Using next-generation sequencing for molecular reconstruction of past Arctic vegetation and climate

Palaeoenvironments and former climates are typically inferred from pollen and macrofossil records. This approach is time-consuming and suffers from low taxonomic resolution and biased taxon sampling. Here, we test an alternative DNA-based approach utilizing the P6 loop in the chloroplast trnL (UAA) intron; a short (13–158 bp) and variable region with highly conserved flanking sequences. For taxonomic reference, a whole trnL intron sequence database was constructed from recently collected material of 842 species, representing all widespread and/or ecologically important taxa of the species-poor arctic flora. The P6 loop alone allowed identification of all families, most genera (>75%) and one-third of the species, thus providing much higher taxonomic resolution than pollen records. The suitability of the P6 loop for analysis of samples containing degraded ancient DNA from a mixture of species is demonstrated by high-throughput parallel pyrosequencing of permafrost-preserved DNA and reconstruction of two plant communities from the last glacial period. Our approach opens new possibilities for DNA-based assessment of ancient as well as modern biodiversity of many groups of organisms using environmental samples.     

Development of SPECT imaging agents for the norepinephrine transporters: [123I]INER

A series of reboxetine analogs was synthesized and evaluated for in vitro binding as racemic mixtures. The best candidate (INER) was synthesized as the optically pure (S,S) enantiomer, labeled with iodine-123 and its in vivo binding determined by SPECT imaging in baboons. The in vivo specificity, selectivity, and kinetics of [123I]INER make it a promising agent for imaging NET in vivo by noninvasive SPECT imaging.     

CCR2 receptor antagonists: optimization of biaryl sulfonamides to increase activity in whole blood

A series of biarylsulfonamides was identified as hCCR2 receptor antagonist but suffered from high plasma protein binding resulting in a >100 fold shift in activity in a functional GTPγS assay run in tandem in the presence and absence of human serum albumin. Introduction of an aryl amide with ethylenediamine linker led to compounds with reduced shifts and improved activity in whole blood.     

DNA-based faecal dietary analysis: a comparison of qPCR and high throughput sequencing approaches

The genetic analysis of faecal material represents a relatively non-invasive way to study animal diet and has been widely adopted in ecological research. Due to the heterogeneous nature of faecal material the primary obstacle, common to all genetic approaches, is a means to dissect the constituent DNA sequences. Traditionally, bacterial cloning of PCR amplified products was employed; less common has been the use of species-specific quantitative PCR (qPCR) assays. Currently, with the advent of High-Throughput Sequencing (HTS) technologies and indexed primers it has become possible to conduct genetic audits of faecal material to a much greater depth than previously possible. To date, no studies have systematically compared the estimates obtained by HTS with that of qPCR. What are the relative strengths and weaknesses of each technique and how quantitative are deep-sequencing approaches that employ universal primers? Using the locally threatened Little Penguin (Eudyptula minor) as a model organism, it is shown here that both qPCR and HTS techniques are highly correlated and produce strikingly similar quantitative estimates of fish DNA in faecal material, with no statistical difference. By designing four species-specific fish qPCR assays and comparing the data to the same four fish in the HTS data it was possible to directly compare the strengths and weaknesses of both techniques. To obtain reproducible quantitative data one of the key, and often overlooked, steps common to both approaches is ensuring that efficient DNA isolation methods are employed and that extracts are free of inhibitors. Taken together, the methodology chosen for long-term faecal monitoring programs is largely dependent on the complexity of the prey species present and the level of accuracy that is desired. Importantly, these methods should not be thought of as mutually exclusive, as the use of both HTS and qPCR in tandem will generate datasets with the highest fidelity.     

Apical location of ferroportin 1 in airway epithelia and its role in iron detoxification in the lung

Ferroportin 1 (FPN1; aka MTP1, IREG1, and SLC40A1), which was originally identified as a basolateral iron transporter crucial for nutritional iron absorption in the intestine, is expressed in airway epithelia and upregulated when these cells are exposed to iron. Using immunofluorescence labeling and confocal microscopic imaging techniques, we demonstrate that in human and rodent lungs, FPN1 localizes subcellularly to the apical but not basolateral membrane of the airway epithelial cells. The role of airway epithelial cells in iron mobilization in the lung was studied in an in vitro model of the polarized airway epithelium. Normal human bronchial epithelial cells, grown on membrane supports until differentiated, were exposed to iron, and the efficiency and direction of iron transportation were studied. We found that these cells can efficiently take up iron across the apical but not basolateral surface in a concentration-dependent manner. Most of the iron taken up by the cells is then released into the medium within 8 h in the form of less reactive protein-bound complexes including ferritin and transferrin. Interestingly, iron release also occurred across the apical but not basolateral membrane. Our findings indicate that FPN1, depending on its subcellular location, could have distinct functions in iron homeostasis in different cells and tissues. Although it is responsible for exporting nutrient iron from enterocytes to the circulation in the intestine, it could play a role in iron detoxification in airway epithelial cells in the lung.     

Reciprocal upregulation of urokinase plasminogen activator and its inhibitor, PAI-2, by Borrelia burgdorferi affects bacterial penetration and host-inflammatory response

The mammalian plasminogen activation system (PAS) is a complex system involved in multiple physiological and pathological processes. Borrelia burgdorferi interacts with certain components of the PAS. Here we further investigate this interaction to determine its effect on bacterial dissemination and host cell migration in vitro. We show that stimulation of monocytic cells with B. burgdorferi induces the transient production and secretion of urokinase plasminogen activator (uPA), shortly followed by its physiological inhibitor, plasminogen activator inhibitor-2 (PAI-2). Mono Mac 6 (MM6) cells as well as peripheral blood monocytes enhanced transmigration of B. burgdorferi across a barrier coated with fibronectin mediated by uPA. Moreover, the induction of PAI-2 or the addition of recombinant PAI-2 did not have a significant effect on the uPA-potentiated transmigration of B. burgdorferi. In contrast, the induction of PAI-2 by B. burgdorferi resulted in significantly diminished invasion by monocytic cells across a reconstituted basement membrane (matrigel), which could be partially restored by treatment with purified uPA. These results show that the PAS plays a twofold role in the pathogenesis of B. burgdorferi infection, both by enhancing bacterial dissemination and by diminishing host-cell inflammatory migration.     

Environmental epigenetics: prospects for studying epigenetic mediation of exposure–response relationships

Changes in epigenetic marks such as DNA methylation and histone acetylation are associated with a broad range of disease traits, including cancer, asthma, metabolic disorders, and various reproductive conditions. It seems plausible that changes in epigenetic state may be induced by environmental exposures such as malnutrition, tobacco smoke, air pollutants, metals, organic chemicals, other sources of oxidative stress, and the microbiome, particularly if the exposure occurs during key periods of development. Thus, epigenetic changes could represent an important pathway by which environmental factors influence disease risks, both within individuals and across generations. We discuss some of the challenges in studying epigenetic mediation of pathogenesis and describe some unique opportunities for exploring these phenomena.     

Ultrasonic degradation, purification and analysis of structure and antioxidant activity of polysaccharide from Porphyra yezoensis Udea

The chemical structure and antioxidant of natural and ultrasonic degraded polysaccharides from Porphyra yezoensis Udea was investigated. The degraded polysaccharide (PYPS_UD) was purified, and F2 (a homogeneous fraction) was obtained. FT-IR, ¹H and ¹³C NMR spectral analysis revealed that F2 have typical porphyran structure. It has a backbone of alternating (1 → 4)-3,6-anhydro-α-l-galactopyranose) units and (1 → 3)-linked β-d-galactose or (1 → 4)-linked α-l-galactose 6-sulfate units. The result ascertained ultrasound degradation did not change the main structure of polysaccharides in the test conditions. Antioxidant proved that the activity of scavenging superoxide and hydroxyl radical is F₂ > V_C > PSPY_UD > PSPY. It was possible that ultrasonic treatment is an effective way for enhancing PSPY's antioxidant activity ascribing to decreasing molecular weight of polysaccharides.     

Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA

Analyses of degraded DNA are typically hampered by contamination, especially when employing universal primers such as commonly used in environmental DNA studies. In addition to false-positive results, the amplification of contaminant DNA may cause false-negative results because of competition, or bias, during the PCR. In this study, we test the utility of human-specific blocking primers in mammal diversity analyses of ancient permafrost samples from Siberia. Using quantitative PCR (qPCR) on human and mammoth DNA, we first optimized the design and concentration of blocking primer in the PCR. Subsequently, 454 pyrosequencing of ancient permafrost samples amplified with and without the addition of blocking primer revealed that DNA sequences from a diversity of mammalian representatives of the Beringian megafauna were retrieved only when the blocking primer was added to the PCR. Notably, we observe the first retrieval of woolly rhinoceros (Coelodonta antiquitatis) DNA from ancient permafrost cores. In contrast, reactions without blocking primer resulted in complete dominance by human DNA sequences. These results demonstrate that in ancient environmental analyses, the PCR can be biased towards the amplification of contaminant sequences to such an extent that retrieval of the endogenous DNA is severely restricted. The application of blocking primers is a promising tool to avoid this bias and can greatly enhance the quantity and the diversity of the endogenous DNA sequences that are amplified.     

Islands in the ice: detecting past vegetation on Greenlandic nunataks using historical records and sedimentary ancient DNA meta-barcoding

Nunataks are isolated bedrocks protruding through ice sheets. They vary in age, but represent island environments in 'oceans' of ice through which organism dispersals and replacements can be studied over time. The J.A.D. Jensen's Nunataks at the southern Greenland ice sheet are the most isolated nunataks on the northern hemisphere - some 30 km from the nearest biological source. They constitute around 2 km(2) of ice-free land that was established in the early Holocene. We have investigated the changes in plant composition at these nunataks using both the results of surveys of the flora over the last 130 years and through reconstruction of the vegetation from the end of the Holocene Thermal Maximum (5528 ± 75 cal year BP) using meta-barcoding of plant DNA recovered from the nunatak sediments (sedaDNA). Our results show that several of the plant species detected with sedaDNA are described from earlier vegetation surveys on the nunataks (in 1878, 1967 and 2009). In 1967, a much higher biodiversity was detected than from any other of the studied periods. While this may be related to differences in sampling efforts for the oldest period, it is not the case when comparing the 1967 and 2009 levels where the botanical survey was exhaustive. As no animals and humans are found on the nunataks, this change in diversity over a period of just 42 years must relate to environmental changes probably being climate-driven. This suggests that even the flora of fairly small and isolated ice-free areas reacts quickly to a changing climate.