Determination of ethyl glucuronide in human serum by capillary zone electrophoresis and an immunoassay

Ethyl glucuronide (EtG) is a marker of recent alcohol consumption. For the optimization of the analysis of EtG by CZE with indirect absorbance detection, the use of capillaries with permanent and dynamic wall coatings, the composition of the BGE, and various sample preparation procedures, including dilution with water, ultrafiltration, protein precipitation, and SPE, were investigated. Two validated screening assays for the determination of EtG in human serum, a CZE-based approach and an enzyme immunoassay (EIA), are described. The CZE assay uses a coated capillary, 2,4-dimethylglutaric acid as an internal standard, and a pH 4.65 BGE comprising 9 mM nicotinic acid, epsilon-aminocaproic acid and 10% v/v ACN. Proteins are removed via precipitation with ACN prior to analysis and the LOQ is 0.50 mg/L. The EIA is based upon commercial reagents which are promoted for the determination of urinary EtG. Krebs-Ringer solution containing 5% BSA is used as a calibration matrix. All samples are ultrafiltered prior to analysis of the ultrafiltrate on a Mira Plus analyzer. Assay calibration ranged between 0 and 2 mg/L and the upper reference limit was determined to be 0.05 mg/L. Both assays proved to be suitable for the analysis of samples from different individuals. For EtG levels above 0.50 mg/L, good agreement was observed for the comparison of the results of the two methods.

Novel functional profiling approach combining reverse phase protein microarrays and human 3-D ex vivo tissue cultures: expression of apoptosis-related proteins in human colon cancer

Cancer is caused by a complex pattern of molecular perturbations. To understand the biology of cancer, it is thus important to look at the activation state of key proteins and signaling networks. The limited amount of available sample material from patients and the complexity of protein expression patterns make the use of traditional protein analysis methods particularly difficult. In addition, the only approach that is currently available for performing functional studies is the use of serial biopsies, which is limited by ethical constraints and patient acceptance. The goal of this work was to establish a 3-D ex vivo culture technique in combination with reverse-phase protein microarrays (RPPM) as a novel experimental tool for use in cancer research. The RPPM platform allows the parallel profiling of large numbers of protein analytes to determine their relative abundance and activation level. Cancer tissue and the respective corresponding normal tissue controls from patients with colorectal cancer were cultured ex vivo. At various time points, the cultured samples were processed into lysates and analyzed on RPPM to assess the expression of carcinoembryonic antigen (CEA) and 24 proteins involved in the regulation of apoptosis. The methodology displayed good robustness and low system noise. As a proof of concept, CEA expression was significantly higher in tumor compared with normal tissue (p<0.0001). The caspase 9 expression signal was lower in tumor tissue than in normal tissue (p<0.001). Cleaved Caspase 8 (p=0.014), Bad (p=0.007), Bim (p=0.007), p73 (p=0.005), PARP (p<0.001), and cleaved PARP (p=0.007) were differentially expressed in normal liver and normal colon tissue. We demonstrate here the feasibility of using RPPM technology with 3-D ex vivo cultured samples. This approach is useful for investigating complex patterns of protein expression and modification over time. It should allow functional proteomics in patient samples with various applications such as pharmacodynamic analyses in drug development.

Cellular and network mechanisms of rhythm generation in spinal cord circuits

The generation of rhythmic electrical activity is a prominent feature of spinal cord circuits that is used for locomotion and also for circuit refinement during development. The mechanisms involved in rhythm generation in spinal cord networks are not fully understood. It is for example not known whether spinal cord rhythms are driven by pacemaker neurons and if yes, which neurons are involved in this function. We studied the mechanisms involved in rhythm generation in slice cultures from fetal rats that were grown on multielectrode arrays (MEAs). We combined multisite extracellular recordings from the MEA electrodes with intracellular patch clamp recordings from single neurons. We found that spatially restricted oscillations of activity appeared in most of the cultures spontaneously. Such activity was based on intrinsic activity in a percentage of the neurons that could activate the spinal networks through recurrent excitation. The local oscillator networks critically involved NMDA, AMPA and GABA / glycine receptors at subsequent phases of the oscillation cycle. Intrinsic spiking in individual neurons (in the absence of functional synaptic coupling) was based on persistent sodium currents. Intrinsic firing as well as persistent sodium currents were increased by 5-HT through 5-HT2 receptors. Comparing neuronal activity to muscle activity in co-cultures of spinal cord slices with muscle fibers we found that a percentage of the intrinsically spiking neurons were motoneurons. These motoneurons were electrically coupled among each other and they could drive the spinal networks through cholinergic recurrent excitation. These findings open the possibility that during development rhythmic activity in motoneurons is not only involved in circuit refinement downstream at the neuromuscular endplates but also upstream at the level of spinal cord circuits.

Prosthetically driven, computer-guided implant planning for the edentulous maxilla: a model study

OBJECTIVES: To analyze computer-assisted diagnostics and virtual implant planning and to evaluate the indication for template-guided flapless surgery and immediate loading in the rehabilitation of the edentulous maxilla. MATERIALS AND METHODS: Forty patients with an edentulous maxilla were selected for this study. The three-dimensional analysis and virtual implant planning was performed with the NobelGuide software program (Nobel Biocare, Göteborg, Sweden). Prior to the computer tomography aesthetics and functional aspects were checked clinically. Either a well-fitting denture or an optimized prosthetic setup was used and then converted to a radiographic template. This allowed for a computer-guided analysis of the jaw together with the prosthesis. Accordingly, the best implant position was determined in relation to the bone structure and prospective tooth position. For all jaws, the hypothetical indication for (1) four implants with a bar overdenture and (2) six implants with a simple fixed prosthesis were planned. The planning of the optimized implant position was then analyzed as follows: the number of implants was calculated that could be placed in sufficient quantity of bone. Additional surgical procedures (guided bone regeneration, sinus floor elevation) that would be necessary due the reduced bone quality and quantity were identified. The indication of template-guided, flapless surgery or an immediate loaded protocol was evaluated. RESULTS: Model (a) - bar overdentures: for 28 patients (70%), all four implants could be placed in sufficient bone (total 112 implants). Thus, a full, flapless procedure could be suggested. For six patients (15%), sufficient bone was not available for any of their planned implants. The remaining six patients had exhibited a combination of sufficient or insufficient bone. Model (b) - simple fixed prosthesis: for 12 patients (30%), all six implants could be placed in sufficient bone (total 72 implants). Thus, a full, flapless procedure could be suggested. For seven patients (17%), sufficient bone was not available for any of their planned implants. The remaining 21 patients had exhibited a combination of sufficient or insufficient bone. DISCUSSION: In the maxilla, advanced atrophy is often observed, and implant placement becomes difficult or impossible. Thus, flapless surgery or an immediate loading protocol can be performed just in a selected number of patients. Nevertheless, the use of a computer program for prosthetically driven implant planning is highly efficient and safe. The three-dimensional view of the maxilla allows the determination of the best implant position, the optimization of the implant axis, and the definition of the best surgical and prosthetic solution for the patient. Thus, a protocol that combines a computer-guided technique with conventional surgical procedures becomes a promising option, which needs to be further evaluated and improved.

Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins

Lipoproteins are a heterogeneous population of blood plasma particles composed of apolipoproteins and lipids. Lipoproteins transport exogenous and endogenous triglycerides and cholesterol from sites of absorption and formation to sites of storage and usage. Three major classes of lipoproteins are distinguished according to their density: high-density (HDL), low-density (LDL) and very low-density lipoproteins (VLDL). While HDLs contain mainly apolipoproteins of lower molecular weight, the two other classes contain apolipoprotein B and apolipoprotein (a) together with triglycerides and cholesterol. HDL concentrations were found to be inversely related to coronary heart disease and LDL/VLDL concentrations directly related. Although many studies have been published in this area, few have concentrated on the exact protein composition of lipoprotein particles. Lipoproteins were separated by density gradient ultracentrifugation into different subclasses. Native gel electrophoresis revealed different gel migration behaviour of the particles, with less dense particles having higher apparent hydrodynamic radii than denser particles. Apolipoprotein composition profiles were measured by matrix-assisted laser desorption/ionization-mass spectrometry on a macromizer instrument, equipped with the recently introduced cryodetector technology, and revealed differences in apolipoprotein composition between HDL subclasses. By combining these profiles with protein identifications from native and denaturing polyacrylamide gels by liquid chromatography-tandem mass spectrometry, we characterized comprehensively the exact protein composition of different lipoprotein particles. We concluded that the differential display of protein weight information acquired by macromizer mass spectrometry is an excellent tool for revealing structural variations of different lipoprotein particles, and hence the foundation is laid for the screening of cardiovascular disease risk factors associated with lipoproteins.

Albrecht von Haller. Leben - Werk- Epoche

Zum 300. Geburtstag des Dichters, Sammlers, Professors und Experimentalforschers am 16. Oktober 2008.Der Universalgelehrte Albrecht von Haller (1708-1777) ist eine der zentralen Gestalten des Jahrhunderts der Aufklärung. Als Dichter und Gelehrter, Sammler und Enzyklopädist, Universitätsprofessor und Experimentalforscher, Gesellschaftspräsident und Korrespondent, profilierter Autor und mächtiger Rezensent, Magistrat und orthodoxer Christ spiegelt er zahlreiche Strömungen, Ereignisse und Verhältnisse seiner Zeit.Der Band führt in die einzelnen Wirkungsgebiete Hallers ein. In drei Abschnitten Leben und Umfeld, Werk und Wirkung und Haller in seiner Zeit liefern die Beiträge vielfältige und überraschende Einblicke in die Problemlagen und Entwicklungen seiner Epoche.

Aseptic intussuscepted incidental appendectomy: three successful cases

BACKGROUND: Incidental appendectomy remains a controversial issue. We aimed to collect experience using a modified surgical technique that could be applied securely in infants. METHODS: We performed aseptic intussuscepted incidental appendectomy (AIIA) in three patients using a technique that is thought to assure appendix necrosis along with intact cecal wall. RESULTS: There was no perioperative morbidity due to AIIA in the three patients. In two infants the necrotic appendix was found in the diaper. One infant died secondary to diaphragmatic hernia. Autopsy with histological examination revealed that the cecum was intact along with appendix necrosis. CONCLUSIONS: Modified AIIA could securely be performed in the 3 reported cases. We advocate prospective evaluation of the method.

Engaging Local Communities in Low Emissions Land-Use Planning: a Case Study from Laos

Reducing Emissions from Deforestation and Forest Degradation and enhancing forest carbon stocks (REDD+) is a performance-based payment mechanism currently being debated in international and national environmental policy and planning forums. As the mechanism is based on conditionality, payments must reflect land stewards’ level of compliance with carbon-efficient management practices. However, lack of clarity in land governance and carbon rights could undermine REDD+ implementation. Strategies are needed to avoid perverse incentives resulting from the commoditization of forest carbon stocks and, importantly, to identify and secure the rights of legitimate recipients of future REDD+ payments. We propose a landscape-level approach to address potential conflicts related to carbon tenure and REDD+ benefit sharing. We explore various land-tenure scenarios and their implications for carbon ownership in the context of a research site in northern Laos. Our case study shows that a combination of relevant scientific tools, knowledge, and participatory approaches can help avoid the marginalization of rural communities during the REDD+ process. The findings demonstrate that participatory land-use planning is an important step in ensuring that local communities are engaged in negotiating REDD+ schemes and that such negotiations are transparent. Local participation and agreements on land-use plans could provide a sound basis for developing efficient measurement, reporting, and verification systems for REDD+.

Class II treatment by extraction of maxillary first molars or Herbst appliance: dentoskeletal and soft tissue effects in comparison

AIM To compare dentoskeletal and soft tissue treatment effects of two alternative Class II division 1 treatment modalities (maxillary first permanent molar extraction versus Herbst appliance). METHODS One-hundred-fifty-four Class II division 1 patients that had either been treated with extractions of the upper first molars and a lightwire multibracket (MB) appliance (n = 79; 38 girls, 41 boys) or non-extraction by means of a Herbst-MB appliance (n = 75; 35 girls, 40 boys). The groups were matched on age and sex. The average age at the start of treatment was 12.7 years for the extraction and for 13.0 years for the Herbst group. Pretreatment (T1) and posttreatment (T2) lateral cephalograms were retrospectively analyzed using a standard cephalometric analysis and the sagittal occlusal analysis according to Pancherz. RESULTS The SNA decrease was 1.10° (p = 0.001) more pronounced in the extraction group, the SNB angle increased 1.49° more in the Herbst group (p = 0.000). In the extraction group, a decrease in SNB angle (0.49°) was observed. The soft tissue profile convexity (N-Sn-Pog) decreased in both groups, which was 0.78° more (n. s.) pronounced in the Herbst group. The nasolabial angle increased significantly more (+ 2.33°, p = 0.025) in the extraction group. The mechanism of overjet correction in the extraction group was predominantly dental (65% dental and 35% skeletal changes), while in the Herbst group it was predominantly skeletal (58% skeletal and 42% dental changes) in origin. CONCLUSION Both treatment methods were successful and led to a correction of the Class II division 1 malocclusion. Whereas for upper first molar extraction treatment more dental and maxillary effects can be expected, in case of Herbst treatment skeletal and mandibular effects prevail.

Long-term clinical, microbiological, and radiographic outcomes of Brånemark™ implants installed in augmented maxillary bone for fixed full-arch rehabilitation

PURPOSE The purpose of this study was to document the long-term outcome of Brånemark implants installed in augmented maxillary bone and to identify parameters that are associated with peri-implant bone level. MATERIAL AND METHODS Patients of a periodontal practice who had been referred to a maxillofacial surgeon for iliac crest bone grafting in the atrophic maxilla were retrospectively recruited. Five months following grafting, they received 7-8 turned Brånemark implants. Following submerged healing of another 5 months, implants were uncovered and restorative procedures for fixed rehabilitation were initiated 2-3 months thereafter. The primary outcome variable was bone level defined as the distance from the implant-abutment interface to the first visible bone-to-implant contact. Secondary outcome variables included plaque index, bleeding index, probing depth, and levels of 40 species in subgingival plaque samples as identified by means of checkerboard DNA-DNA hybridization. RESULTS Nine out of 16 patients (eight females, one male; mean age 59) with 71 implants agreed to come in for evaluation after on average 9 years (SD 4; range 3-13) of function. One implant was deemed mobile at the time of inspection. Clinical conditions were acceptable with 11% of the implants showing pockets ≥ 5 mm. Periodontopathogens were encountered frequently and in high numbers. Clinical parameters and bacterial levels were highly patient dependent. The mean bone level was 2.30 mm (SD 1.53; range 0.00-6.95), with 23% of the implants demonstrating advanced resorption (bone level > 3 mm). Regression analysis showed a significant association of the patient (p < .001) and plaque index (p = .007) with bone level. CONCLUSIONS The long-term outcome of Brånemark implants installed in iliac crest-augmented maxillary bone is acceptable; however, advanced peri-implant bone loss is rather common and indicative of graft resorption. This phenomenon is patient dependent and seems also associated with oral hygiene.