in the local contact topology of individual grains. We demonstrate the method for dense granular materials under quasistatic biaxial shear. In these systems, the initially stable and solidlike response eventually gives way to liquidlike behavior and global failure. This crossover in mechanical behavior, akin to a phase transition, is marked by bursts of kinetic energy and frictional dissipation. Mechanisms underlying this release of energy include the buckling of major load-bearing structures known as force chains. These columns of grains represent major repositories for stored strain energy. Stored energy initially accumulates at all of the contacts along the force chain, but is released collectively when the chain overloads and buckles. The exact quantification of the buildup and release of energy in force chains, and the manner in which force chain buckling propagates in the sample (i.e., diffuse and systemwide versus localized into shear bands), requires detailed knowledge of contact forces. To date, however, the forces at grain contacts continue to elude measurement in natural granular materials like sand. Here, using data from computer simulations, we show that a proxy for the fluctuating kinetic energy in dense granular materials can be suitably constructed solely from the evolving properties of the grain's local contact topology. Our approach directly relates the evolution of fabric to energy flux and makes possible research into the propagation of failure from measurements of grain contacts in real granular materials.