Intervertebral Disc Repair using Fleece-Membrane Composite Silk Material and Genipin-enhanced Fibrin Hydrogel

Introduction: Treating low back pain (LBP) has become an increasing challenge, as it is one of the main factors causing pain and is accompanied by high costs for the individual and the society. LBP can be caused by trauma of the intervertebral disc (IVD) or IVD degeneration. In the case of disc herniation the inner gelatinous part of the IVD, called nucleus pulposus, is pressed through the fibrous, annulus fibrosus that forms the outer part of the IVD. Today’s gold standard for treatment is extensive surgery as removal of the IVD and fusion of the vertebrae. In order to find a more gentle way to treat LBP and restore the native IVD we use a novel silk fleece-membrane composite from genetically modified silk worms whose silk contains a growth factor (GDF-6) that is associated with pushing stem cells towards a disc like phenotype (1). By combining it with a genipin-enhanced fibrin hydrogel we tested its suitability in organ culture on prior injured bovine IVD in our custom built two-degree of freedom bioreactor to mimic natural loading conditions. Material & Methods: Bovine IVDs of 12-17 months old animals were isolated by first removing all surrounding tissue followed by cutting out the IVDs as previously described (2). Culturing of discs occurred in high glucose Dulbecco's Modified Eagle Medium (HG-DMEM) supplemented with 5% serum as previously described (2). On the next day injury was induced using a 2mm biopsy punch (Polymed, Switzerland). The formed cavity was filled with (0.4%) genipin-enhanced human based fibrin hydrogel (35-55mg/mL human fibrinogen, Baxter, Austria) and sealed with a silk fleece-membrane composite (Spintec Engineering, Germany). Different culture conditions were applied: free swelling, static diurnal load of 0.2MPa for 8h/d and complex loading at 0.2MPa compression combined with ± 2° torsion at 0.2Hz for 8h/d (2). After 14 days of culture cell activity was determined with resazurin assay. Additionally, glycosaminoglycan (dimethyl-methylene blue), DNA (Hoechst) and collagen content (hydroxy- proline) were determined. Finally, real-time qPCR of major IVD marker and inflammation genes was performed to judge integrity of IVDs. Results: The fibrin hydrogel is able to keep the silk seal in place throughout the 14 days of in organ culture under all conditions. Additionally, cell activity showed optimistic results and we could not confirm negative effects of the repaired discs regarding overexpression of inflammation markers. Conclusions: The genipin-enhanced fibrin hydrogel in combination with the silk fleece- membrane composite seems to be a promising approach for IVD repair. Currently we assess the capability of GDF-6 incorporated in our silk composites on human mesenchymal stem cells and later on in organ culture. References 1. Clarke LE, McConnell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA. Growth differentiation factor 6 and transforming growth factor-beta differentially mediate mesenchymal stem cell differentiation, composition and micromechanical properties of nucleus pulposus constructs. Arthritis Res Ther 2014, Mar 12;16(2):R67. 2. Chan SC, Gantenbein-Ritter B. Preparation of intact bovine tail intervertebral discs for organ culture. J Vis Exp 2012, Feb 2;60(60):e3490. Acknowledgements. This work is funded by the Gebert Rüf Foundation, project number GRS-028/13.

Shifts in microbial communities do not explain the response of grassland ecosystem function to plant functional composition and rainfall change

Ecosystem functioning in grasslands is regulated by a range of biotic and abiotic factors, and the role of microbial communities in regulating ecosystem function has been the subject of much recent scrutiny. However, there are still knowledge gaps regarding the impacts of rainfall and vegetation change upon microbial communities and the implications of these changes for ecosystem functioning. We investigated this issue using data from an experimental mesotrophic grassland study in south-east England, which had been subjected to four years of rainfall and plant functional composition manipulations. Soil respiration, nitrogen and phosphorus stocks were measured, and the abundance and community structure of soil microbes were characterised using quantitative PCR and multiplex-TRFLP analysis, respectively. Bacterial community structure was strongly related to the plant functional composition treatments, but not the rainfall treatment. However, there was a strong effect of both rainfall change and plant functional group upon bacterial abundance. There was also a weak interactive effect of the two treatments upon fungal community structure, although fungal abundance was not affected by either treatment. Next, we used a statistical approach to assess whether treatment effects on ecosystem function were regulated by the microbial community. Our results revealed that ecosystem function was influenced by the experimental treatments, but was not related to associated changes to the microbial community. Overall, these results indicate that changes in fungal and bacterial community structure and abundance play a relatively minor role in determining grassland ecosystem function responses to precipitation and plant functional composition change, and that direct effects on soil physical and chemical properties and upon plant and microbial physiology may play a more important role.

Litter nutrients and retranslocation in a central African rain forest dominated by ectomycorrhizal trees

In the strongly seasonal, but annually very wet, parts of the tropics, low-water availability in the short dry season leads to a semi-deciduous forest, one which is also highly susceptible to nutrient loss from leaching in the long wet season. Patterns in litterfall were compared between forest with low (LEM) and high (HEM) abundances of ectomycorrhizal trees in Korup National Park, Cameroon, over 26 months in 1990–92. Leaf litter was sorted into 26 abundant species which included six ectomycorrhizal species, and of these three were the large grove-forming trees Microberlinia bisulcata, Tetraberlinia bifoliolata and Tetraberlinia moreliana. Larger-tree species shed their leaves with pronounced peaks in the dry season, whereas other species had either weaker dependence, showed several peaks per year, or were wet-season shedders. Although total annual litterfall differed little between forest types, in the HEM forest (dominated by M. bisulcata) the dry-season peak was more pronounced and earlier than that in the LEMforest. Species differed greatly in their mean leaf litterfall nutrient concentrations, with an approx. twofold range for nitrogen and phosphorus, and 2.5–3.5-fold for potassium, magnesium and calcium. In the dry season, LEM and HEM litter showed similar declines in P and N concentration, and increases in K and Mg; some species, especially M. bisculcata, showed strong dry-wet season differences. The concentration of P (but not N) was higher in the leaf litter of ectomycorrhizal than nonectomycorrhizal species. Retranslocation of N and P was lower among the ectomycorrhizal than nonectomycorrhizal species by approx. twofold. It is suggested that, within ectomycorrhizal groves on this soil low in P, a fast decomposition rate with minimal loss of mineralized P is possible due to the relatively high litter P not limiting the cycle at this stage, combined with an efficient recapture of released P by the surface organic layer of ectomycorrhizas and fine roots. This points to a feedback between two essential controlling steps (retranslocation and mineralization) in a tropical rain forest ecosystem dominated by ectomycorrhizal trees.

Influence of catchment quality and altitude on the water and sediment composition of 68 small lakes in Central Europe

68 lakes (63 Swiss, 2 French and 3 Italian) located in an altitudinal range between 334 and 2339m spanning a wide range of land-use have been investigated. The aim of the study was to discuss influences of geographic location, vegetation and land-use in the catchment area on the water and sediment chemistry of small lakes. Detailed quantitative description of land-use, vegetation, and climate in the watershed of all lakes was established. Surface and bottom water samples collected from each lake were analyzed for major ions and nutrients. Correlations were interpreted using linear regression analysis. Chemical parameters of water and sediment reflect the characteristics of the catchment areas. All lakes were alkaline since they were situated on calcareous bedrock. Concentrations of nitrogen and phosphorus strongly increase with increasing agricultural land-use. Na and K, however, are positively correlated with the amount of urbanization within the catchment area. These elements as well as dissolved organic carbon (DOC), Mg, Ca, and alkalinity, increase when the catchment is urbanized or used for agriculture. Total nitrogen and organic carbon in the sediments decrease distinctly if large parts of the catchment consist of bare land. No correlations between sediment composition and maximum water depth or altitude of the lakes were found.¶Striking differences in the water compositions of lakes above and below approximately 700 m of altitude were observed. Concentrations of total nitrogen and nitrate, total phosphorus, DOC, Na, K, Mg, Ca, and alkalinity are distinctly higher in most lakes below 700 m than above, and the pH of the bottom waters of these lakes is generally lower. Estimates of total nitrogen concentrations, even in remote areas, indicate that precipitation is responsible for increased background concentrations. At lower altitudes nitrogen concentrations in lakes is explained by the nitrogen loaded rain from urban areas deposited on the catchment, and with high percentages of agricultural land-use in the watershed.

Impact of Liquid Swine Manure Application and Cover Crops on Ground Water Quality

The primary objective of this project was to determine the impact of appropriate rates of swine manure applications to corn and soybeans based on nitrogen and phosphorus requirements of crops, soil phosphorus accumulation, and the potential of nitrate and phosphorus leaching to groundwater. Another purpose of this long-term experimental study was to develop and recommend appropriate manure and nutrient management practices to producers to minimize the water contamination potential and enhance the use of swine manure as inorganic fertilizer. A third component of this study was to determine the potential effects of rye as a cover crop to reduce nitrate loss to shallow ground water.

On the composition of oceanic and littoral manganese nodules (published online 2015)

The following analyses were made some years ago, principally with the object of ascertaining the state of oxidation of the manganese in the nodules. The nodules examined came from three different localities, two of them oceanic and the third littoral. Samples marked I., II., and III. are from nodules brought up in the trawl on board the "Challenger," on 13th March 1874, in lat. 42° 42' S., long. 134° 10' E. The depth of the water was 2600 fathoms, and the temperature of the bottom water 0·2° C. The density of the bottom water was 1·02570 at 15·56° C. Being from a high southern latitude, and therefore near the source of surface aeration, the water is highly charged with atmospheric gases, especially oxygen. It contained, per litre, 18·4 c.c. of mixed nitrogen and oxygen, of which 31·81 per cent, was oxygen, and 27·33 c.c, or 53·7 milligrammes, loosely-bound carbonic acid. The position of the station is about 400 miles south-west of the nearest part of the Australian coast, and about 500 miles west of Tasmania. It was the deepest water observed in the Antarctic voyage between the Cape of Good Hope and Melbourne. The haul was a very abundant one, and a few notes which I made at the time may be interesting: -"The water was found unexpectedly deep, the bottom being red clay, with some Foraminifera.

Propiedades químicas de suelos desarrollados sobre distintos materiales originales bajo bosques de Austrocedrus chilensis

En este trabajo se caracterizaron las propiedades químicas del horizonte A de los suelos desarrollados a partir de distintos materiales originarios, sobre los cuales habitan bosques de Austrocedrus chilensis. Se seleccionaron cinco sitios, ubicados en el Valle 16 de Octubre y en el Parque Nacional Los Alerces de la Provincia del Chubut, Argentina. De cada sitio se tomaron muestras compuestas del horizonte A para la caracterización de las propiedades químicas. Todos los suelos analizados presentaron elevados contenidos de materia orgánica y nitrógeno. Los suelos originados a partir de materiales glaciarios presentaron los máximos valores de bases de intercambio, capacidad de intercambio catiónico y contenido de fósforo, diferenciándose significativamente de los suelos volcánicos. Dentro de los suelos volcánicos se detectaron diferencias en función de la granulometría del material: los suelos derivados de ceniza (< 2mm) tendieron a presentar mayores valores de capacidad de intercambio catiónico y bases de intercambio que los suelos de pumita (> 2mm).

Specific conductivity and hydrogen ions measured on two ice core (NEEM and NGRIP)

A stratigraphy-based chronology for the North Greenland Eemian Ice Drilling (NEEM) ice core has been derived by transferring the annual layer counted Greenland Ice Core Chronology 2005 (GICC05) and its model extension (GICC05modelext) from the NGRIP core to the NEEM core using 787 match points of mainly volcanic origin identified in the electrical conductivity measurement (ECM) and dielectrical profiling (DEP) records. Tephra horizons found in both the NEEM and NGRIP ice cores are used to test the matching based on ECM and DEP and provide five additional horizons used for the timescale transfer. A thinning function reflecting the accumulated strain along the core has been determined using a Dansgaard-Johnsen flow model and an isotope-dependent accumulation rate parameterization. Flow parameters are determined from Monte Carlo analysis constrained by the observed depth-age horizons. In order to construct a chronology for the gas phase, the ice age-gas age difference (Delta age) has been reconstructed using a coupled firn densification-heat diffusion model. Temperature and accumulation inputs to the Delta age model, initially derived from the water isotope proxies, have been adjusted to optimize the fit to timing constraints from d15N of nitrogen and high-resolution methane data during the abrupt onset of Greenland interstadials. The ice and gas chronologies and the corresponding thinning function represent the first chronology for the NEEM core, named GICC05modelext-NEEM-1. Based on both the flow and firn modelling results, the accumulation history for the NEEM site has been reconstructed. Together, the timescale and accumulation reconstruction provide the necessary basis for further analysis of the records from NEEM.

Age determination and n-Alkane concentration of Arctic Ocean sediment cores

We have reconstructed the surface water environment of the Arctic Ocean over the last ? 50,000 years using measurements of the organic nitrogen and carbon isotope ratios, carbonate and total organic carbon concentrations (TOC), and terrestrial biomarkers (lignin and long-chain n-alkanes) in four multicores. Variations in nitrogen isotope ratios that are concordant with TOC and carbonate concentrations (representing foraminifera and excluding ice-rafted-debris) reflect differences in relative nutrient utilization of phytoplankton in the surface waters. However, d15N variations also appear to be dependent on the stratification of the water column and therefore potentially track the exchange of nutrients between deep and surface waters. Low Last Glacial Maximum (LGM) d15N values and higher Holocene values are opposite to those recorded in the Southern Ocean. The Arctic Ocean with higher nutrient utilization today compared to the LGM therefore acts as a counterpart to the Southern Ocean, although the global impact on carbon dioxide variations compared to the Southern Ocean is probably low.

Biotic and abiotic parameters and cynobacteria abundance during an experimental set-up with varying pCO2 conditions

Heterocystous cyanobacteria of the genus Nodularia form extensive blooms in the Baltic Sea and contribute substantially to the total annual primary production. Moreover, they dispense a large fraction of new nitrogen to the ecosystem when inorganic nitrogen concentration in summer is low. Thus, it is of ecological importance to know how Nodularia will react to future environmental changes, in particular to increasing carbon dioxide (CO2) concentrations and what consequences there might arise for cycling of organic matter in the Baltic Sea. Here, we determined carbon (C) and dinitrogen (N2) fixation rates, growth, elemental stoichiometry of particulate organic matter and nitrogen turnover in batch cultures of the heterocystous cyanobacterium Nodularia spumigena under low (median 315 µatm), mid (median 353 µatm), and high (median 548 µatm) CO2 concentrations. Our results demonstrate an overall stimulating effect of rising pCO2 on C and N2 fixation, as well as on cell growth. An increase in pCO2 during incubation days 0 to 9 resulted in an elevation in growth rate by 84 ± 38% (low vs. high pCO2) and 40 ± 25% (mid vs. high pCO2), as well as in N2 fixation by 93 ± 35% and 38 ± 1%, respectively. C uptake rates showed high standard deviations within treatments and in between sampling days. Nevertheless, C fixation in the high pCO2 treatment was elevated compared to the other two treatments by 97% (high vs. low) and 44% (high vs. mid) at day 0 and day 3, but this effect diminished afterwards. Additionally, elevation in carbon to nitrogen and nitrogen to phosphorus ratios of the particulate biomass formed (POC : POP and PON : POP) was observed at high pCO2. Our findings suggest that rising pCO2 stimulates the growth of heterocystous diazotrophic cyanobacteria, in a similar way as reported for the non-heterocystous diazotroph Trichodesmium. Implications for biogeochemical cycling and food web dynamics, as well as ecological and socio-economical aspects in the Baltic Sea are discussed.

(Table 1) Bioclimatic subzones and soil characteristics along the Yamal Arctic Transect, northwestern Siberia

Sustainability of tundra vegetation under changing climate on the Yamal Peninsula, northwestern Siberia, home to the world's largest area of reindeer husbandry, is of crucial importance to the local native community. An integrated investigation is needed for better understanding of the effects of soils, climate change and grazing on tundra vegetation in the Yamal region. In this study we applied a nutrient-based plant community model - ArcVeg - to evaluate how two factors (soil organic nitrogen (SON) levels and grazing) interact to affect tundra responses to climate warming across a latitudinal climatic gradient on the Yamal Peninsula. Model simulations were driven by field-collected soil data and expected grazing patterns along the Yamal Arctic Transect (YAT), within bioclimate subzones C (high arctic), D (northern low arctic) and E (southern low arctic). Plant biomass and NPP (net primary productivity) were significantly increased with warmer bioclimate subzones, greater soil nutrient levels and temporal climate warming, while they declined with higher grazing frequency. Temporal climate warming of 2 °C caused an increase of 665 g/m**2 in total biomass at the high SON site in subzone E, but only 298 g/m**2 at the low SON site. When grazing frequency was also increased, total biomass increased by only 369 g/m**2 at the high SON site in contrast to 184 g/m**2 at the low SON site in subzone E. Our results suggest that high SON can support greater plant biomass and plant responses to climate warming, while low SON and grazing may limit plant response to climate change. In addition to the first order factors (SON, bioclimate subzones, grazing and temporal climate warming), interactions among these significantly affect plant biomass and productivity in the arctic tundra and should not be ignored in regional scale studies.

Geochemistry and soil characteristics of sediment core GeoB4234

Stable isotopes of sedimentary nitrogen and organic carbon are widely used as proxy variables for biogeochemical parameters and processes in the water column. In order to investigate alterations of the primary isotopic signal by sedimentary diagenetic processes, we determined concentrations and isotopic compositions of inorganic nitrogen (IN), organic nitrogen (ON), total nitrogen (TN), and total organic carbon (TOC) on one short core recovered from sediments of the eastern subtropical Atlantic, between the Canary Islands and the Moroccan coast. Changes with depth in concentration and isotopic composition of the different fractions were related to early diagenetic conditions indicated by pore water concentrations of oxygen, nitrate, and ammonium. Additionally, the nature of the organic matter was investigated by Rock-Eval pyrolysis and microscopic analysis. A decrease in ON during aerobic organic matter degradation is accompanied by an increase of the 15N/14N ratio. Changes in the isotopic composition of ON can be described by Rayleigh fractionation kinetics which are probably related to microbial metabolism. The influence of IN depleted in 15N on the bulk sedimentary (TN) isotope signal increases due to organic matter degradation, compensating partly the isotopic changes in ON. In anoxic sediments, fixation of ammonium between clay lattices results in a decrease of stable nitrogen isotope ratio of IN and TN. Changes in the carbon isotopic composition of TOC have to be explained by Rayleigh fractionation in combination with different remineralization kinetics of organic compounds with different isotopic composition. We have found no evidence for preferential preservation of terrestrial organic carbon. Instead, both TOC and refractory organic carbon are dominated by marine organic matter. Refractory organic carbon is depleted in 13C compared to TOC.

Synergistic effects of pCO2 and iron availability on nutrient consumption ratio of the Bering Sea phytoplankton community

Little is known concerning the effect of CO2 on phytoplankton ecophysiological processes under nutrient and trace element-limited conditions, because most CO2 manipulation experiments have been conducted under elements-replete conditions. To investigate the effects of CO2 and iron availability on phytoplankton ecophysiology, we conducted an experiment in September 2009 using a phytoplankton community in the iron limited, high-nutrient, low-chlorophyll (HNLC) region of the Bering Sea basin . Carbonate chemistry was controlled by the bubbling of the several levels of CO2 concentration (180, 380, 600, and 1000 ppm) controlled air, and two iron conditions were established, one with and one without the addition of inorganic iron. We demonstrated that in the iron-limited control conditions, the specific growth rate and the maximum photochemical quantum efficiency (Fv/Fm) of photosystem (PS) II decreased with increasing CO2 levels, suggesting a further decrease in iron bioavailability under the high-CO2 conditions. In addition, biogenic silica to particulate nitrogen and biogenic silica to particulate organic carbon ratios increased from 2.65 to 3.75 and 0.39 to 0.50, respectively, with an increase in the CO2 level in the iron-limited controls. By contrast, the specific growth rate, Fv/Fm values and elemental compositions in the iron-added treatments did not change in response to the CO2 variations, indicating that the addition of iron canceled out the effect of the modulation of iron bioavailability due to the change in carbonate chemistry. Our results suggest that high-CO2 conditions can alter the biogeochemical cycling of nutrients through decreasing iron bioavailability in the iron-limited HNLC regions in the future.

Biogenic and lithogenic silica, silicic acid, production of biogenic silica, and irradiance measurements during the Ross Sea Bloom Project 1994-1996

The development of the seasonal phytoplankton bloom in the Ross Sea was studied during two cruises. The first, conducted in November-December 1994, investigated the initiation and rapid growth of the bloom, whereas the second (December 1995-January 1996) concentrated on the bloom's maximum biomass period and the subsequent decline in biomass. Central to the understanding of the controls of growth and the summer decline of the bloom is a quantitative assessment of the growth rate of phytoplankton. Growth rates were estimated over two time scales with different methods. The first estimated daily growth rates from isotropic incorporation under simulated in situ conditions, including 14C, 15N and 32Si uptake measurements combined with estimates of standing stocks of particulate organic carbon, nitrogen and biogenic silica. The second method used daily to weekly changes in biomass at selected locations, with net growth rates being estimated from changes in standing stocks of phytoplankton. In addition, growth rates were estimated in large-volume experiments under optimal irradiances. Growth rates showed distinct temporal patterns. Early in the growing season, short-term estimates suggested that growth rates of in situ assemblages were less than maximum (relative to the temperature-limited maximum) and were likely reduced due to low irradiance regimes encountered under the ice. Growth rates increased thereafter and appeared to reach their maximum as biomass approached the seasonal peak, but decreased markedly in late December. Differences between the major taxonomic groups present were also noted, especially from the isotopic tracer experiments. The haplophyte Phaeocystic antarctica was dominant in 1994 throughout the growing season, and it exhibited the greatest growth rates (mean 0.41/day) during spring. Diatom standing stocks were low early in the growing season, and growth rates averaged 0.100/day. In summer diatoms were more abundant, but their growth rates remained much lower (mean of 0.08/day) than the potential maximum. Understanding growth rate controls is essential to the development of predictive models of the carbon cycle and food webs in Antarctic waters.

Collection of data on particulate and dissolved soil phosphorus in the Jena Experiment (Main Experiment, time series since 2002)

This data set contains two time series of measurements of dissolved phosphorus (organic, inorganic and total with a biweekly resolution) and dissolved inorganic phosphorus with a seasonal resolution. In addition, data on phosphorus from soil samples measured in 2007 and fractionated by different acid-extrations (Hedley fractions) are provided. All data measured at the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. 1. Dissolved phosphorus in soil solution: Suction plates installed on the field site in 10, 20, 30 and 60 cm depth were used to sample soil pore water. Cumulatively extracted soil solution was collected every two weeks from October 2002 to May 2006. The biweekly samples from 2002, 2003 and 2004 were analyzed for dissolved organic phosphorus (DOP), dissolved inorganic phosphorus (PO4P) and dissolved total phosphorus (TDP) by Continuous Flow Analyzer (CFA SAN ++, SKALAR [Breda, The Netherlands]). 2. Seasonal values of dissolved inorganic phosphorus in soil solution were calculated as volume-weighted mean values of the biweekly measurements (spring = March to May, summer = June to August, fall = September to November, winter = December to February). 3. Phosphorus fractions in soil: Five independent soil samples per plot were taken in a depth of 0-15 cm using a soil corer with an inner diameter of 1 cm. The five samples per plot were combined to one composite sample per plot. A four-step sequential P fractionation (Hedley fractions) was applied and concentrations of P fractions in soil were measured photometrically (molybdenum blue-reactive P) with a Continuous Flow Analyzer (Bran&Luebbe, Germany).