Uranyl salts as photochemical agents for cleavage of DNA and probing of protein-DNA contacts

Single-strand DNA nicks are induced by uranyl nitrate or uranyl acetate in combination with long-wavelength (lambda approximately 420 nm) ultraviolet irradiation. The nicks occur randomly with respect to the DNA sequence. Using the lambda-repressor/ORI operator DNA system it is shown that uranyl salts can be used to photofootprint protein contacts with the DNA backbone.     

Psoralen photofootprinting of protein-binding sites on DNA

Using a BAL31 exonuclease assay to determine the sites of 4,5',8-trimethylpsoralen photocrosslinking in DNA we have shown that 5'-TA sites which are accessible to psoralen DNA interstrand photocrosslinking in naked DNA become inaccessible when protein, in casu, lambda-repressor E. coli or RNA polymerase are bound at their recognition DNA sequences (OR1 operator or deo1 promoter, respectively). These results show that psoralens can be used as photofootprinting reagents to study specific protein-DNA interactions.     

A specific and efficient photoreaction between E. coli RNA polymerase and T+1 in the lacUV5 or deoP1 promoter.

Upon irradiation of the RNA polymerase-lacUV5 or deoP1 promoter complex with short wavelength ultraviolet light (lambda less than or equal to 300 nm) the polymerase is covalently crosslinked at an efficiency of greater than 10% to the first transcribed base of the template DNA strand when this is a thymine. The temperature dependence of this RNA polymerase-T+1 photoreaction strongly indicates a relation to the formation of the open complex. It is suggested that open complex formation is preceded or accompanied by a specific contact between the RNA polymerase and the first transcribed base of the DNA template.     

Lack of steady-state in the global biogeochemical Si cycle: emerging evidence from lake Si sequestration

Weathering of silicate minerals releases dissolved silicate (DSi) to the soil-vegetation system. Accumulation and recycling of this DSi by terrestrial ecosystems creates a pool of reactive Si on the continents that buffers DSi export to the ocean. Human perturbations to the functioning of the buffer have been a recent research focus, yet a common assumption is that the continental Si cycle is at steady-state. However, we have no good idea of the timescales of ecosystem Si pool equilibration with their environments. A review of modelling and geochemical considerations suggests the modern continental Si cycle is in fact characterised in the long-term by an active accumulation of reactive Si, at least partially attributable to lakes and reservoirs. These lentic systems accumulate Si via biological conversion of DSi to biogenic silica (BSi). An analysis of new and published data for nearly 700 systems is presented to assess their contribution to the accumulating continental pool. Surface sediment BSi concentrations (n = 692) vary between zero and >60 % SiO₂ by weight, apparently independently of lake size, location or water chemistry. Using sediment core BSi accumulation rates (n = 109), still no relationships are found with lake or catchment parameters. However, issues associated with single-core accumulation rates should in any case preclude their use in elemental accumulation calculations. Based on lake/reservoir mass-balances (n = 34), our best global-scale estimate of combined lake and reservoir Si retention is 1.53 TMol year^?1, or 21–27 % of river DSi export. Again, no scalable relationships are apparent, suggesting Si retention is a complex process that varies from catchment to catchment. The lake Si sink has implications for estimation of weathering flux generation from river chemistry. The size of the total continental Si pool is poorly constrained, as is its accumulation rate, but lakes clearly contribute substantially. A corollary to this emerging understanding is that the flux and isotopic composition of DSi delivered to the ocean has likely varied over time, partly mediated by a fluctuating continental pool, including in lakes.     

Temporal trends and variability in a high-arctic ecosystem in Greenland: multidimensional analyses of limnic and terrestrial ecosystems

The high arctic is undergoing a faster change in climate than most other regions of the planet, with already observed ecological consequences. Combined with the characteristics of high-arctic ecosystems, such as low species redundancy, high seasonality and weather extremes, shifts in individual species performance and phenology may lead to altered interaction dynamics through trophic mismatch and cascades. An ecosystem approach is therefore desirable in the attempt to understand the multidimensional impacts of climate. Here, we present ecosystem-wide trend analyses of a long-term dataset on terrestrial and limnic biota with focus on the distribution of observed trends and associated variation across the ecosystem. We used 114 time series drawn from 11 abiotic variables, 19 terrestrial and 7 limnic biotic species/taxa and compared temporal trends, changes and abrupt shifts in the variation within and across the two biota. A total of 36 % of the time series analysed showed a significant trend during the study period with a higher frequency of trends occurring within performance variables. Overall, the changes tended to be negative, indicating advances in phenology but reduced species performance. General system variance was also higher in the limnic biota than in the terrestrial biota, both exhibiting increasing variance up through the trophic system. Overall, our results suggest that multiple biotic responses to the climatic changes in this high-arctic ecosystem are not synchronised across trophic levels and may differ qualitatively and quantitatively between terrestrial and limnic biota.     

Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells reveal differential expression of EMT factors

Cancer cells secrete soluble factors and various extracellular vesicles, including exosomes, into their tissue microenvironment. The secretion of exosomes is speculated to facilitate local invasion and metastatic spread. Here, we used an in vivo metastasis model of human bladder carcinoma cell line T24 without metastatic capacity and its two isogenic derivate cell lines SLT4 and FL3, which form metastases in the lungs and liver of mice, respectively. Cultivation in CLAD1000 bioreactors rather than conventional culture flasks resulted in a 13‐ to 16‐fold increased exosome yield and facilitated quantitative proteomics of fractionated exosomes. Exosomes from T24, SLT4, and FL3 cells were partitioned into membrane and luminal fractions and changes in protein abundance related to the gain of metastatic capacity were identified by quantitative iTRAQ proteomics. We identified several proteins linked to epithelial–mesenchymal transition, including increased abundance of vimentin and hepatoma‐derived growth factor in the membrane, and casein kinase II α and annexin A2 in the lumen of exosomes, respectively, from metastatic cells. The change in exosome protein abundance correlated little, although significant for FL3 versus T24, with changes in cellular mRNA expression. Our proteomic approach may help identification of proteins in the membrane and lumen of exosomes potentially involved in the metastatic process.     

Using invertebrate remains and pigments in the sediment to infer changes in trophic structure after fish introduction in Lake Fogo: a crater lake in the Azores

Fish introduction may have marked effects on the trophic dynamics and ecological state of former fishless lakes, but due to scarcity of historical data this can seldom be documented. We used remains of cladoceran, chironomid and pigment assemblages in the sediment archive to unravel the effect of introduction of carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss) and a cyprinid (Chondrostoma oligolepis) in Lake Fogo, the Azores (Portugal). The stratigraphical record showed two major shifts in community assemblage coinciding with the time of introduction of carp (AD ca. 1890) and trout (AD 1941), respectively. Carp introduction was followed by an abrupt and major decline in the abundance of chironomids, a shift in the cladoceran community from a benthic to a more pelagic dominated community, and Daphnia size was significantly reduced. Pigment assemblages also indicated a shift from a benthic to a pelagic dominated ecosystem, as cryptophytes became markedly more abundant at the expense of benthic diatoms. Trout introduction was followed by a return to a more benthic cladoceran and benthic algae (pigments) dominated state, which we attribute to trout predation on carp leading to improved water clarity. A steady increase in the abundance of pigments and cladoceran remains followed, suggesting enhanced productivity, which may be attributed to enhanced atmospheric nitrogen deposition and introduction of C. oligolepis. We conclude that fish introduction has profoundly altered the trophic dynamics and the relative importance of benthic and pelagic production in this species poor and natural fishless lake in the Azores, and likely in most others lakes at the archipelago islands as fish stocking has been a widespread practice.     

Community structure and diel migration of zooplankton in shallow brackish lakes: role of salinity and predators

Diel horizontal migration (DHM), where zooplankton moves towards macrophytes during daytime to avoid planktivorous fish, has been reported as a common migration pattern of zooplankton in shallow temperate freshwater lakes. However, in shallow eutrophic brackish lakes, macrophytes seem not to have the same refuge effect, as these lakes may remain turbid even at relatively high macrophyte abundances. To investigate the extent to which macrophytes serve as a refuge for zooplankton at different salinities, we introduced artificial plants mimicking submerged macrophytes in the littoral zone of four shallow lakes, with salinities ranging from almost freshwater (0.3) to oligohaline waters (3.8). Furthermore, we examined the effects of different salinities on the community structure. Diel samples of zooplankton were taken from artificial plants, from areas where macrophytes had been removed (intermediate areas) and, in two of the lakes, also in open water. Fish and macroinvertebrates were sampled amongst the artificial plants and in intermediate areas to investigate their influence on zooplankton migration. Our results indicated that diel vertical migration (DVM) was the most frequent migration pattern of zooplankton groups, suggesting that submerged macrophytes were a poor refuge against predation at all salinities under study. Presumably, this pattern was the result of the relatively high densities of small planktivorous fish and macroinvertebrate predators within the submerged plants. In addition, we found major differences in the composition of zooplankton, fish and macroinvertebrate communities at the different salinities and species richness and diversity of zooplankton decreased with increasing salinity. At low salinities both planktonic/free-swimming and benthic/plant-associated cladocerans occurred, whilst only benthic ones occurred at the highest salinity. The low zooplankton biomass and overall smaller-bodied zooplankton specimens may result in a lower grazing capacity on phytoplankton, and enhance the turbid state in nutrient rich shallow brackish lakes.     

Implications of Climate-enforced Temperature Increases on Freshwater Pico- and Nanoplankton Populations Studied in Artificial Ponds During 16 Months

Global warming scenarios foresee increases in air temperatures of 3–5 °C in Northern European regions within the next 70 years. To evaluate the potential effects of global warming on shallow eutrophic lakes, a flow-through experiment combining three temperature scenarios and two nutrient levels was conducted in 24 outdoor mesocosms. Eight mesocosms were unheated and acted as controls, while sixteen were heated – eight according to the Intergovernmental Panel on Climate Change’s (IPCC) climate scenario A2 down-scaled to regional level (2.5–4.4 °C, depending on season) and eight according to scenario A2+ with an additional 50% temperature increase. Half of the mesocosms were enriched with nitrogen and phosphorus to simulate increased runoff from terrestrial sources due to the increased precipitation predicted by the A2 scenario. The other half were un-enriched and received only natural nutrient input from the groundwater that fed all the mesocosms. The abundance and development pattern of the microbial communities within the mesocosms were tracked during a 16-month period. Generally, the results showed that the abundances of picoalgae, bacteria and heterotrophic nanoflagellates changed in a similar manner over time; abundances being lower in winter than in summer. Warming in itself had no effect on abundance, albeit it significantly modified the positive effect of the nutrients. Only at ambient temperatures did the whole microbial assemblage respond positively to nutrients. In the A2 scenario, only picoalgae responded to nutrients, while in the A2+ scenario all but the heterotrophic nanoflagellates showed a response. Elevated winter temperatures seemed not to be more important for the microbial assemblage than elevated summer temperatures. Our results demonstrate that the direct effects of warming were far less important than the nutrient effect. The results furthermore reveal that warming and nutrients in combination set off complex interactions. In consequence, global warming may possibly have pronounced effects on aquatic ecosystems if accompanied by increased nutrient loading.