Annual cycles of apoptosis,DNA strand breaks,heat shock proteins,and metallothionen isoforms in dab (Limanda limanda): influences of natural factors and consequences for biological effect monitoring

The present study was undertaken to investigate the influence of natural and anthropogenic stressors on the induction of apoptosis, metallothionein (MT) isoforms, heat shock proteins and DNA strand breaks in the marine flatfish dab (Limbanda limanda) Seasonal changes and possible physiological influences were evaluated over a 1-year period at a fixed location northwest of Helgoland in the German Bight. These results were compared with data from sampling sites in the North Sea and the Baltic Sea. Annual cycles could be observed for all parameters except for Cd. The data revealed that changes in biomarker are not only linked to physiological processes related to reproduction but also to factors like water temperature changes, lipid content and zinc content. Cd and organochlorines had no influence on biomarkers whereas an influence of Cd on MT levels revealed in the regional observations was possibly masked by the major changes in Zn content during the annual cycle. Due to different abiotic factors we supposed that the annual cycles at each sampling site in the North and the Baltic Sea might be shifted temporally and therefore measurements at different locations during a small time window of a few weeks may lead to misinterpretation in biomarker research.     

Seasonal variation in pathways of CH4 production and in CH4 oxidation in rice fields determined by stable carbon isotopes and specific inhibitors

Flooded rice fields, which are an important source of the atmospheric methane, have become a model system for the study of interactions between various microbial processes. We used a combination of stable carbon isotope measurements and application of specific inhibitors in order to investigate the importance of various methanogenic pathways and of CH₄ oxidation for controlling CH₄ emission. The fraction of CH₄ produced from acetate and H₂/CO₂ was calculated from the isotopic signatures of acetate, carbon dioxide (CO₂) and methane (CH₄) measured in porewater, gas bubbles, in the aerenchyma of the plants and/or in incubation experiments. The calculated ratio between both pathways reflected well the ratio determined by application of methyl fluoride (CH₃F) as specific inhibitor of acetate‐dependent methanogenesis. Only at the end of the season, the theoretical ratio of acetate: H₂ = 2 : 1 was reached, whereas at the beginning H₂/CO₂‐dependent methanogenesis dominated. The isotope discrimination was different between rooted surface soil and unrooted deep soil. Root‐associated CH₄ production was mainly driven by H₂/CO₂. Porewater CH₄ was found to be a poor proxy for produced CH₄. The fraction of CH₄ oxidised was calculated from the isotopic signature of CH₄ produced in vitro compared to CH₄ emitted in situ, corrected for the fractionation during the passage from the aerenchyma to the atmosphere. Isotope mass balances and in situ inhibition experiments with difluoromethane (CH₂F₂) as specific inhibitor of methanotrophic bacteria agreed that CH₄ oxidation was quantitatively important at the beginning of the season, but decreased later. The seasonal pattern was consistent with the change of potential CH₄ oxidation rates measured in vitro. At the end of the season, isotope techniques detected an increase of oxidation activity that was too small to be measured with the flux‐based inhibitor technique. If porewater CH₄ was used as a proxy of produced CH₄, neither magnitude nor seasonal pattern of in situ CH₄ oxidation could be reproduced. An oxidation signal was also found in the isotopic signature of CH₄ from gas bubbles that were released by natural ebullition. In contrast, bubbles stirred up from the bulk soil had preserved the isotopic signature of the originally produced CH₄.     

Fam60a defines a variant Sin3a‐Hdac complex in embryonic stem cells required for self‐renewal

Sin3a is the central scaffold protein of the prototypical Hdac1/2 chromatin repressor complex, crucially required during early embryonic development for the growth of pluripotent cells of the inner cell mass. Here, we compare the composition of the Sin3a‐Hdac complex between pluripotent embryonic stem (ES) and differentiated cells by establishing a method that couples two independent endogenous immunoprecipitations with quantitative mass spectrometry. We define the precise composition of the Sin3a complex in multiple cell types and identify the Fam60a subunit as a key defining feature of a variant Sin3a complex present in ES cells, which also contains Ogt and Tet1. Fam60a binds on H3K4me3‐positive promoters in ES cells, together with Ogt, Tet1 and Sin3a, and is essential to maintain the complex on chromatin. Finally, we show that depletion of Fam60a phenocopies the loss of Sin3a, leading to reduced proliferation, an extended G1‐phase and the deregulation of lineage genes. Taken together, Fam60a is an essential core subunit of a variant Sin3a complex in ES cells that is required to promote rapid proliferation and prevent unscheduled differentiation.     

Micromachined sensor for lactate monitoring in saliva

A miniaturised sensor for continuous lactate measurement in saliva was developed and tested. The sensor was built using silicon microfabrication technologies. The size of the chip is 5.5 mmx6.4 mmx0.7 mm and features a working, a counter and an Iridium reference electrode. The chip has a cavity whose floor is perforated by fine pores. The cavity contains the enzyme lactate oxidase (LOD), which is immobilised in an agarose gel. Prior to the amperometric detection of the reaction product hydrogen peroxide at the working electrode, the analyte lactate has to pass the pores to reach the cavity with the lactate oxidase by diffusion. To test the silicon sensor, capillary blood and saliva samples were obtained during standardised ergometer tests. Salivary lactate concentrations were determined with the sensor and compared to photometrically derived data from a lab-automate. In addition the saliva data were compared to standard capillary blood lactate concentrations measured with a pocket photometer. Lactate concentration versus load graphs were plotted and compared visually showing very similar progressions. The novel approach enables a location independent, permanent real-time measurement of the lactate concentration during exercise.     

An improved method for two-dimensional fluorescence monitoring of complex bioreactors

An improved method for deconvoluting complex spectral maps from bidimensional fluorescence monitoring is presented, relying on a combination of principal component analysis (PCA) and feedforward artificial neural networks (ANN). With the aim of reducing ANN complexity, spectral maps are first subjected to PCA, and the scores of the retained principal components are subsequently used as ANN input vector. The method is presented using the case study of an extractive membrane biofilm reactor, where fluorescence maps of a membrane-attached biofilm were analysed, which were collected under different reactor operating conditions. During ANN training, the spectral information is associated with process performance indicators. Originally, 231 excitation/emission pairs per fluorescence map were used as ANN input vector. Using PCA, each fluorescence map could be represented by a maximum of six principal components, thereby catching 99.5% of its variance. As a result, the dimension of the ANN input vector and hence the complexity of the artificial neural network was significantly reduced, and ANN training speed was increased. Correlations between principal components and ANN predicted process performance parameters were good with correlation coefficients in the order of 0.7 or higher.     

Continuous cultivation of human hamstring tenocytes on microcarriers in a spinner flask bioreactor system

Tendon healing is a time consuming process leading to the formation of a functionally altered reparative tissue. Tissue engineering‐based tendon reconstruction is attracting more and more interest. The aim of this study was to establish tenocyte expansion on microcarriers in continuous bioreactor cultures and to study tenocyte behavior during this new approach. Human hamstring tendon‐derived tenocytes were expanded in monolayer culture before being seeded at two different seeding densities (2.00 and 4.00 × 10⁶ cells/1000 cm² surface) on Cytodex? type 3 microcarriers. Tenocytes' vitality, growth kinetics and glucose/lactic acid metabolism were determined dependent on the seeding densities and stirring velocities (20 or 40 rpm) in a spinner flask bioreactor over a period of 2 weeks. Gene expression profiles of tendon extracellular matrix (ECM) markers (type I/III collagen, decorin, cartilage oligomeric protein [COMP], aggrecan) and the tendon marker scleraxis were analyzed using real time detection polymerase chain reaction (RTD‐PCR). Type I collagen and decorin deposition was demonstrated applying immunolabeling. Tenocytes adhered on the carriers, remained vital, proliferated and revealed an increasing glucose consumption and lactic acid formation under all culture conditions. “Bead‐to‐bead” transfer of cells from one microcarrier to another, a prerequisite for continuous tenocyte expansion, was demonstrated by scanning electron microscopy. Type I and type III collagen gene expression was mainly unaffected, whereas aggrecan and partly also decorin and COMP expression was significantly downregulated compared to monolayer cultures. Scleraxis gene expression revealed no significant regulation on the carriers. In conclusion, tenocytes could be successfully expanded on microcarriers. Therefore, bioreactors are promising tools for continuous tenocyte expansion. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:142–151, 2014