The study of the relevance of macro- and microelements in the hair of young wrestlers depending on the style of wrestling.

While participating in an intensive training process, the athlete's body requires not only energy, but also specific macro- and microelements. The purpose of this study was to show the meaning of monitoring the level of mineral trace elements in athletes-wrestlers during physical activity. As an experimental group, 66 male wrestlers aged 18-20 years with at least 3 years of intensive wrestling experience were examined. The control group consisted of 92 young cadets of military school aged 18-20 years, who had previous sports training, but were not engaged in wrestling. To determine the quantitative content of trace elements, the hair was cut from the back of the head for the entire length in an amount of at least 0.1 g. an examined using the mass spectrometer ICP-MS Agilent 7900. Strong positive correlations were found for sodium with potassium and rubidium, magnesium with calcium, potassium with rubidium, and rubidium with caesium among wrestlers. Wrestlers were found to have higher levels of a number of macro- and microelements, including toxic ones.

Improved survival prognostication of node-positive malignant melanoma patients utilizing shotgun proteomics guided by histopathological characterization and genomic data.

Metastatic melanoma is one of the most common deadly cancers, and robust biomarkers are still needed, e.g. to predict survival and treatment efficiency. Here, protein expression analysis of one hundred eleven melanoma lymph node metastases using high resolution mass spectrometry is coupled with in-depth histopathology analysis, clinical data and genomics profiles. This broad view of protein expression allowed to identify novel candidate protein markers that improved prediction of survival in melanoma patients. Some of the prognostic proteins have not been reported in the context of melanoma before, and few of them exhibit unexpected relationship to survival, which likely reflects the limitations of current knowledge on melanoma and shows the potential of proteomics in clinical cancer research.

Correlation of histopathologic characteristics to protein expression and function in malignant melanoma.

BACKGROUND: Metastatic melanoma is still one of the most prevalent skin cancers, which upon progression has neither a prognostic marker nor a specific and lasting treatment. Proteomic analysis is a versatile approach with high throughput data and results that can be used for characterizing tissue samples. However, such analysis is hampered by the complexity of the disease, heterogeneity of patients, tumors, and samples themselves. With the long term aim of quest for better diagnostics biomarkers, as well as predictive and prognostic markers, we focused on relating high resolution proteomics data to careful histopathological evaluation of the tumor samples and patient survival information. PATIENTS AND METHODS: Regional lymph node metastases obtained from ten patients with metastatic melanoma (stage III) were analyzed by histopathology and proteomics using mass spectrometry. Out of the ten patients, six had clinical follow-up data. The protein deep mining mass spectrometry data was related to the histopathology tumor tissue sections adjacent to the area used for deep-mining. Clinical follow-up data provided information on disease progression which could be linked to protein expression aiming to identify tissue-based specific protein markers for metastatic melanoma and prognostic factors for prediction of progression of stage III disease. RESULTS: In this feasibility study, several proteins were identified that positively correlated to tumor tissue content including IF6, ARF4, MUC18, UBC12, CSPG4, PCNA, PMEL and MAGD2. The study also identified MYC, HNF4A and TGFB1 as top upstream regulators correlating to tumor tissue content. Other proteins were inversely correlated to tumor tissue content, the most significant being; TENX, EHD2, ZA2G, AOC3, FETUA and THRB. A number of proteins were significantly related to clinical outcome, among these, HEXB, PKM and GPNMB stood out, as hallmarks of processes involved in progression from stage III to stage IV disease and poor survival. CONCLUSION: In this feasibility study, promising results show the feasibility of relating proteomics to histopathology and clinical outcome, and insight thus can be gained into the molecular processes driving the disease. The combined analysis of histological features including the sample cellular composition with protein expression of each metastasis enabled the identification of novel, differentially expressed proteins. Further studies are necessary to determine whether these putative biomarkers can be utilized in diagnostics and prognostic prediction of metastatic melanoma.

Fully Enzymatic Membraneless Glucose|Oxygen Fuel Cell That Provides 0.275 mA cm(-2) in 5 mM Glucose, Operates in Human Physiological Solutions, and Powers Transmission of Sensing Data.

Coimmobilization of pyranose dehydrogenase as an enzyme catalyst, osmium redox polymers [Os(4,4'-dimethoxy-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) or [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) as mediators, and carbon nanotube conductive scaffolds in films on graphite electrodes provides enzyme electrodes for glucose oxidation. The recombinant enzyme and a deglycosylated form, both expressed in Pichia pastoris, are investigated and compared as biocatalysts for glucose oxidation using flow injection amperometry and voltammetry. In the presence of 5 mM glucose in phosphate-buffered saline (PBS) (50 mM phosphate buffer solution, pH 7.4, with 150 mM NaCl), higher glucose oxidation current densities, 0.41 mA cm(-2), are obtained from enzyme electrodes containing the deglycosylated form of the enzyme. The optimized glucose-oxidizing anode, prepared using deglycosylated enzyme coimmobilized with [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) and carbon nanotubes, was coupled with an oxygen-reducing bilirubin oxidase on gold nanoparticle dispersed on gold electrode as a biocathode to provide a membraneless fully enzymatic fuel cell. A maximum power density of 275 µW cm(-2) is obtained in 5 mM glucose in PBS, the highest to date under these conditions, providing sufficient power to enable wireless transmission of a signal to a data logger. When tested in whole human blood and unstimulated human saliva maximum power densities of 73 and 6 µW cm(-2) are obtained for the same fuel cell configuration, respectively.

Semi-automated biobank sample processing with a 384 high density sample tube robot used in cancer and cardiovascular studies.

BACKGROUND: In the postgenomic era, it has become evident that analysis of genetic and protein expression changes alone is not sufficient to understand most disease processes in e.g. cardiovascular and cancer disease. Biobanking has been identified as an important area for development and discovery of better diagnostic tools and new treatment modalities. Biobanks are developed in order to integrate the collection of clinical samples from both healthy individuals and patients and provide valuable information that will make possible improved patient care. Modern healthcare developments are intimately linked to information based on studies of patient samples from biobank archives in large scale studies. Today biobanks form important national, as well as international, networks that share and combine global resources. METHODS: We have developed and validated a novel biobanking workflow process that utilizes 384-tube systems with a high speed sample array robot with unique processing principles. RESULTS: The 384-tube format and robotic processing is incorporated into a cancer and cardiovascular diagnostic/prognostic research program with therapeutic interventions. Our biobank practice has gained acceptance within many hospitals and research units and is based on high-density sample storage with small aliquot sample volumes. The previous standard of 5-10 mL sample volume tubes is being replaced by smaller volumes of 50-70 µL blood fractions that typically result in hundreds of thousands of aliquot fractions in 384-tube systems. CONCLUSIONS: Our novel biobanking workflow process is robust and well suited for clinical studies.

A protein deep sequencing evaluation of metastatic melanoma tissues.

Malignant melanoma has the highest increase of incidence of malignancies in the western world. In early stages, front line therapy is surgical excision of the primary tumor. Metastatic disease has very limited possibilities for cure. Recently, several protein kinase inhibitors and immune modifiers have shown promising clinical results but drug resistance in metastasized melanoma remains a major problem. The need for routine clinical biomarkers to follow disease progression and treatment efficacy is high. The aim of the present study was to build a protein sequence database in metastatic melanoma, searching for novel, relevant biomarkers. Ten lymph node metastases (South-Swedish Malignant Melanoma Biobank) were subjected to global protein expression analysis using two proteomics approaches (with/without orthogonal fractionation). Fractionation produced higher numbers of protein identifications (4284). Combining both methods, 5326 unique proteins were identified (2641 proteins overlapping). Deep mining proteomics may contribute to the discovery of novel biomarkers for metastatic melanoma, for example dividing the samples into two metastatic melanoma "genomic subtypes", ("pigmentation" and "high immune") revealed several proteins showing differential levels of expression. In conclusion, the present study provides an initial version of a metastatic melanoma protein sequence database producing a total of more than 5000 unique protein identifications. The raw data have been deposited to the ProteomeXchange with identifiers PXD001724 and PXD001725.

Engineering of pyranose dehydrogenase for application to enzymatic anodes in biofuel cells.

In the search for improved glucose oxidising enzymes for biofuel cells, a number of Agaricus meleagris (Am) pyranose dehydrogenase mutants (mPDHs) exhibiting different degrees of glycosylation were produced using site-directed mutagenesis and electrochemically characterised. The response of electrodes modified with different mPDHs is compared in a mediated electron transfer mode, where the electrodes are modified with each of the mutants covalently attached to redox polymers based on polyvinylimidazole-bound osmium complexes using a cross-linking agent. Coating of each of the enzymes onto the graphite electrode surface is also used to screen for their capacity for direct electron transfer. The double mutant PDH exhibits the highest response to glucose at physiological pH in both direct and mediated electron transfer modes, producing a Jmax of ≈800 µA cm(-2) at room temperature and when "wired" to the Os-polymer having the highest formal potential. From the results obtained the double mPDH is proposed as the most suitable candidate for application to bioanode fabrication.

Further insights into the catalytical properties of deglycosylated pyranose dehydrogenase from Agaricus meleagris recombinantly expressed in Pichia pastoris.

The present study focuses on fragmented deglycosylated pyranose dehydrogenase (fdgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris . Fragmented deglycosylated PDH is formed from the deglycosylated enzyme (dgPDH) when it spontaneously loses a C-terminal fragment when stored in a buffer solution at 4 °C. The remaining larger fragment has a molecular weight of ∼46 kDa and exhibits higher volumetric activity for glucose oxidation compared with the deglycosylated and glycosylated (gPDH) forms of PDH. Flow injection amperometry and cyclic voltammetry were used to assess and compare the catalytic activity of the three investigated forms of PDH, "wired" to graphite electrodes with two different osmium redox polymers: [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) [Os(dmbpy)PVI] and [Os(4,4'-dimethoxy-2,2'-bipyridine)2(poly-(vinylimidazole))10Cl](+) [Os(dmobpy)PVI]. When "wired" with Os(dmbpy)PVI, the graphite electrodes modified with fdgPDH showed a pronounced increase in the current density with Jmax 13- and 6-fold higher than that observed for gPDH- and dgPDH-modified electrodes, making the fragmented enzyme extraordinarily attractive for further biotechnological applications. An easier access of the substrate to the active site and improved communication between the enzyme and mediator matrix are suggested as the two main reasons for the excellent performance of the fdgPDH when compared with that of gPDH and dgPDH. Three of the four glycosites in PDH: N(75), N(175), and N(252) were assigned using mass spectrometry in conjunction with endoglycosidase treatment and tryptic digestion. Determination of the asparagine residues carrying carbohydrate moieties in PDH can serve as a solid background for production of recombinant enzyme lacking glycosylation.

The enzyme thermistor--a realistic biosensor concept. A critical review.

This review describes principles and features of thermal biosensors and microbiosensors in flow injection analysis. Examples are given that illustrate the great versatility and excellent operational stability offered by thermal biosensors. The examples are mostly from work with the original type of enzyme thermistor operating with an enzyme column, but there will also be work described involving miniaturised devices including thermal lab-on-chip constructions and other types of sensing materials, such as MIPs (molecularly imprinted polymers) for both affinity and catalytic reactions. Several recently presented thermal biosensor concepts are reviewed including a thermal-electrochemical hybrid sensor for lactose based on immobilised cellobiose dehydrogenase. Another recent method is the determination of fructose using a fructose-6-phosphate kinase column. Operation with complex sample matrices such as blood, plasma and milk and how to avoid non-specific temperature effects are considered.

A novel combined thermometric and amperometric biosensor for lactose determination based on immobilised cellobiose dehydrogenase.

A novel method for lactose determination in milk is proposed. It is based on oxidation of lactose by cellobiose dehydrogenase (CDH) from the basidiomycete Phanerochaete chrysosporium, immobilised in an enzyme reactor. The reactor was prepared by cross-linking CDH onto aminopropyl-silanised controlled pore glass (CPG) beads using glutaraldehyde. The combined biosensor worked in flow injection analysis (FIA) mode and was developed for simultaneous monitoring of the thermometric signal associated with the enzymatic oxidation of lactose using p-benzoquinone as electron acceptor and the electrochemically generated current associated with the oxidation of the hydroquinone formed. A highly reproducible linear response for lactose was obtained between 0.05 mM and 30 mM. For a set of more than 500 samples an R.S.D. of less than 10% was achieved. The assay time was ca. 2 min per sample. The sensor was applied for the determination of lactose in dairy milk samples (milk with a fat content of 1.5% or 3% and also "lactose free" milk). No sample preparation except dilution with buffer was needed. The proposed method is rapid, suitable for repeated use and allows the possibility to compare results from two different detection methods, thus providing a built-in quality assurance. Some differences in the response observed between the methods indicate that the dual approach can be useful in mechanistic studies of redox enzymes. In addition, a dual system opens up interesting possibilities for studies of enzyme properties and mechanisms.

Lectin typing of Campylobacter jejuni using a novel quartz crystal microbalance technique.

Seven Campylobacter jejuni strains were characterised by a lectin typing assay. The typing system was based on a quartz crystal microbalance technique (QCM) with four commercially available lectins (wheat germ agglutinin, Maackia amurensis lectin, Lens culinaris agglutinin, and Concanavalin A), which were chosen for their differing carbohydrate specificities. Initially, the gold surfaces of the quartz crystals were modified with 11-mercaptoundecanoic acid followed by lectin immobilisation using a conventional amine-coupling technique. Bacterial cells were applied for lectin typing without preliminary treatment, and resonant frequency and dissipation responses were recorded. The adhesion of microorganisms on lectin surfaces was confirmed by atomic force microscopy. Scanning was performed in the tapping mode and the presence of bacteria on lectin-coated surfaces was successfully demonstrated. A significant difference in the dissipation response was observed for different C. jejuni strains which made it possible to use this parameter for discriminating between bacterial strains. In summary, the QCM technique proved a powerful tool for the recognition and discrimination of C. jejuni strains. The approach may also prove applicable to strain discrimination of other bacterial species, particularly pathogens.

Tracking the small with the smallest--using nanotechnology in tracking zooplankton.

A major problem when studying behavior and migration of small organisms is that many of the questions addressed for larger animals are not possible to formulate due to constraints on tracking smaller animals. In aquatic ecosystems, this problem is particularly problematic for zoo- and phytoplankton, since tracking devices are too heavy to allow the organism to act naturally. However, recent advances in nanotechnology have made it possible to track individual animals and thereby to focus on important and urgent questions which previously have not been possible to address. Here we report on a novel approach to track movement and migratory behavior of millimeter sized aquatic animals, particularly Daphnia magna, using the commercially available nanometer sized fluorescent probes known as quantum dots. Experimental trials with and without quantum dots showed that they did not affect behavior, reproduction or mortality of the tested animals. Compared to previously used methods to label small animals, the nano-labeling method presented here offers considerable improvements including: 24 h fluorescence, studies in both light and darkness, much improved optical properties, potential to study large volumes and even track animals in semi-natural conditions. Hence, the suggested method, developed in close cooperation between biologists, chemists and physicists, offers new opportunities to routinely study zooplankton responses to light, food and predation, opening up advancements within research areas such as diel vertical/horizontal migration, partial migration and other differences in intra- and interspecific movements and migration.

Fructose-selective calorimetric biosensor in flow injection analysis.

A highly selective, interference free biosensor for the measurement of fructose in real syrup samples was developed. The assay is based on the phosphorylation of D(-)fructose to fructose-6-phosphate by hexokinase and subsequent conversion of fructose-6-phosphate to fructose-1,6-biphosphate by fructose-6-phosphate-kinase. The heat liberated in the second reaction is monitored using an enzyme thermistor. The major advantages of this biosensor are rapid and selective measurement of fructose without the need to eliminate glucose and inexpensive FIA-based, mediator-free calorimetric measurement suitable for regular fructose analysis. This method was optimised for parameters, such as pH, ionic strength, interference, operational stability and shelf life. Good and reproducible linearity (0.5-6.0 mM) with a detection limit of 0.12 mM was obtained. Fructose determination in commercial syrup samples and spiked samples confirmed the reliability of this set-up and technique. The biosensor gave reproducible results with good overall stability for continuous measurements over a period of three months besides a useful shelf life of six months. The method could be used for routine fructose monitoring in food samples.

A study of glycoprotein-lectin interactions using quartz crystal microbalance.

A study of biospecific interactions between lectins and glycoproteins using a quartz crystal microbalance biosensor with dissipation monitoring (QCM-D) was reported. Four lectins were covalently immobilised on the thiol-modified gold electrode of the QCM chips in order to obtain sensing surfaces. The frequency shift served as analytical signal and the dissipation shift provided additional information about the viscoelastic properties of the glycoprotein-lectin complex formed on the surface of the QCM chip. The working conditions of the assay were optimised. The interaction between different lectins and glycoproteins was characterised by specific frequency shifts and each glycoprotein displayed its own unique lectin-binding pattern. This lectin pattern can serve as a finger print for the discrimination between various glycoproteins. The biosensor enabled quantitative determination of glycoproteins in the concentration range of 50 microg mL(-1) to 1 mg mL(-1) with good linearity and R.S.D. of less than 6.0%. An additional advantage of the proposed biosensor was the possibility to re-use the same lectin surfaces during a long period of time (2 month) without changes in analytical response. This was experimentally achieved by the application of a proper regeneration solution (10 mM glycine-HCl, pH 2.5). The lectin-based quartz crystal microbalance technique is suitable both for rapid screening and for quantitative assay of serum glycoproteins.

Why do we trust? Moving beyond individual to dyadic perceptions.

Trust between coworkers is critical to the success of organizations and teams. This is especially true for those who are geographically dispersed and who must interact virtually. The authors studied the factors related to the development of trust between pairs of coworkers (dyads) in a new product development team. Some of the members were colocated, and others worked virtually. Using the actor-partner interdependence model, the authors found reciprocal effects for propensity to trust and trust in dyads. They found that propensity has greater influence on trust for virtual dyads and that trust has less influence on organizational citizenship when partners are virtual. Trustworthiness was shown to fully mediate the influence of trusting predisposition on trust.

Development of fluorescence polarization immunoassay for the rapid detection of 6-chloronicotinic acid: main metabolite of neonicotinoid insecticides.

A fluorescence polarization immunoassay (FPIA) for the quantitative determination of 6-chloronicotinic acid (6-CNA) using polyclonal antibody was developed. The 6-CNA-protein (bovine serum albumin and soybean trypsin inhibitor) conjugates and fluorescein-labeled 6-CNA derivative (tracer) were prepared and used as the immunogens and tracer, respectively. The synthesized tracer was purified by thin layer chromatography (TLC) and showed a good binding to antiserum (73/5) which was obtained from the immunized rabbit (No. 73) with 6-CNA-BSA conjugate. The detection limit (10% inhibition) of FPIA was 4 microg/mL, and IC(50) value was 32 microg/mL. The FPIA showed a cross-reaction for 5-amino-2-chloropyridine (60%), but no cross-reaction for other pesticides was observed. Recoveries for spiked apple, urine, soil, and water samples (5, 50, and 500 ppm) averaging between 78.6 +/- 8.8 and 114 +/- 18% were reasonable and in good agreement with the amounts spiked. Although the developed FPIA possesses low sensitivity, this assay is more simple and quick than other analytical methods, such as high performance liquid chromatography and gas chromatography. Thus, the developed FPIA method could be a useful tool for express screening 6-CNA in agricultural, environmental, and biological samples.