Application of chemically modified oligonucleotides in nanopore sensing and DNA nano – biotechnology

This thesis describes how targeted chemical modification can enhance the properties of nucleic acids for use in (i) nanopore analytics and (ii) nanobiotechnology. In nanopore analytics, individual molecules are detected as they pass a nanoscale pore to give rise to detectable blockades in ionic current. Despite progress in the sensing of a multitude of molecular species, the analytical resolution in the sensing of DNA is poor as individual bases in passing strands cannot be resolved due to the high speed of translocation. Here a new approach is presented which slows down single stranded DNA and enables the detection of multiple separate bases. Chemical tags are attached to bases, which cause a steric blockade each time a modified base passes a narrow pore. The resulting characteristic current signatures are specific for the chemical composition and the size of the tags. The unique electrical signatures can be exploited to encode sequence information as demonstrated for the discrimination between drug resistance-conferring point mutations. In addition, the generation of nucleotides with tailored properties may help develop a fast nanopore approach to size highly repetitive DNA sequences for forensic applications. In DNA nanobiotechnology, oligonucleotides are self-assembled via hybridization to generate higher-order structures of defined geometry. Here, the functional range of DNA nanostructures is expanded by chemically modifying the constituent nucleic acids. Firstly, tetrahedron-shaped nanostructures are demonstrated to act as a scaffold to assemble a multitude of different chemical groups at tunable stoichiometry and at geometrically defined sites. The new molecular entities exhibit functional properties beneficial in biosensing and diagnostics. In addition, an approach is presented to achieve self-assembly between DNA-strands via covalently attached tags that form reversible yet tight metal chelate complexes. This chemical strategy to form supramolecular structures can potentially be extended to protein or peptide networks of interest in basic science and technology

Biodiversity of nematode assemblages from the region of the Clarion-Clipperton Fracture Zone, an area of commercial mining interest

BACKGROUND: The possibility for commercial mining of deep-sea manganese nodules is currently under exploration in the abyssal Clarion-Clipperton Fracture Zone. Nematodes have potential for biomonitoring of the impact of commercial activity but the natural biodiversity is unknown. We investigate the feasibility of nematodes as biomonitoring organisms and give information about their natural biodiversity. RESULTS: The taxonomic composition (at family to genus level) of the nematode fauna in the abyssal Pacific is similar, but not identical to, the North Atlantic. Given the immature state of marine nematode taxonomy, it is not possible to comment on the commonality or otherwise of species between oceans. The between basin differences do not appear to be directly linked to current ecological factors. The abyssal Pacific region (including the Fracture Zone) could be divided into two biodiversity subregions that conform to variations in the linked factors of flux to the benthos and of sedimentary characteristics. Richer biodiversity is associated with areas of known phytodetritus input and higher organic-carbon flux. Despite high reported sample diversity, estimated regional diversity is less than 400 species. CONCLUSION: The estimated regional diversity of the CCFZ is a tractable figure for biomonitoring of commercial activities in this region using marine nematodes, despite the immature taxonomy (i.e. most marine species have not been described) of the group. However, nematode ecology is in dire need of further study

Interhemispheric structure and variability of the 5-day planetary wave from meteor radar wind measurements

A study of the quasi-5-day wave (5DW) was performed using meteor radars at conjugate latitudes in the Northern and Southern hemispheres. These radars are located at Esrange, Sweden (68° N) and Juliusruh, Germany (55° N) in the Northern Hemisphere, and at Tierra del Fuego, Argentina (54° S) and Rothera Station, Antarctica (68° S) in the Southern Hemisphere. The analysis was performed using data collected during simultaneous measurements by the four radars from June 2010 to December 2012 at altitudes from 84 to 96 km. The 5DW was found to exhibit significant short-term, seasonal, and interannual variability at all sites. Typical events had planetary wave periods that ranged between 4 and 7 days, durations of only a few cycles, and infrequent strongly peaked variances and covariances. Winds exhibited rotary structures that varied strongly among sites and between events, and maximum amplitudes up to ~ 20 m s−1. Mean horizontal velocity covariances tended to be largely negative at all sites throughout the interval studied

Interhemispheric structure and variability of the 5-day planetary wave from meteor radar wind measurements

A study of the quasi-5-day wave (5DW) was performed using meteor radars at conjugate latitudes in the Northern and Southern hemispheres. These radars are located at Esrange, Sweden (68° N) and Juliusruh, Germany (55° N) in the Northern Hemisphere, and at Tierra del Fuego, Argentina (54° S) and Rothera Station, Antarctica (68° S) in the Southern Hemisphere. The analysis was performed using data collected during simultaneous measurements by the four radars from June 2010 to December 2012 at altitudes from 84 to 96 km. The 5DW was found to exhibit significant short-term, seasonal, and interannual variability at all sites. Typical events had planetary wave periods that ranged between 4 and 7 days, durations of only a few cycles, and infrequent strongly peaked variances and covariances. Winds exhibited rotary structures that varied strongly among sites and between events, and maximum amplitudes up to ~ 20 m s−1. Mean horizontal velocity covariances tended to be largely negative at all sites throughout the interval studied

Global observations of 2 day wave coupling to the diurnal tide in a high‐altitude forecast‐assimilation system

We examine wave components in a high-altitude forecast-assimilation system that arise from nonlinear interaction between the diurnal tide and the westward traveling quasi 2 day wave. The process yields a westward traveling “sum” wave with zonal wave number 4 and a period of 16 h, and an eastward traveling “difference” wave with zonal wave number 2 and a period of 2 days. While the eastward 2 day wave has been reported in satellite temperatures, the westward 16 h wave lies outside the Nyquist limits of resolution of twice daily local time satellite sampling. Hourly output from a high-altitude forecast-assimilation model is used to diagnose the nonlinear quadriad. A steady state primitive equation model forced by tide-2 day wave advection is used to intepret the nonlinear wave products. The westward 16 h wave maximizes in the midlatitude winter mesosphere and behaves like an inertia-gravity wave. The nonlinearly generated component of the eastward 2 day wave maximizes at high latitudes in the lower thermosphere, and only weakly penetrates to low latitudes. The 16 h and the eastward 2 day waves are of comparable amplitude and alias to the same apparent frequency when viewed from a satellite perspective

Genetic consequences of multiple translocations of the banded hare-qallaby in Western Australia

Many Australian mammal species now only occur on islands and fenced mainland havens free from invasive predators. The range of one species, the banded hare-wallaby (Lagostrophus fasciatus), had contracted to two offshore islands in Western Australia. To improve survival, four conservation translocations have been attempted with mixed success, and all occurred in the absence of genetic information. Here, we genotyped seven polymorphic microsatellite markers in two source (Bernier Island and Dorre Island), two historic captive, and two translocated L. fasciatus populations to determine the impact of multiple translocations on genetic diversity. Subsequently, we used population viability analysis (PVA) and gene retention modelling to determine scenarios that will maximise demographic resilience and genetic richness of two new populations that are currently being established. One translocated population (Wadderin) has undergone a genetic bottleneck and lost 8.1% of its source population’s allelic diversity, while the other (Faure Island) may be inbred. We show that founder number is a key parameter when establishing new L. fasciatus populations and 100 founders should lead to high survival probabilities. Our modelling predicts that during periodic droughts, the recovery of source populations will be slower post-harvest, while 75% more animals—about 60 individuals—are required to retain adequate allelic diversity in the translocated population. Our approach demonstrates how genetic data coupled with simulations of stochastic environmental events can address central questions in translocation programmes

Model thrombi formed under flow reveal the role of factor XIII-mediated cross-linking in resistance to fibrinolysis

Background: Activated factor XIII (FXIIIa), a transglutaminase, introduces fibrin-fibrin and fibrin-inhibitor cross-links, resulting in more mechanically stable clots. The impact of cross-linking on resistance to fibrinolysis has proved challenging to evaluate quantitatively. Methods: We used a whole blood model thrombus system to characterize the role of cross-linking in resistance to fibrinolytic degradation. Model thrombi, which mimic arterial thrombi formed in vivo, were prepared with incorporated fluorescently labeled fibrinogen, in order to allow quantification of fibrinolysis as released fluorescence units per minute. Results: A site-specific inhibitor of transglutaminases, added to blood from normal donors, yielded model thrombi that lysed more easily, either spontaneously or by plasminogen activators. This was observed both in the cell/platelet-rich head and fibrin-rich tail. Model thrombi from an FXIII-deficient patient lysed more quickly than normal thrombi; replacement therapy with FXIII concentrate normalized lysis. In vitro addition of purified FXIII to the patient's preprophylaxis blood, but not to normal control blood, resulted in more stable thrombi, indicating no further efficacy of supraphysiologic FXIII. However, addition of tissue transglutaminase, which is synthesized by endothelial cells, generated thrombi that were more resistant to fibrinolysis; this may stabilize mural thrombi in vivo. Conclusions: Model thrombi formed under flow, even those prepared as plasma 'thrombi', reveal the effect of FXIII on fibrinolysis. Although very low levels of FXIII are known to produce mechanical clot stability, and to achieve ?-dimerization, they appear to be suboptimal in conferring full resistance to fibrinolysis

On the longitudinal structure of the transient day-to-day variation of the semidiurnal tide in the mid-latitude lower thermosphere - I. Winter season

The longitudinal structure of the day-to-day variations of semidiurnal tide amplitudes is analysed based on coordinated mesosphere/lower thermosphere wind measurements at several stations during three winter campaigns. Possible excitation sources of these variations are discussed. Special attention is given to a nonlinear interaction between the semidiurnal tide and the day-to-day mean wind variations. Data processing includes the S-transform analysis which takes into account transient behaviour of secondary waves. It is shown that strong tidal modulations appear during a stratospheric warming and may be caused by aperiodic mean wind variations during this event

Multiple Imputation for Incomplete Data in Epidemiologic Studies

Epidemiologic studies are frequently susceptible to missing information. Omitting observations with missing variables remains a common strategy in epidemiologic studies, yet this simple approach can often severely bias parameter estimates of interest if the values are not missing completely at random. Even when missingness is completely random, complete-case analysis can reduce the efficiency of estimated parameters, because large amounts of available data are simply tossed out with the incomplete observations. Alternative methods for mitigating the influence of missing information, such as multiple imputation, are becoming an increasing popular strategy in order to retain all available information, reduce potential bias, and improve efficiency in parameter estimation. In this paper, we describe the theoretical underpinnings of multiple imputation, and we illustrate application of this method as part of a collaborative challenge to assess the performance of various techniques for dealing with missing data (Am J Epidemiol. 2018;187(3):568–575). We detail the steps necessary to perform multiple imputation on a subset of data from the Collaborative Perinatal Project (1959–1974), where the goal is to estimate the odds of spontaneous abortion associated with smoking during pregnancy

Principled Approaches to Missing Data in Epidemiologic Studies

Principled methods with which to appropriately analyze missing data have long existed; however, broad implementation of these methods remains challenging. In this and 2 companion papers (Am J Epidemiol. 2018;187(3):576–584 and Am J Epidemiol. 2018;187(3):585–591), we discuss issues pertaining to missing data in the epidemiologic literature. We provide details regarding missing-data mechanisms and nomenclature and encourage the conduct of principled analyses through a detailed comparison of multiple imputation and inverse probability weighting. Data from the Collaborative Perinatal Project, a multisite US study conducted from 1959 to 1974, are used to create a masked data-analytical challenge with missing data induced by known mechanisms. We illustrate the deleterious effects of missing data with naive methods and show how principled methods can sometimes mitigate such effects. For example, when data were missing at random, naive methods showed a spurious protective effect of smoking on the risk of spontaneous abortion (odds ratio (OR) = 0.43, 95% confidence interval (CI): 0.19, 0.93), while implementation of principled methods multiple imputation (OR = 1.30, 95% CI: 0.95, 1.77) or augmented inverse probability weighting (OR = 1.40, 95% CI: 1.00, 1.97) provided estimates closer to the “true” full-data effect (OR = 1.31, 95% CI: 1.05, 1.64). We call for greater acknowledgement of and attention to missing data and for the broad use of principled missing-data methods in epidemiologic research