Simultaneous determination of natural and synthetic steroid estrogens and their conjugates in aqueous matrices by liquid chromatography / mass spectrometry

An analytical method for the simultaneous determination of nine free and conjugated steroid estrogens was developed with application to environmental aqueous matrices. Solid phase extraction (SPE) was employed for isolation and concentration, with detection by liquid chromatography/mass spectrometry (LC/MS) using electrospray ionisation (ESI) in the negative mode. Method recoveries for various aqueous matrices (wastewater, lake and drinking water) were determined, recoveries proving to be sample dependent. When spiked at 50 ng/l concentrations in sewage influent, recoveries ranged from 62-89 % with relative standard deviations (RSD) < 8.1 %. In comparison, drinking water spiked at the same concentrations had recoveries between 82-100 % with an RSD < 5%. Ion suppression is a known phenomenon when using ESI; hence its impact on method recovery was elucidated for raw sewage. Both ion suppression from matrix interferences and the extraction procedure has bearing on the overall method recovery. Analysis of municipal raw sewage identified several of the analytes of interest at ng/l concentrations, estriol (E3) being the most abundant. Only one conjugate, estrone 3-sulphate (E1-3S) was observe

Increased variability in ApcMin/+ intestinal tissue can be measured with microultrasound

Altered tissue structure is a feature of many disease states and is usually measured by microscopic methods, limiting analysis to small areas. Means to rapidly and quantitatively measure the structure and organisation of large tissue areas would represent a major advance not just for research but also in the clinic. Here, changes in tissue organisation that result from heterozygosity in Apc, a precancerous situation, are comprehensively measured using microultrasound and three-dimensional high-resolution microscopy. Despite its normal appearance in conventionally examined cross-sections, both approaches revealed a significant increase in the variability of tissue organisation in Apc heterozygous tissue. These changes preceded the formation of aberrant crypt foci or adenoma. Measuring these premalignant changes using microultrasound provides a potential means to detect microscopically abnormal regions in large tissue samples, independent of visual examination or biopsies. Not only does this provide a powerful tool for studying tissue structure in experimental settings, the ability to detect and monitor tissue changes by microultrasound could be developed into a powerful adjunct to screening endoscopy in the clinic

The implementation of the serial trial intervention for pain and challenging behaviour in advanced dementia patients (STA OP!): a clustered randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Pain (physical discomfort) and challenging behaviour are highly prevalent in nursing home residents with dementia: at any given time 45-80% of nursing home residents are in pain and up to 80% have challenging behaviour. In the USA Christine Kovach developed the serial trial intervention (STI) and established that this protocol leads to less discomfort and fewer behavioural symptoms in moderate to severe dementia patients. The present study will provide insight into the effects of implementation of the Dutch version of the STI-protocol (STA OP!) in comparison with a control intervention, not only on behavioural symptoms, but also on pain, depression, and quality of life. This article outlines the study protocol.</p> <p>Methods/Design</p> <p>The study is a cluster randomized controlled trial in 168 older people (aged >65 years) with mild or moderate dementia living in nursing homes. The clusters, Dutch nursing homes, are randomly assigned to either the intervention condition (training and implementation of the STA OP!-protocol) or the control condition (general training focusing on challenging behaviour and pain, but without the step-wise approach). Measurements take place at baseline, after 3 months (end of the STA OP! training period) and after 6 months.</p> <p>Primary outcome measures are symptoms of challenging behaviour (measured with the Cohen-Mansfield Agitation Inventory (CMAI) and the Neuropsychiatric Inventory-Nursing Home version (NPI-NH)), and pain (measure with the Dutch version of the Pain Assessment Checklist for Seniors (PACSLAC-D) and the Minimum Data Set of the Resident Assessment Instrument (MDS-RAI) pain scale). Secondary outcome measures include symptoms of depression (Cornell and MDS-RAI depression scale), Quality of Live (Qualidem), changes in prescriptions of analgesics and psychotropic drugs, and the use of non-pharmacological comfort interventions (e.g. snoezelen, reminiscence therapy).</p> <p>Discussion</p> <p>The transfer from the American design to the Dutch design involved several changes due to the different organisation of healthcare systems. Specific strengths and limitations of the study are discussed.</p> <p>Trial registration</p> <p>Netherlands Trial Register (NTR): <a href="http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=1967">NTR1967</a></p

Differences in External and Internal Cortical Strain with Prosthesis in the Femur

The contact between a femoral stem prosthesis and the internal surface of the cortical bone with the stress in the interface is of crucial importance with respect to loosening. However, there are no reports of strain patterns at this site, and the main aim of the current study was to investigate differences of internal and external cortical strain in the proximal femur after insertion of a stem prosthesis. The external cortical strain of a human cadaveric femur was measured with strain gauges before and after implantation of a stem prosthesis. By use of optical fibres embedded longitudinally in the endosteal cortex, deformations at the implant–internal cortex interface could also be measured. The main external deformation during loading of the intact femur occurred as compression of the medial cortex; both at the proximal and distal levels. The direction of the principal strain on the medial and lateral aspects was close to the longitudinal axis of the bone. After resection of the femoral neck and insertion of a stem prosthesis, the changes in external strain values were greatest medially at the proximal level, where the magnitude of deformation in compression was reduced to about half the values measured on the intact specimen. Otherwise, there were rather small changes in external principal strain. However, by comparing vertical strain in the external and internal cortex of the proximal femur, there were great differences in values and patterns at all positions. The transcortical differences in strain varied from compression on one side to distraction on the other and vice versa in some of the positions with a correlation coefficient of 0.07. Our results show that differences exist between the external and internal cortical strain when loading a stem prosthesis. Hence, strain at the internal cortex does not correspond and can not be deducted from measured strain at the external cortex

GLUT4 and UBC9 Protein Expression Is Reduced in Muscle from Type 2 Diabetic Patients with Severe Insulin Resistance

Subgroups of patients with type 2 diabetes mellitus demand large insulin doses to maintain euglycemia. These patients are characterized by severe skeletal muscle insulin resistance and the underlying pathology remains unclear. The purpose of this study was to examine protein expression of the principal glucose transporter, GLUT4, and associated proteins in skeletal muscle from type 2 diabetic patients characterized by severe insulin resistance.Seven type 2 diabetic patients with severe insulin resistance (mean insulin dose 195 IU/day) were compared with seven age matched type 2 diabetic patients who did not require insulin treatment, and with an age matched healthy control group. Protein expression of GLUT4 and associated proteins was assessed in muscle and fat biopsies using standard western blotting techniques.GLUT4 protein expression was significantly reduced by ∼30 pct in skeletal muscle tissue from severely insulin resistant type 2 diabetic subjects, compared with both healthy controls and type 2 diabetic subjects that did not require insulin treatment. In fat tissue, GLUT4 protein expression was reduced in both diabetic groups. In skeletal muscle, the reduced GLUT4 expression in severe insulin resistance was associated with decreased ubiquitin-conjugating enzyme 9 (UBC9) expression while expression of GLUT1, TBC1D1 and AS160 was not significantly different among type 2 diabetic patients and matched controls.Type 2 diabetic patients with severe insulin resistance have reduced expression of GLUT4 in skeletal muscle compared to patients treated with oral antidiabetic drugs alone. GLUT4 protein levels may therefore play a role in the pathology behind type 2 diabetes mellitus among subgroups of patients, and this may explain the heterogeneous response to insulin treatment. This new finding contributes to the understanding of the underlying mechanisms for the development of extreme insulin resistance

The APEX Quantitative Proteomics Tool: Generating protein quantitation estimates from LC-MS/MS proteomics results

Mass spectrometry (MS) based label-free protein quantitation has mainly focused on analysis of ion peak heights and peptide spectral counts. Most analyses of tandem mass spectrometry (MS/MS) data begin with an enzymatic digestion of a complex protein mixture to generate smaller peptides that can be separated and identified by an MS/MS instrument. Peptide spectral counting techniques attempt to quantify protein abundance by counting the number of detected tryptic peptides and their corresponding MS spectra. However, spectral counting is confounded by the fact that peptide physicochemical properties severely affect MS detection resulting in each peptide having a different detection probability. Lu et al. (2007) described a modified spectral counting technique, Absolute Protein Expression (APEX), which improves on basic spectral counting methods by including a correction factor for each protein (called O(i) value) that accounts for variable peptide detection by MS techniques. The technique uses machine learning classification to derive peptide detection probabilities that are used to predict the number of tryptic peptides expected to be detected for one molecule of a particular protein (O(i)). This predicted spectral count is compared to the protein's observed MS total spectral count during APEX computation of protein abundances. Results: The APEX Quantitative Proteomics Tool, introduced here, is a free open source Java application that supports the APEX protein quantitation technique. The APEX tool uses data from standard tandem mass spectrometry proteomics experiments and provides computational support for APEX protein abundance quantitation through a set of graphical user interfaces that partition thparameter controls for the various processing tasks. The tool also provides a Z-score analysis for identification of significant differential protein expression, a utility to assess APEX classifier performance via cross validation, and a utility to merge multiple APEX results into a standardized format in preparation for further statistical analysis. Conclusion: The APEX Quantitative Proteomics Tool provides a simple means to quickly derive hundreds to thousands of protein abundance values from standard liquid chromatography-tandem mass spectrometry proteomics datasets. The APEX tool provides a straightforward intuitive interface design overlaying a highly customizable computational workflow to produce protein abundance values from LC-MS/MS datasets.National Institute of Allergy and Infectious Diseases (NIAID) N01-AI15447National Institutes of HealthNational Science Foundation, the Welsh and Packard FoundationsInternational Human Frontier Science ProgramCenter for Systems and Synthetic Biolog

Association of PPARγ2 polymorphisms with carcass and meat quality traits in a Pietrain x Jinhua F2 population

The PPARγ2 gene is a key regulator of both proliferation and preadipocyte differentiation in mammals. Herein its genotype and allele frequencies were analyzed using PCR-SSCP in eight pig breeds (N = 416). Two kinds of polymorphisms of the PPARγ2 gene were detected, including a previously reported shift SNP A177G (Met59Val) in exon 1 and a novel silent mutation G876A in exon 5. The results revealed that European pig breeds carry a higher allele A frequency at the A177G locus and a fixed GG genotype at the G876A locus. Allele A at the G876A locus was only found in Jinhua pigs. The association between haplotype (A177G/G876A) and carcass and meat quality traits was analyzed in a Pietrain x Jinhua F2 population (N = 248). The PPARγ2 gene was found to be significantly associated with backfat thickness at the shoulder (p < 0.05), 6–7th ribs (p < 0.01), last rib (p < 0.01), gluteus medius (p <0.05) and ham weight (p < 0.01). Significant effects of different haplotypes on ham weight and backfat thickness at the 6–7th ribs, last rib, and gluteus medius were also observed

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein

Visuomotor Cerebellum in Human and Nonhuman Primates

In this paper, we will review the anatomical components of the visuomotor cerebellum in human and, where possible, in non-human primates and discuss their function in relation to those of extracerebellar visuomotor regions with which they are connected. The floccular lobe, the dorsal paraflocculus, the oculomotor vermis, the uvula–nodulus, and the ansiform lobule are more or less independent components of the visuomotor cerebellum that are involved in different corticocerebellar and/or brain stem olivocerebellar loops. The floccular lobe and the oculomotor vermis share different mossy fiber inputs from the brain stem; the dorsal paraflocculus and the ansiform lobule receive corticopontine mossy fibers from postrolandic visual areas and the frontal eye fields, respectively. Of the visuomotor functions of the cerebellum, the vestibulo-ocular reflex is controlled by the floccular lobe; saccadic eye movements are controlled by the oculomotor vermis and ansiform lobule, while control of smooth pursuit involves all these cerebellar visuomotor regions. Functional imaging studies in humans further emphasize cerebellar involvement in visual reflexive eye movements and are discussed