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Cellular homeostasis and responses to stimuli are mediated by complex signaling network events dominated by changes in protein phosphorylation states. Understanding information flow in the network is essential for correlating signaling changes to cell physiology. Tyrosine phosphorylation constitutes only a small portion of all protein phosphorylation, but its importance is manifested by the significant role it plays in diseases such as cancer. A peptide-based immunoassay microarray, designed to provide site specificity, quantification, broad coverage, and accessibility, is described that profiles 45 tyrosine phosphorylation sites across 34 proteins. Epidermal growth factor–stimulated A431 cells in the absence and presence of kinase inhibitors analyzed by microarrays showed biologically validated tyrosine phosphorylation changes and unanticipated activation of other targets. The approach is scalable for increasing the breadth of content as well as for interrogating other types of protein posttranslational modifications. (Journal of Biomolecular Screening 2008:626-637)

Aberrant epidermal growth factor receptor (EGFR, ErbB1) signaling is implicated in cell transformation, motility, and invasion in a variety of cell types, and EGFR is the target of several anticancer drugs. However, the kinetics of EGFR signaling and the individual contributions of site-specific phosphorylation events remain largely unknown. A peptide-based, multiplex immunoassay approach was developed to simultaneously measure both total and phosphorylated protein in a single sample. The approach involves the proteolytic digestion of proteins prior to the isolation and quantitation of site-specific phosphorylation events within an individual protein. Quantitation of phosphorylated and total proteins, in picomolar to nanomolar concentrations, were interpolated from standard curves generated with synthetic peptides that correspond to the peptide targets used in the immunoassays. In this study, a bead-based, nine-plex immunoassay measuring total and phosphorylated protein was constructed to measure temporal, site-specific phosphorylation of key members of the EGFR pathway (ErbB1 receptor, MEK1, MEK2, ERK1, and ERK2) in A431 cells stimulated with epidermal growth factor. The effect of MEK inhibition on this pathway was determined using a known MEK kinase inhibitor, SL327. The results reported herein are the first quantitative measurements of site-specific phosphorylation events and total proteins in a single sample, at the same time representing a new paradigm for standardized protein and phosphorylation analysis using multiplexed, peptide-based, sandwich immunoassays.

Background The Modified Mini-Mental State Examination-revised (3MS-R) is a brief cognitive test designed to detect cognitive impairment, which is often used in Canada and USA. Objective To assess the accuracy of the 3MS-R in identifying Alzheimer's disease (AD) in comparison with the conventional Mini-Mental State Examination (MMSE) in a German-speaking population. Subjects The study refers to 31 patients with early AD and 5 patients with moderate dementia of AD etiology, as well as to 46 age-matched cognitively normal participants. Method The 3MS-R and the MMSE were validated against an expert diagnosis based on a comprehensive diagnostic workup. The 3MS scores were adjusted for educational attainment. Statistical analysis was performed using the Receiver Operating Characteristics (ROC). Results ROC curves demonstrated the superiority of the ACE over the MMSE in identifying AD (Area under the Curve: 3MS-R vs. MMSE: 0.995 vs.0.953). The optimal cut-off score for the 3MS for detecting AD was 88 and had a sensitivity of 98% and a specificity of 94%. The German version of the 3MS-R is a short and practical but accurate test battery for the identification of AD. The effectiveness of the German version of the test in detecting other forms of dementias or mild cognitive impairment could be a task for future studies.

We assessed the diagnostic accuracy of the German version of the Addenbrooke’s Cognitive Examination (ACE) in identifying early Alzheimer’s disease (AD) and mild vascular dementia (VaD) in comparison with the conventional Mini-Mental State Examination (MMSE). The study refers to 50 patients with mild dementia of AD, 26 patients with mild dementia of vascular etiology and to 54 cognitively normal subjects. The ACE and MMSE were validated against an expert diagnosis based on a comprehensive diagnostic workup. Statistical analysis was performed using the receiver operator characteristics method. The optimal cut-off score for the ACE for detecting dementia in patients with early AD was 85/86, which had a sensitivity of 93% and a specificity of 86%. The optimal cut-off for the ACE for the identification of dementia in patients with mild VaD was also 85/86 and it had a sensitivity of 93% and a specificity of 100%. The ĸ values imply a substantial agreement between the diagnoses made by the ACE and the MMSE. The German version of the ACE is a short and practical but accurate test battery for the identification of AD and VaD, assessing a broad range of cognitive functions and providing a wide profile of cognitive functions/dysfunctions.

Identification of proteins previously separated by one-dimensional (1-D) or two-dimensional gel electrophoresis requires significant manipulations to digest the proteins into their respective peptides and to extract them from the gel prior to mass analysis. This article describes the simultaneous transfer and digestion of proteins directly from 1-D gels onto a membrane ready for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric (MS) analysis. Protein transfer and digestion efficiencies are estimated to be more than 95%. The effectiveness of this procedure is demonstrated by identifying 110 unique proteins derived from a lysate of Escherichia coli and 149 proteins derived from a mouse liver homogenate separated by 1-D sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Using crude mouse liver homogenates, four distinct glutathione S -transferase classes, ranging from 23 to 27 kDa, are identified from a separating gel, indicating the discriminating potential for this method. A Visual Basic program allowed visualization of the identified proteins according to their respective positions on the 1-D gels. In many cases, two or more proteins could be identified within a single band of the SDS gel. The “digital” images generated resemble Western blots without the use of antibodies or signal amplification techniques.

Cell migration and invasion are crucial steps in many normal, as well as, aberrant physiological processes, in particular, the highly lethal metastatic progression of cancer. Once the normally restrictive basement membrane boundaries of the tumor have been breached, invasive cells can gain access to the vasculature and lymphatic system permitting systemic spread of the disease. Invasion is a complex multistep process involving cell adhesion, directed migration, and proteolytic degradation of surrounding extracellular matrix (ECM) barriers. Multiple factors present in the local microenvironment, including soluble growth factors or chemokines and ECM-anchored integrins and proteases, act coordinately to regulate the invasive potential. Understanding the cellular and molecular mechanisms underlying invasion is essential to the development of therapeutics that target tumor metastasis. Therefore, techniques for the quantification and dynamic visualization of penetrating cells have become central to oncology research. The most widely employed method for analyzing cell invasion in vitro is a modified Boyden chamber assay using Matrigel, a basement membrane matrix preparation. While amenable to semi-quantitative “screening” assays, these systems suffer from the inability to establish continuous gradients. As a result, this method, such gradients cannot be stably maintained under static culturing conditions, precluding longer-term real-time cell analysis. Moreover, these platforms are not strictly designed for microscope-based visualization and thus cannot be employed for dynamic mechanistic studies through imaging analysis at the inter- and intra-cellular levels. Here we present a two-tiered platform for the analysis of cell invasion. Cell culture insert plates (24 and 96-well capacity), provide the ideal tool for initial compound screening for functional response (inhibit invasion) and toxicity. For selected compounds, mechanistic studies were then performed using a microfluidic-based system. The platform consists of a microfluidic culture plate and environmental control system; the latter regulates matrix loading, media perfusion, the establishment of stable continuous concentration gradients within the plate, as well as temperature and gas control. Most significantly, the plate can be paired with an inverted microscope enabling dynamic analysis of cell movement in real-time. Fluorescent visualization further permits selective discrimination of unique cell types in heterogeneous samples as well as changes in expression patterns of labeled proteins. In summary, the combined workflow platform presented here provides a framework for studying the effects of signaling molecules and growth factors on the migratory and invasive propensities of tumors cells. Citation Format: Amedeo J. Cappione, Timothy Nadler. A comprehensive workflow for screening and real-time analysis of cell invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5083.

A novel approach to three-dimensional inter-surface clearance calculation for twin-shaft rotary displacement machines based on a NURBS representation of both rotor surfaces is presented. The calculation is split up in separate steps. At first, a tree data structure of both surfaces is created by using bounding volumes. Based on the tree structure, an efficient identification of nearest pairs of bounding volumes is possible. In the next step, a contour line with a fixed, user defined distance is created with the marching squares algorithm. The contour line encloses a stripe-shaped region which is triangulated by means of dynamic programming so that the total length of the triangle edges is minimized. For each edge, a closest point on the opposite surface is calculated with the Newton algorithm. The resulting intermesh clearance is represented by the resulting set of closest points. A comparison with of the approach to other common methods shows several advantages. The use of NURBS surfaces guarantees a good mathematical accuracy for miscellaneous rotor profiles as well as the possibility to communicate directly with common CAD-systems. Finally the new approach for the intermesh clearance calculation is compared to another calculation method and the advantages and improvements are shown.

To attain lower pressures in the fine and adjacent high vacuum roots vacuum pumps are used in conjunction with a forevacuum pump in almost any application in the so-called "blower” operating range. At this point, the already advanced screw compressor technology may be applied to “blower” applications too.

Antifibronectin, monoclonal antibody was monitored through 52 h of production. Samples were automatically drawn from a bioreactor into the injection valve of an HPLC system without prior sample preparation. The hybridoma cell line was nonadherent, so whole cells were injected directly onto the perfusable protein A affinity column. There was only a modest column back pressure (ca. 1700 psi at a linear flow rate of 1.5 cm/s) after over 75 injections over the 52-h experiment. These experiments demonstrate the utility of high-speed chromatography for rapid process monitoring.

The Modified Mini-Mental State Examination-revised (3MS-R) is a brief cognitive test designed to detect cognitive impairment, which is often used in Canada and USA.To assess the accuracy of the 3MS-R in identifying Alzheimer's disease (AD) in comparison with the conventional Mini-Mental State Examination (MMSE) in a German-speaking population.The study refers to 31 patients with early AD and 5 patients with moderate dementia of AD etiology, as well as to 46 age-matched cognitively normal participants.The 3MS-R and the MMSE were validated against an expert diagnosis based on a comprehensive diagnostic workup. The 3MS scores were adjusted for educational attainment. Statistical analysis was performed using the Receiver Operating Characteristics (ROC).ROC curves demonstrated the superiority of the ACE over the MMSE in identifying AD (Area under the Curve: 3MS-R vs. MMSE: 0.995 vs.0.953). The optimal cut-off score for the 3MS for detecting AD was 88 and had a sensitivity of 98% and a specificity of 94%. The German version of the 3MS-R is a short and practical but accurate test battery for the identification of AD. The effectiveness of the German version of the test in detecting other forms of dementias or mild cognitive impairment could be a task for future studies.

The frequent occurrence of both rheumatoid arthritis and autoimmune thyroiditis was already investigated with in part many conflicting results. We investigated a number of 792 patients (383 of them suffering from rheumatoid arthritis and 409 with osteoarthritis). In all patients antithyroid peroxidase and antithyroglobulin antibodies were determined. Patients with rheumatoid arthritis showed a significantly higher occurrence of circulating thyroid antibodies than those with osteoarthritis (9.1% versus 3.7%, p = 0.0016). We conclude that there exists a cumulate coincidence of both diseases. Patients suffering from rheumatoid arthritis should undergo a thyroid examination especially for the presence of autoimmune thyroiditis.

Influences of non-steroidal anti-inflammatory drugs (NSAID) on concentrations of thyroid hormones are known for a long time. These effects could be explained with interference between NSAIDs and thyroid hormone binding. We investigated the effects of a single dose of aceclofenac on thyroid function and thyroid hormone binding in 18 healthy volunteers. Serum levels of free thyroid hormones (FT3, FT4) and thyrotropin (TSH) were measured with commercial available kids and thyroid hormone binding was estimated with a specially modified horizontal argarose-gel-electrophoresis prior to and 2 hours after receiving a single dose of aceclofenac. We found a significant decrease in T3 binding on TBG and a significant increase of albumin-bound T3. All other investigated thyroid hormone binding parameters, FT3 and FT4, showed no significant changes. We conclude that aceclofenac leads to a significant redistribution of T3 protein binding. These effects seem to be explained by T3 displacement from TBG induced by aceclofenac.

Effects of gender and age on quantitative sacroiliac joint imaging are discussed controversially. In most investigations the number of controls has been small and might not exactly reflect the change of sacroiliac/sacral (SI/S) ratios related to different age and gender. The aim of our study was to evaluate the changes SI/S ratios according to age and gender. In 125 patients without any history of either diseases or complaints of the sacroiliac joints a bone scintigraphy was obtained and the SI/S ratios were calculated. We observed a significant negative correlation of SI/S index with age. After separation into 4 different age groups a significant decline of the ratios could be shown. There were no significant differences between male and female patients. We conclude that the influence of age on SI/S ratios is substantial. But also many other factors like patient position, algorithm of SI/S ratio calculation, the time interval between application of the radiopharmaceutical and data acquisition may exert effects on SI/S ratios. It seems necessary for each department to evaluate their own age related reference values of SI/S ratios.

It is important to determine the amount of IgG multimers in immunoglobulin-containing pharmaceuticals because these aggregates can cause adverse reactions in patients. Previous methods for determining aggregates either suffered from interference of other proteins or required fraction collection and sample purification. A new, automated two-dimensional approach has been developed in which size-exclusion chromatography is performed in the first dimension followed by protein A affinity chromatography in the second dimension. This method is robust in that the aggregates are not disturbed by a preliminary purification step. Further, the presence of contaminating proteins has no effect on the analysis since affinity chromatography is used to determine the presence of IgG in the second dimension. The entire automated two-dimensional analysis can be performed in ca. 1 h.

KTP waveguides are being investigated for frequency upconversion of strained-layer InGaAs lasers in the 900 - 1100 nm range. Phasematching for the lowest-order mode interaction can be obtained using the modal dispersion properties of these diffused waveguides and modeling calculations have been used to determine the waveguide parameters required for phasematching to occur at a particular wavelength. Ion-exchange in pure RbNO3 has been used to form a waveguide that permits phasematched frequency doubling at wavelengths near 1040 nm.

Deconvolution is a mathematical technique for improving the resolution of spectra. Deconvolution can be done in either signal space or frequency space. Both methods were attempted after special adaptations of the algorithms were carried out in order to make them specific for the near-infrared (NIR) spectrum. Signal-space deconvolution works well when a line is subtracted from the spectrum before a modified Jansson's algorithm is applied. Frequency-space deconvolution requires the addition of a polynomial tail to the spectrum, the addition of a constant to the bandpass divisor, and a Gaussian apodization function.

Results and experiences in 51 transhepatic endless drainage operations according to the method of Goetze-Dick are dealt with, the indication as well as the technique of the operation are discussed in detail. The advantage of this method is based on the fact, that the drain can be easily changed. So patients with inoperable obstructive jaundice can often be given many month of worthwhile life. The extracorporal part of the drain is hardly found to trouble the patients. Also benign intrahepatic obstructions, where no bile duct is left to be anastomized, can be operated on satisfactorily with this method. In spite of the blunt penetration of the liver necessitated by this technique, intra- and postoperative complications are rare. Postoperative care is paramount in avoiding cholangitis.

The legume-Rhizobium endosymbiosis may be the most highly evolved and perhaps ultimate association between a microbe and a plant in which the two partners can still grow independently. Undoubtedly the strong selective pressure on this association is the resulting nutritional complementation: the plant can be considerd a carbon-rich, nitrogen-deficient phototroph and the Rhizobium a carbon-deficient, nitrogen-fixing heterotroph. The resulting symbiosis which occurs in a specialized organ, the root nodule, makes the plant autotrophic with respect to the availability of reduced nitrogen, a limiting factor in plant nutrition. This unique intracellular association contributes significantly towards the yield of the agriculturally important legume crops.

Nodule-specific root proteins – so called nodulins – were identified in root nodules of pea plants by an immunological assay. Nodulin patterns were examined at different stages of nodule development. About 30 nodulins were detectable during development. Some were preferentially synthesized before nitrogen fixation started, whereas the majority were synthesized concomitantly with leghaemoglobin. Some of the nodulins were located within the peribacteroid membrane. Ineffective Rhizobium strains (a natural nod+fix- and a pop -fix-) appeared to be useful in studying the expression of nodulin genes. Synthesis of some nodulins was repressed in ineffective root nodules, indicating that nodulins are essential for the establishment of nitrogen fixation. In both types of ineffective root nodules, leghaemoglobin synthesis was not completely repressed. Low amounts of leghaemoglobin were always detected in young ineffective root nodules whereas in old nodules no leghaemoglobin was present.

Es werden 3 Operationsmethoden vorgestellt: 1. Die Rectotomia posterior als hinterer Zugang bei Tumoren des Rectum bis 12 cm Hohe. 2. Die Operationstechnik einer freien Transplantation autologer Dickdarmmuskulatur als Sphincterersatz bei endstandiger Colostomie. 3. Ein Gerat zur Herstellung tiefer maschineller Dickdarmanastomosen.

Bericht uber 49 Falle von intra- und extrahepatischen Stenosen der Gallenwege (1970–1977), bei denen durch eine transhepatische Endlosdrainage nach Goetze-Dick der Galleabflus wieder hergestellt wurde. Hauptindikation ist ein Verschlusikterus durch inoperable Tumoren der Gallenblase (21 Falle), der Gallengange (12 Falle) und des Pankreas (8 Falle). In 5 Fallen wurde eine benigne intrahepatisch gelegene Stenose mit Verschlusikterus durch die Endlosdrainage nach Dick beseitigt. Auf Indikationsstellung, Fruh- und Spatkomplikationen sowie auf die postoperative Nachsorge wird naher eingegangen.