Cation Effects on the Acidic Oxygen Reduction Reaction at Carbon Surfaces.

Hydrogen peroxide (H2O2) is a widely used green oxidant. Until now, research has focused on the development of efficient catalysts for the two-electron oxygen reduction reaction (2e- ORR). However, electrolyte effects on the 2e- ORR have remained little understood. We report a significant effect of alkali metal cations (AMCs) on carbons in acidic environments. The presence of AMCs at a glassy carbon electrode shifts the half wave potential from -0.48 to -0.22 VRHE. This cation-induced enhancement effect exhibits a uniquely sensitive on/off switching behavior depending on the voltammetric protocol. Voltammetric and in situ X-ray photoemission spectroscopic evidence is presented, supporting a controlling role of the potential of zero charge of the catalytic enhancement. Density functional theory calculations associate the enhancement with stabilization of the *OOH key intermediate as a result of locally induced field effects from the AMCs. Finally, we developed a refined reaction mechanism for the H2O2 production in the presence of AMCs.

Establishment of a resazurin-based aortic valve tissue viability assay for dynamic culture in a microphysiological system.

BACKGROUND/AIM: Tissue pathogenesis of aortic valve (AV) stenosis is research focus in cardiac surgery. Model limitations of conventional 2D culture of human or porcine valvular interstitial/endothelial cells (VIC/VECs) isolated from aortic valve tissues but also limited ability of (small) animal models to reflect human (patho)physiological situation in AV position raise the need to establish an in vitro setup using AV tissues. Resulting aim is to approximate (patho)physiological conditions in a dynamic pulsatile Microphysiological System (MPS) to culture human and porcine AV tissue with preservation of tissue viability but also defined ECM composition. MATERIALS/METHODS: A tissue incubation chamber (TIC) was designed to implement human or porcine tissues (3×5 mm2) in a dynamic pulsatile culture in conventional cell culture ambience in a MPS. Cell viability assays based on lactate dehydrogenase (LDH)-release or resazurin-conversion were tested for applicability in the system and applied for a culture period of 14 days with interval evaluation of tissue viability on every other day. Resazurin-assay setup was compared in static vs. dynamic culture using varying substance saturation settings (50-300µM), incubation times and tissue masses and was consequently adapted. RESULTS: Sterile dynamic culture of human and porcine AV tissue segments was established at a pulsatile flow rate range of 0.9-13.4µl/s. Implementation of tissues was realized by stitching the material in a thermoplastic polyurethane (TPU)-ring and insertion in the TIC-MPS-system. Culture volume of 2 ml caused LDH dilution not detectable in standard membrane integrity assay setup. Therefore, detection of resazurin-conversion of viable tissue was investigated. Optimal incubation time for viability conversion was determined at two hours at a saturated concentration of 300µM resazurin. Measurement in static conditions was shown to offer comparable results as dynamic condition but allowing optimal handling and TIC sterilization protocols for long term culture. Preliminary results revealed favourable porcine AV tissue viability over a 14 day period confirmed via resazurin-assay comparing statically cultured tissue counterparts. CONCLUSIONS: Human and porcine AV tissue can be dynamically cultured in a TIC-MPS with monitoring of tissue viability using an adapted resazurin-assay setup. Preliminary results reveal advantageous viability of porcine AV tissues after dynamic TIC-MPS culture compared to static control.

Tired of feeling tired - The role of circulating inflammatory biomarkers and long-term cancer related fatigue in breast cancer survivors.

BACKGROUND: Low-grade inflammation has been associated with cancer related fatigue (CRF). However, most studies focused on CRF during or shortly after treatment. Longitudinal studies are rare with inconsistent results. We assessed the association of inflammatory biomarkers with total CRF and all subdomains (physical, cognitive, affective) in long-term breast cancer survivors. METHOD: Patients recruited between 2002 and 2005 provided information on CRF at first follow-up (FU1) (N = 1292) and second follow-up (FU2) (N = 1205), after a median of 6.2 years and 11.7 years, respectively. Associations of 11 inflammatory biomarkers with CRF at FU1 and at FU2 were assessed using linear regression models. Logistic regression models were used to compare patients fatigued at both time-points and those never fatigued (N = 932). RESULTS: C-reactive protein (CRP) was significantly associated with total CRF at FU1 (ß = 1.47, 95%CI = 0.62-2.31, p = 0.0007), at FU2 (ß = 1.98, 95 %CI = 0.96-2.99, p = 0.0001) and with persistent CRF (OR = 1.29, 95%CI = 1.13-1.47, p < 0.0001). IL-6 levels were associated with total CRF at FU1 (ß = 1.01, 95%CI = 0.43-1.59, p = 0.0006), but not with CRF at FU2 or persistent CRF. No association remained significant after adjustment for relevant covariates. DISCUSSION: CRP and Il-6 were associated with risk of CRF in long-term breast cancer survivors, but were not independent of other known risk factors, suggesting that currently studied inflammatory markers are not suitable to identify patients at risk of long-term CRF.

Prognostic gene expression signature for high-grade serous ovarian cancer.

BACKGROUND: Median overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is ∼4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC. PATIENTS AND METHODS: Expression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies. RESULTS: Expression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years. CONCLUSION: The OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.

Validation of inflammatory genetic variants associated with long-term cancer related fatigue in a large breast cancer cohort.

BACKGROUND: Studies to date have reported several associations between single nucleotide polymorphisms (SNPs) and cancer related fatigue (CRF), but have been limited by small sample sizes, missing adjustment for relevant covariates or multiple testing, as well as varying CRF definitions, i.e. time and method of assessment. This study aimed to validate previously reported associations using the largest independent breast cancer sample to date and to evaluate further functional cytokine variants in relation to total CRF and all relevant CRF subdomains (physical, cognitive, and affective CRF). METHOD: 45 candidate SNPs in inflammatory pathway genes were selected based on previous reports (16 SNPs) or regulatory function (29 SNPs). Breast cancer patients recruited between 2002 and 2005 provided information on CRF at first follow-up (FU1) (N = 1389) and second follow-up (FU2) (N = 950), a median of 6.2 years and 11.7 years respectively after diagnosis. SNP associations were assessed using linear regression models on CRF scores separately for FU1 and FU2. Additionally, patients with persistent fatigue (fatigued at both time-points) were compared to those never fatigued using logistic regression models (N = 684). All analyses were adjusted for relevant covariates. Secondary analyses were conducted for CRF subdomains. RESULTS: For total CRF none of the previously reported associations were confirmed after correction for multiple testing. The p-value distribution of all SNPs was not different than the one expected by chance. Analyses of CRF subdomains yielded a significant association between TNF-α rs3093662 and persistent physical CRF (Odds Ratio (OR) = 3.23, 95% Confidence Interval (CI) = 1.71-6.10, p = 0.0003). CONCLUSION: We were unable to confirm previously reported findings, suggesting that individual SNPs are unlikely to be of clinical utility. Further investigations in well powered studies are warranted, which consider genetic heterogeneity according to subdomains of CRF.

Highly efficient Tsuji-Trost allylation in water catalyzed by Pd-nanoparticles.

Palladium nanoparticles stabilized by poly(vinylpyrrolidone) catalyze Tsuji-Trost allylations in water with very high turnover numbers. The di-allylation of methylene active compounds and the allylation of bio-based phenols was performed in high yield. The allylation of lignin showed a high selectivity towards the phenolic OH groups.

Insights into Nitrate-Reducing Fe(II) Oxidation Mechanisms through Analysis of Cell-Mineral Associations, Cell Encrustation, and Mineralogy in the Chemolithoautotrophic Enrichment Culture KS.

Most described nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOB) are mixotrophic and depend on organic cosubstrates for growth. Encrustation of cells in Fe(III) minerals has been observed for mixotrophic NRFeOB but not for autotrophic phototrophic and microaerophilic Fe(II) oxidizers. So far, little is known about cell-mineral associations in the few existing autotrophic NRFeOB. Here, we investigate whether the designated autotrophic Fe(II)-oxidizing strain (closely related to Gallionella and Sideroxydans) or the heterotrophic nitrate reducers that are present in the autotrophic nitrate-reducing Fe(II)-oxidizing enrichment culture KS form mineral crusts during Fe(II) oxidation under autotrophic and mixotrophic conditions. In the mixed culture, we found no significant encrustation of any of the cells both during autotrophic oxidation of 8 to 10 mM Fe(II) coupled to nitrate reduction and during cultivation under mixotrophic conditions with 8 to 10 mM Fe(II), 5 mM acetate, and 4 mM nitrate, where higher numbers of heterotrophic nitrate reducers were present. Two pure cultures of heterotrophic nitrate reducers (Nocardioides and Rhodanobacter) isolated from culture KS were analyzed under mixotrophic growth conditions. We found green rust formation, no cell encrustation, and only a few mineral particles on some cell surfaces with 5 mM Fe(II) and some encrustation with 10 mM Fe(II). Our findings suggest that enzymatic, autotrophic Fe(II) oxidation coupled to nitrate reduction forms poorly crystalline Fe(III) oxyhydroxides and proceeds without cellular encrustation while indirect Fe(II) oxidation via heterotrophic nitrate-reduction-derived nitrite can lead to green rust as an intermediate mineral and significant cell encrustation. The extent of encrustation caused by indirect Fe(II) oxidation by reactive nitrogen species depends on Fe(II) concentrations and is probably negligible under environmental conditions in most habitats.IMPORTANCE Most described nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOB) are mixotrophic (their growth depends on organic cosubstrates) and can become encrusted in Fe(III) minerals. Encrustation is expected to be harmful and poses a threat to cells if it also occurs under environmentally relevant conditions. Nitrite produced during heterotrophic denitrification reacts with Fe(II) abiotically and is probably the reason for encrustation in mixotrophic NRFeOB. Little is known about cell-mineral associations in autotrophic NRFeOB such as the enrichment culture KS. Here, we show that no encrustation occurs in culture KS under autotrophic and mixotrophic conditions while heterotrophic nitrate-reducing isolates from culture KS become encrusted. These findings support the hypothesis that encrustation in mixotrophic cultures is caused by the abiotic reaction of Fe(II) with nitrite and provide evidence that Fe(II) oxidation in culture KS is enzymatic. Furthermore, we show that the extent of encrustation caused by indirect Fe(II) oxidation by reactive nitrogen species depends on Fe(II) concentrations and is probably negligible in most environmental habitats.

Geochemistry and Mineralogy of Western Australian Salt Lake Sediments: Implications for Meridiani Planum on Mars.

UNLABELLED: Hypersaline lakes are characteristic for Western Australia and display a rare combination of geochemical and mineralogical properties that make these lakes potential analogues for past conditions on Mars. In our study, we focused on the geochemistry and mineralogy of Lake Orr and Lake Whurr. While both lakes are poor in organic carbon (<1%), the sediments' pH values differ and range from 3.8 to 4.8 in Lake Orr and from 5.4 to 6.3 in Lake Whurr sediments. Lake Whurr sediments were dominated by orange and red sediment zones in which the main Fe minerals were identified as hematite, goethite, and tentatively jarosite and pyrite. Lake Orr was dominated by brownish and blackish sediments where the main Fe minerals were goethite and another paramagnetic Fe(III)-phase that could not be identified. Furthermore, a likely secondary Fe(II)-phase was observed in Lake Orr sediments. The mineralogy of these two salt lakes in the sampling area is strongly influenced by events such as flooding, evaporation, and desiccation, processes that explain at least to some extent the observed differences between Lake Orr and Lake Whurr. The iron mineralogy of Lake Whurr sediments and the high salinity make this lake a suitable analogue for Meridiani Planum on Mars, and in particular the tentative identification of pyrite in Lake Whurr sediments has implications for the interpretation of the Fe mineralogy of Meridiani Planum sediments. KEY WORDS: Western Australia-Salt lakes-Jarosite-Hematite-Pyrite-Mars analogue. Astrobiology 16, 525-538.

Formation of chloroform and tetrachloroethene by Sinorhizobium meliloti strain 1021.

UNLABELLED: The mechanisms and organisms involved in the natural formation of volatile organohalogen compounds (VOX) are largely unknown. We provide evidence that the common and widespread soil bacterium Sinorhizobium meliloti strain 1021 is capable of producing up to 3338·6 ± 327·8 ng l(-1) headspace volume of chloroform (CHCl3 ) and 807·8 ± 13·5 ng l(-1)  headspace volume of tetrachloroethene (C2 Cl4 ) within 1 h when grown in soil extract medium. Biotic VOX formation has been suggested to be linked to the activity of halogenating enzymes such as haloperoxidases. We tested if the observed VOX formation by S. meliloti can be attributed to one of its chloroperoxidases (Smc01944) that is highly expressed in the presence of H2 O2. However, addition of 10 mmol l(-1) H2 O2 to the S. meliloti cultures decreased VOX formation by 52% for chloroform and 25% for tetrachloroethene, while viable cell numbers decreased by 23%. Interestingly, smc01944 gene expression increased 450-fold. The quantification of extracellular chlorination activity in cell suspension experiments did not provide evidence for a role of S. meliloti chloroperoxidases in the observed VOX formation. This suggests that a momentarily unknown mechanism which requires no H2 O2 might be responsible for the VOX formation by S. meliloti. Regardless of the underlying mechanism our results suggest that the soil bacterium S. meliloti might be an important source of VOX in soils. SIGNIFICANCE AND IMPACT OF THE STUDY: Volatile organohalogen compounds (VOX) strongly influence atmospheric chemistry and Earth's climate. Besides anthropogenic emissions they are naturally produced by either abiotic or biotic pathways in various environments. Particularly in soils, microbial processes drive the natural halogen cycle but the direct link to microbial VOX formation has not been studied in detail yet. In this study we provide evidence that the common and widespread soil bacterium Sinorhizobium meliloti strain 1021 forms chloroform and tetrachloroethene. The potential contribution of S. meliloti to soil VOX release could significantly influence soil and atmospheric chemistry.

[Deterministic record linkage with indirect identifiers: data of the Berlin Myocardial Infarction Registry and the AOK Nordost for patients with myocardial infarction]. / Deterministisches Record Linkage mit indirekten Identifikatoren: Daten des Berliner Herzinfarktregisters und der AOK Nordost zum Herzinfarkt.

AIM OF THE STUDY: How can 2 pseudonymised data sets be linked? Using the example of data from the Berlin Myocardial Infarction Registry and from a German sickness fund (AOK Nordost) we will demonstrate how record linkage can be achieved without personal identifiers. METHODS: In different steps the method of deterministic record linkage with indirect identifiers: age, sex, hospital admission date and time, will be explained. RESULTS: We were able to show that 80.6% of the expected maximum number of patients were matched with our approach. As a result we had no duplicate matches in the linkage process, where one AOK patient was linked to 2 or more BMIR patients or vice versa. The matching variables produced enough uniqueness to be used as indirect patient identifiers. CONCLUSION: Deterministic record linkage with the following indirect indicators: age, sex, hospital admission date and time was possible in our study of patients with myocardial infarction in a circumscribed geographical region, which limited the number of cases and avoided mismatches.

Predominance of biotic over abiotic formation of halogenated hydrocarbons in hypersaline sediments in Western Australia.

Volatile halogenated organic compounds (VOX) contribute to ozone depletion and global warming. There is evidence of natural VOX formation in many environments ranging from forest soils to salt lakes. Laboratory studies have suggested that VOX formation can be chemically stimulated by reactive Fe species while field studies have provided evidence for direct biological (enzymatic) VOX formation. However, the relative contribution of abiotic and biotic processes to global VOX budgets is still unclear. The goals of this study were to quantify VOX release from sediments from a hypersaline lake in Western Australia (Lake Strawbridge) and to distinguish between the relative contributions of biotic and abiotic VOX formation in microbially active and sterilized microcosms. Our experiments demonstrated that the release of organochlorines from Lake Strawbridge sediments was mainly biotic. Among the organochlorines detected were monochlorinated, e.g., chloromethane (CH3Cl), and higher chlorinated VOX compounds such as trichloromethane (CHCl3). Amendment of sediments with either Fe(III) oxyhydroxide (ferrihydrite) or a mixture of lactate/acetate or both ferrihydrite and lactate/acetate did not stimulate VOX formation. This suggests that although microbial Fe(III) reduction took place, there was no stimulation of VOX formation via Fe redox transformations or the formation of reactive Fe species under our experimental conditions.

Microbially enhanced dissolution of HgS in an acid mine drainage system in the California Coast Range.

Mercury sulfides (cinnabar and metacinnabar) are the main ores of Hg and are relatively stable under oxic conditions (Ksp = 10⁻54 and 10⁻5² , respectively). However, until now their stability in the presence of micro-organisms inhabiting acid mine drainage (AMD) systems was unknown. We tested the effects of the AMD microbial community from the inoperative Hg mine at New Idria, CA, present in sediments of an AMD settling pond adjacent to the main waste pile and in a microbial biofilm on the surface of this pond, on the solubility of crystalline HgS. A 16S rRNA gene clone library revealed that the AMD microbial community was dominated by Fe-oxidizing (orders Ferritrophicales and Gallionellas) and S-oxidizing bacteria (Thiomonas sp.), with smaller amounts (≤ 6%) being comprised of the orders Xanthomondales and Rhodospirillales. Though the order Ferritrophicales dominate the 16S rRNA clones (>60%), qPCR results of the microbial community indicate that the Thiomonas sp. represents ~55% of the total micro-organisms in the top 1 cm of the AMD microbial community. Although supersaturated with respect to cinnabar and metacinnabar, microcosms inoculated with the AMD microbial community were capable of releasing significantly more Hg into solution compared to inactivated or abiotic controls. Four different Hg-containing materials were tested for bacterially enhanced HgS dissolution: pure cinnabar, pure metacinnabar, mine tailings, and calcine material (processed ore). In the microcosm with metacinnabar, the presence of the AMD microbial community resulted in an increase of dissolved Hg concentrations up to 500 µg L ⁻¹during the first 30 days of incubation. In abiotic control microcosms, dissolved Hg concentrations did not increase above 100 ng L⁻¹ . When Hg concentrations were below 50 µg L⁻¹ , the Fe-oxidizing bacteria in the AMD microbial community were still capable of oxidizing Fe(II) to Fe(III) in the AMD solution, whereas concentrations above 50 µg L⁻¹ resulted in inhibition of microbial iron oxidation. Our experiments show that the AMD microbial community contributes to the dissolution of mercury sulfide minerals. These findings have major implications for risk assessment and future management of inoperative Hg mines worldwide.

Interventional treatment and outcome in elderly patients with stable coronary artery disease. Results from the German ALKK registry.

BACKGROUND: The number of elderly and very elderly patients undergoing percutaneous coronary interventions (PCI) is increasing. We therefore analyzed data from the German ALKK registry (Arbeitsgemeinschaft Leitende Krankenhausärzte; Working Group of Hospital Cardiologists) to determine differences in procedural features, antithrombotic treatment, and in-hospital outcome in patients with coronary artery disease (CAD) according to age in a large series of patients. METHODS AND RESULTS: The present analysis was based on the data of 35,534 consecutive patients undergoing elective PCI who were enrolled in the ALKK registry. Of these 27,145 (76.4 %) were younger than 75 years, 7,645 (21.5 %) were aged between 75 and 84 years, and 744 (2.1 %) patients were older than 85 years. Mean age was 68.5 years (60.9-74.5 years), and 25,784 patients (72.6 %) were male. Overall intraprocedural events were very low (1.1 %) and there was no significant difference between the three age groups [< 75 years (1.1 %); 75-< 85 years (1.2 %); ≥ 85 years (0.5 %) (p = not significant)]. Rates of in-hospital death, stroke and transient ischemic attack (TIA), as well as the combined endpoint in-hospital major adverse cardiac and cerebrovascular events (MACCE) were also very low (0.6 % vs. 0.9 % vs. 0.9 %; p < 0.001) but significantly higher in elderly patients with no further increase in the very elderly patient group. CONCLUSION: We found no differences in this registry in intraprocedural complications during elective PCI between younger and elderly patients. Although in-hospital MACCE were somewhat higher in the elderly, the overall event rate was low and thus elderly patients should not be deprived from this therapy because of age alone.

Modeling of WalkMECH: a fully-passive energy-efficient transfemoral prosthesis prototype.

In this paper we present the port-based model of WalkMECH, a fully-passive transfemoral prosthesis prototype that has been designed and realized for normal walking. The model has been implemented in a simulation environment so to analyze the performance of the prosthetic leg in walking experiments and so to enhance the mechanics of the system. The accuracy of the model has been validated by experimental tests with a unilateral amputee participant.

Multiple cis-regulatory elements are involved in the complex regulation of the sieve element-specific MtSEO-F1 promoter from Medicago truncatula.

The sieve element occlusion (SEO) gene family includes several members that are expressed specifically in immature sieve elements (SEs) in the developing phloem of dicotyledonous plants. To determine how this restricted expression profile is achieved, we analysed the SE-specific Medicago truncatula SEO-F1 promoter (PMtSEO-F1) by constructing deletion, substitution and hybrid constructs and testing them in transgenic tobacco plants using green fluorescent protein as a reporter. This revealed four promoter regions, each containing cis-regulatory elements that activate transcription in SEs. One of these segments also contained sufficient information to suppress PMtSEO-F1 transcription in the phloem companion cells (CCs). Subsequent in silico analysis revealed several candidate cis-regulatory elements that PMtSEO-F1 shares with other SEO promoters. These putative sieve element boxes (PSE boxes) are promising candidates for cis-regulatory elements controlling the SE-specific expression of PMtSEO-F1.

Design of a fully-passive transfemoral prosthesis prototype.

In this study, we present the mechanical design of a prototype of a fully-passive transfemoral prosthesis for normal walking. The conceptual working principle at the basis of the design is inspired by the power flow in human gait, with the main purpose of realizing an energy efficient device. The mechanism is based on three elements, which are responsible of the energetic coupling between the knee and ankle joints. The design parameters of the prototype are determined according to the human body and the natural gait characteristics, in order to mimic the dynamic behavior of a healthy leg. Hereby, we present the construction details of the prototype, which realizes the working principle of the conceptual mechanism.

[Eosinophilic oesophagitis. Significance of routine oesophageal biopsies for dysphagia of unknown aetiology]. / Eosinophile Ösophagitis. Signifikanz von Routinebiopsien des Ösophagus bei unklarer Dysphagie.

Eosinophilic oesophagitis (EO), primarily a gastro-enterological disease, should be known to the surgeon and endoscopist as a differential diagnosis of dysphagia. We present a chronic and recurrent case of EO. As frequently seen, macroscopic findings are indicative of the causal illness. The diagnosis is finally made by the histological findings of a macroscopically inconspicuous mucosa of the esophagus, which is found in 10% of cases with EO. Random biopsies are necessary for the diagnosis. A short overview of therapy and course and a review of the literature are given.

Improved protocols for protein and RNA isolation from three-dimensional collagen sandwich cultures of primary hepatocytes.

The sandwich culture is the most widely used long-term culture system for functional primary hepatocytes. Despite its advantages, the currently available protocols for protein and RNA extraction are either time-consuming or contain steps that may skewer the results. This paper describes improved protocols for RNA and protein extraction from sandwich cultures that are easy to perform, require short working time, and use no additional enzymatic reactions that could change the expression profile of the cells. The quality of the RNA is excellent, allowing also applications requiring high purity such as microarrays. In general, the protocols are suited for any cells in 3D collagen culture.

Improved semiquantitative Western blot technique with increased quantification range.

With the development of new interdisciplinary fields such as systems biology, the quantitative analysis of protein expression in biological samples gains more and more importance. Although the most common method for this is ELISA, Western blot also has advantages: The separation of proteins by size allows the evaluation of only specifically bound protein. This work examines the Western blot signal chain, determines some of the parameters relevant for quantitative analysis and proposes a mathematical model of the reaction kinetics. Using this model, a semiquantitative Western blot method for simultaneous quantification of different proteins using a hyperbolic calibration curve was developed. A program was written for the purpose of hyperbolic regression that allows quick determination of the calibration curve coefficients. This program can be used also for approximation of calibration curves in other applications such as ELISA, BCA or Bradford assays.