AAV-mediated in vivo knockdown of luciferase using combinatorial RNAi and U1i

RNA interference (RNAi) has been successfully employed for specific inhibition of gene expression; however, safety and delivery of RNAi remain critical issues. We investigated the combinatorial use of RNAi and U1 interference (U1i). U1i is a gene-silencing technique that acts on the pre-mRNA by preventing polyadenylation. RNAi and U1i have distinct mechanisms of action in different cellular compartments and their combined effect allows usage of minimal doses, thereby avoiding toxicity while retaining high target inhibition. As a proof of concept, we investigated knockdown of the firefly luciferase reporter gene by combinatorial use of RNAi and U1i, and evaluated their inhibitory potential both in vitro and in vivo. Co-transfection of RNAi and U1i constructs showed additive reduction of luciferase expression up to 95% in vitro. We attained similar knockdown when RNAi and U1i constructs were hydrodynamically transfected into murine liver, demonstrating for the first time successful in vivo application of U1i. Moreover, we demonstrated long-term gene silencing by AAV-mediated transduction of murine muscle with RNAi/U1i constructs targeting firefly luciferase. In conclusion, these results provide a proof of principle for the combinatorial use of RNAi and U1i to enhance target gene knockdown in vivo

Determining Signalling Nodes for Apoptosis by a Genetic High-Throughput Screen

With the ever-increasing information emerging from the various sequencing and gene annotation projects, there is an urgent need to elucidate the cellular functions of the newly discovered genes. The genetically regulated cell suicide of apoptosis is especially suitable for such endeavours as it is governed by a vast number of factors.We have set up a high-throughput screen in 96-well microtiter plates for genes that induce apoptosis upon their individual transfection into human cells. Upon screening approximately 100,000 cDNA clones we determined 74 genes that initiate this cellular suicide programme. A thorough bioinformatics analysis of these genes revealed that 91% are novel apoptosis regulators. Careful sequence analysis and functional annotation showed that the apoptosis factors exhibit a distinct functional distribution that distinguishes the cell death process from other signalling pathways. While only a minority of classic signal transducers were determined, a substantial number of the genes fall into the transporter- and enzyme-category. The apoptosis factors are distributed throughout all cellular organelles and many signalling circuits, but one distinct signalling pathway connects at least some of the isolated genes. Comparisons with microarray data suggest that several genes are dysregulated in specific types of cancers and degenerative diseases.Many unknown genes for cell death were revealed through our screen, supporting the enormous complexity of cell death regulation. Our results will serve as a repository for other researchers working with genomics data related to apoptosis or for those seeking to reveal novel signalling pathways for cell suicide

Human impact on the hydroenvironment of Lake Parishan, SW Iran, through the late Holocene

A multiproxy record from Lake Parishan, SW Iran, shows human impact on the lake and its catchment over the last 4000 years. The Parishan record provides evidence of changes in lake hydrology, from ostracod, diatom and isotope analyses, that are directly linked to human activity in the catchment; recorded by pollen and charcoal and supported by regional archaeological and historical data. The lake ostracod fauna is particularly sensitive to human induced catchment alterations and allow us to identify changes in catchment hydrology that are due to more than a simple change in precipitation: evaporation state. Oxygen isotope data from endogenic carbonates follow these faunal changes but also displays a longer trend to more positive values through the period, coincident with regional patterns of water balance for the late Holocene in the eastern Mediterranean

The Falkland Islands’ palaeoecological response to millennial‐scale climate perturbations during the Pleistocene–Holocene transition: implications for future vegetation stability in the southern ocean islands

Oceanic island flora is vulnerable to future climate warming, which is likely to promote changes in vegetation composition, and invasion of non‐native species. Sub‐Antarctic islands are predicted to experience rapid warming during the next century; therefore, establishing trajectories of change in vegetation communities is essential for developing conservation strategies to preserve biological diversity. We present a Late‐glacial‐early Holocene (16 500–6450 cal a bp) palaeoecological record from Hooker's Point, Falkland Islands (Islas Malvinas), South Atlantic. This period spans the Pleistocene‐Holocene transition, providing insight into biological responses to abrupt climate change. Pollen and plant macrofossil records appear insensitive to climatic cooling during the Late‐glacial, but undergo rapid turnover in response to regional warming. The absence of trees throughout the Late‐glacial‐early Holocene enables the recognition of far‐travelled pollen from southern South America. The first occurrence of Nothofagus (southern beech) may reflect changes in the strength and/or position of the Southern Westerly Wind Belt during the Late‐glacial period. Peat inception and accumulation at Hooker's Point is likely to be promoted by the recalcitrant litter of wind‐adapted flora. This recalcitrant litter helps to explain widespread peatland development in a comparatively dry environment, and suggests that wind‐adapted peatlands can remain carbon sinks even under low precipitation regimes

Protein Conformation and Supercharging with DMSO from Aqueous Solution

The efficacy of dimethyl sulfoxide (DMSO) as a supercharging reagent for protein ions formed by electrospray ionization from aqueous solution and the mechanism for supercharging were investigated. Addition of small amounts of DMSO to aqueous solutions containing hen egg white lysozyme or equine myoglobin results in a lowering of charge, whereas a significant increase in charge occurs at higher concentrations. Results from both near-UV circular dichroism spectroscopy and solution-phase hydrogen/deuterium exchange mass spectrometry indicate that DMSO causes a compaction of the native structure of these proteins at low concentration, but significant unfolding occurs at ~63% and ~43% DMSO for lysozyme and myoglobin, respectively. The DMSO concentrations required to denature these two proteins in bulk solution are ~3–5 times higher than the concentrations required for the onset of supercharging, consistent with a significantly increased concentration of this high boiling point supercharging reagent in the ESI droplet as preferential evaporation of water occurs. DMSO is slightly more basic than m-nitrobenzyl alcohol and sulfolane, two other supercharging reagents, based on calculated proton affinity and gas-phase basicity values both at the B3LYP and MP2 levels of theory, and all three of these supercharging reagents are significantly more basic than water. These results provide additional evidence that the origin of supercharging from aqueous solution is the result of chemical and/or thermal denaturation that occurs in the ESI droplet as the concentration of these supercharging reagents increases, and that proton transfer reactivity does not play a significant role in the charge enhancement observed

Revealing the last 13,500 years of environmental history from the multiproxy record of a mountain lake (Lago Enol, northern Iberian Peninsula)

This is the author's accepted manuscript. The final publication is available at Springer via http://dx.doi.org/10.1007/s10933-009-9387-7.We present the Holocene sequence from Lago Enol (43°16′N, 4°59′W, 1,070 m a.s.l.), Cantabrian Mountains, northern Spain. A multiproxy analysis provided comprehensive information about regional humidity and temperature changes. The analysis included sedimentological descriptions, physical properties, organic carbon and carbonate content, mineralogy and geochemical composition together with biological proxies including diatom and ostracod assemblages. A detailed pollen study enabled reconstruction of variations in vegetation cover, which were interpreted in the context of climate changes and human impact. Four distinct stages were recognized for the last 13,500 years: (1) a cold and dry episode that includes the Younger Dryas event (13,500–11,600 cal. year BP); (2) a humid and warmer period characterizing the onset of the Holocene (11,600–8,700 cal. year BP); (3) a tendency toward a drier climate during the middle Holocene (8,700–4,650 cal. year BP); and (4) a return to humid conditions following landscape modification by human activity (pastoral activities, deforestation) in the late Holocene (4,650–2,200 cal. year BP). Superimposed on relatively stable landscape conditions (e.g. maintenance of well established forests), the typical environmental variability of the southern European region is observed at this site.The Spanish Inter-Ministry Commission of Science and Technology (CICYT), the Spanish National Parks agency, the European Commission, the Spanish Ministry of Science, and the European Social Fund

Revealing the last 13,500 years of environmental history from the multiproxy record of a mountain lake (Lago Enol, northern Iberian Peninsula)

This is the author's accepted manuscript. The final publication is available at Springer via http://dx.doi.org/10.1007/s10933-009-9387-7.We present the Holocene sequence from Lago Enol (43°16′N, 4°59′W, 1,070 m a.s.l.), Cantabrian Mountains, northern Spain. A multiproxy analysis provided comprehensive information about regional humidity and temperature changes. The analysis included sedimentological descriptions, physical properties, organic carbon and carbonate content, mineralogy and geochemical composition together with biological proxies including diatom and ostracod assemblages. A detailed pollen study enabled reconstruction of variations in vegetation cover, which were interpreted in the context of climate changes and human impact. Four distinct stages were recognized for the last 13,500 years: (1) a cold and dry episode that includes the Younger Dryas event (13,500–11,600 cal. year BP); (2) a humid and warmer period characterizing the onset of the Holocene (11,600–8,700 cal. year BP); (3) a tendency toward a drier climate during the middle Holocene (8,700–4,650 cal. year BP); and (4) a return to humid conditions following landscape modification by human activity (pastoral activities, deforestation) in the late Holocene (4,650–2,200 cal. year BP). Superimposed on relatively stable landscape conditions (e.g. maintenance of well established forests), the typical environmental variability of the southern European region is observed at this site.The Spanish Inter-Ministry Commission of Science and Technology (CICYT), the Spanish National Parks agency, the European Commission, the Spanish Ministry of Science, and the European Social Fund

Late glacial and Holocene landscape change and rapid climate and coastal impacts in the Canal Beagle, southernmost Patagonia

Palaeoenvironmental data for the Late Glacial and Holocene periods are provided from Caleta Eugenia, in the eastern sector of Canal Beagle, southernmost Patagonia. The record commences at c. 16 200 cal a bp following glacier retreat in response to climatic warming. However, cooler conditions persisted during the Late Glacial period. The onset of more temperate conditions after c. 12 390 cal a bp is indicated by the arrival of southern beech forest and later establishment at c. 10 640 cal a bp, but the woodland growth was restricted by lower levels of effective moisture. The climate signal is then truncated by a rapid marine incursion at c. 8640 cal a bp which lasted until a more gradual emergence of the coast at c. 6600 cal a bp. During this period the pollen record appears to be dominated by the southern beech woodland. A punctuated hydroseral succession follows the isolation of the site from the sea leading to the re‐establishment of a peat bog. Between c. 5770 cal a bp and the present there were several periods of short rapid climatic change leading to drier conditions, probably as a result of late Holocene periods of climatic warming

Late glacial and Holocene landscape change and rapid climate and coastal impacts in the Canal Beagle, southernmost Patagonia

Palaeoenvironmental data for the Late Glacial and Holocene periods are provided from Caleta Eugenia, in the eastern sector of Canal Beagle, southernmost Patagonia. The record commences at c. 16 200 cal a bp following glacier retreat in response to climatic warming. However, cooler conditions persisted during the Late Glacial period. The onset of more temperate conditions after c. 12 390 cal a bp is indicated by the arrival of southern beech forest and later establishment at c. 10 640 cal a bp, but the woodland growth was restricted by lower levels of effective moisture. The climate signal is then truncated by a rapid marine incursion at c. 8640 cal a bp which lasted until a more gradual emergence of the coast at c. 6600 cal a bp. During this period the pollen record appears to be dominated by the southern beech woodland. A punctuated hydroseral succession follows the isolation of the site from the sea leading to the re‐establishment of a peat bog. Between c. 5770 cal a bp and the present there were several periods of short rapid climatic change leading to drier conditions, probably as a result of late Holocene periods of climatic warming

Movement and habitat use of the snapping turtle in an urban landscape

In order to effectively manage urban habitats, it is important to incorporate the spatial ecology and habitat use of the species utilizing them. Our previous studies have shown that the distribution of upland habitats surrounding a highly urbanized wetland habitat, the Central Canal (Indianapolis, IN, USA) influences the distribution of map turtles (Graptemys geographica) and red-eared sliders (Trachemys scripta) during both the active season and hibernation. In this study we detail the movements and habitat use of another prominent member of the Central Canal turtle assemblage, the common snapping turtle, Chelydra serpentina. We find the same major upland habitat associations for C. serpentina as for G. geographica and T. scripta, despite major differences in their activity (e.g., C. serpentina do not regularly engage in aerial basking). These results reinforce the importance of recognizing the connection between aquatic and surrounding terrestrial habitats, especially in urban ecosystems