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When studying and investigating the history of a property or neighbourhood, common resources often sought by researchers include air photos, fire insurance plans and historical maps. These will provide information about what an area looked like, often offering the building’s footprint and surrounding landscape. Timespan studies will no doubt show growth, development and possibly changes to the buildings of interest. But what the rich resources don’t tell the researcher is information about the people connected to those buildings. Who lived or worked there? What did they do for a living? Did they move often? Did they change jobs regularly? These types of questions can’t be answered with just maps alone as they require a detailed census to go along with it. To fill this type of need, Geospatial Centre staff at the University of Waterloo Library embarked on a massive-scale digitization and geo-location city directory project – one that had started in 2019, has involved at least 40 staff members, and still has a couple years to go before completion. This paper will summarize the project thus far, with a focus on the journey of geocoding historical streets.

In recent years, unmanned aerial vehicles (UAVs) have increasingly been used in various application areas, such as in the remote sensing or surveying. For these applications the UAV has to be equipped with a mapping sensor, which is mostly a camera. Furthermore, a georeferencing of the UAV platform and/or the acquired mapping data is required. The most efficient way to realize this georeferencing is the direct georeferencing, which is based on an onboard multi-sensor system. In recent decades, direct georeferencing systems have been researched and used extensively in airborne, ship and land vehicle applications. However, these systems cannot easily be adapted to UAV platforms, which is mainly due to weight and size limitations. In this paper a direct georeferencing system for micro- and mini-sized UAVs is presented, which consists of a dual-frequency geodetic grade OEM GPS board, a low-cost single-frequency GPS chip, a tactical grade IMU and a magnetometer. To allow for cm-level position and sub-degree attitude accuracies, RTK GPS (real-time kinematic) and GPS attitude (GPS compass) determination algorithms are running on this system, as well as a GPS/IMU integration. Beside the direct georeferencing, also the precise time synchronization of the camera, which acts as the main sensor for mobile mapping applications, and the calibration of the lever arm between the camera reference point and the direct georeferencing reference point are explained in this paper. Especially the high accurate time synchronization of the camera is very important, to still allow for high surveying accuracies, when the images are taken during the motion of the UAV. Results of flight tests demonstrate that the developed system, the camera synchronization and the lever arm calibration make directly georeferenced UAV based single point measurements possible, which have cm-level accuracies on the ground.

Due to its perennial nature and size, the acquisition of phenotypic data in grapevine research is almost exclusively restricted to the field and done by visual estimation. This kind of evaluation procedure is limited by time, cost and the subjectivity of records. As a consequence, objectivity, automation and more precision of phenotypic data evaluation are needed to increase the number of samples, manage grapevine repositories, enable genetic research of new phenotypic traits and, therefore, increase the efficiency in plant research. In the present study, an automated field phenotyping pipeline was setup and applied in a plot of genetic resources. The application of the PHENObot allows image acquisition from at least 250 individual grapevines per hour directly in the field without user interaction. Data management is handled by a database (IMAGEdata). The automatic image analysis tool BIVcolor (Berries in Vineyards-color) permitted the collection of precise phenotypic data of two important fruit traits, berry size and color, within a large set of plants. The application of the PHENObot represents an automated tool for high-throughput sampling of image data in the field. The automated analysis of these images facilitates the generation of objective and precise phenotypic data on a larger scale.

In grapevine research the acquisition of phenotypic data is largely restricted to the field due to its perennial nature and size. The methodologies used to assess morphological traits and phenology are mainly limited to visual scoring. Some measurements for biotic and abiotic stress, as well as for quality assessments, are done by invasive measures. The new evolving sensor technologies provide the opportunity to perform non-destructive evaluations of phenotypic traits using different field phenotyping platforms. One of the biggest technical challenges for field phenotyping of grapevines are the varying light conditions and the background. In the present study the Phenoliner is presented, which represents a novel type of a robust field phenotyping platform. The vehicle is based on a grape harvester following the concept of a moveable tunnel. The tunnel it is equipped with different sensor systems (RGB and NIR camera system, hyperspectral camera, RTK-GPS, orientation sensor) and an artificial broadband light source. It is independent from external light conditions and in combination with artificial background, the Phenoliner enables standardised acquisition of high-quality, geo-referenced sensor data.

Methylierung von Cytosin zu 5-Methylcytosin (5mC) dient der epigenetischen Kontrolle der Gentranskription in Saugetieren und hat direkte Auswirkungen auf die Entwicklung und Pathogenese des Menschen. Insbesondere die Diagnose und Prognostik von Krebspatienten profitierte in den letzten Jahren enorm von der Entwicklung neuer Methoden zur Analyse von DNA-Methylierungsmustern. Wir zeigen hier, dass zwei hitzestabile DNA-Polymerasen – die DNA-Polymerase KlenTaq aus Thermus aquaticus und die KOD-DNA-Polymerase aus Thermococcus kodakaraensis – 3′-fehlgepaarte Oligonukleotid-Primer gegenuber 5mC effektiver verlangern als gegenuber nichtmethyliertem C. Wir konnten auserdem eine DNA-Polymerase-Mutante mit weiter verbesserter 5mC/C-Diskriminierung erzeugen. Diese Mutante wurde erfolgreich in einer neuartigen 5mC-Detektierungsmethode eingesetzt, die es ermoglicht, den Methylierungszustand eines Cytosins direkt durch PCR von unbehandelter genomischer DNA zu bestimmen.

The epigenetic control of genes by the methylation of cytosine resulting in 5-methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3′-mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation-specific PCR approach directly from untreated human genomic DNA.

„Untreu“: Eine DNA-abhängige DNA-Polymerase, deren Wildtyp-Vorläufer keine signifikante Reverse-Transkriptase-Aktivität zeigte, wurde beim Einbau eines Nukleotids gegenüber einem RNA-Templat abgefangen und erstmals strukturell charakterisiert. Die signifikant erhöhte Reverse-Transkriptase-Aktivität der DNA- Polymerase-Variante ermöglicht ihre Anwendung in PCR-basierten molekular- diagnostischen Prozessen. published

In many unmanned aerial vehicle (UAV) applications a direct georeferencing is required. The reason can be that the UAV flies autonomous and must be navigated precisely, or that the UAV performs a remote sensing operation, where the position of the camera has to be known at the moment of the recording. In our application, a project called Mapping on Demand, we are motivated by both of these reasons. The goal of this project is to develop a lightweight autonomously flying UAV that is able to identify and measure inaccessible three-dimensional objects by use of visual information. Due to payload and space limitations, precise position and attitude determination of micro- and mini-sized UAVs is very challenging. The limitations do not only affect the onboard computing capacity, but they are also noticeable when choosing the georeferencing sensors. In this article, we will present a new developed onboard direct georeferencing system which is real-time capable, applicable for lightweight UAVs and provides very precise results (position accuracy σ < 5 cm and attitude accuracy σ < 0.5 deg). In this system GPS, inertial sensors, magnetic field sensors, a barometer as well as stereo video cameras are used as georeferencing sensors. We will describe the hardware development and will go into details of the implemented software. In this context especially the RTK-GPS software and the concept of the attitude determination by use of inertial sensors, magnetic field sensors as well as an onboard GPS baseline will be highlighted. Finally, results of first field tests as well as an outlook on further developments will conclude this contribution.

Artificial regulatory RNAs have been attracting more and more interest over the past years. Their small size, combined with versatile applications, make them promising tools for designing complex eukaryotic gene networks. A variety of such regulatory RNA parts have been developed that are capable of efficiently controlling gene expression. A common basis for many of these genetic switches is the specific ligand binding to a RNA domain, followed by a conformational change. Depending on the surroundings, this can affect transcription, translation or even RNA interference (RNAi) efficacy. This article provides a concise summary of the main controllable RNA parts thus far developed for eukaryotic systems.

The protocol presented here allows for the investigation of the formation of unusual nucleic acid structures in the 5'-untranslated region (UTR) of bacteria by correlating gene expression levels to the in vitro stability of the respective structure. In particular, we describe the introduction of G-quadruplex forming sequences close to the ribosome-binding site (RBS) on the mRNA of a reporter gene and the subsequent read-out of the expression levels. Insertion of a stable secondary structure results in the cloaking of RBS and eventually reduced gene expression levels. The structures and stability of the introduced sequences are further characterized by circular dichroism (CD) spectroscopy and thermal melting experiments. The extent of inhibition is then correlated to the stability of the respective quadruplex structure, allowing judgement of whether factors other than thermodynamic stability affect the formation of a given quadruplex sequence in vivo. Measuring gene expression levels takes 2 d including cloning; CD experiments take 5 hours per experiment.

thesis is based on bringing small-molecule reactants in close proximity through DNA conjugation and hybridization, and thereby increasing the effective molarity, which significantly A rates of chemical reactions. Several applications of this approach, including compound discovery from synthetic libraries, reaction discovery, and nucleic-acid sensing, have been described. [1] All of the depicted approaches have in common the fact that the two reactants or a reactant and a catalyst are covalently linked to two DNA or PNA strands and are subsequently brought to react by self hybridization or aligned through hybridization. Roelfes and Feringa reported a Diels– Alder reaction mediated by a catalyst intercalated into a DNA duplex. Binding to the duplex resulted in good enantioselectivity of the reaction. [2] Poulin-Kerstien and Dervan employed binding of two polyamides to double-stranded DNA (dsDNA) to promote a 1,3-dipolar cycloaddition; this resulted in linked polyamides. [2] Previously, we found that proline-modified DNA acts as a catalyst in the aldol reaction between a complementary DNA-tethered aldehyde and various nontethered ketones. [3] The formation of a Watson–Crick duplex between the

Light-dependent gene regulation systems are advantageous as they allow for precise spatio-temporal control of target gene expression. In this paper, we present a novel UV-A and blue-light-inducible gene control system that is based on the light-dependent heterodimerization of the CRY2 and C1BN domains. Upon their interaction, a transcription factor is released from the cell membrane and initiates target gene expression. Capitalizing on that, sun cream UV-A protection properties were measured intracellularly.

Unfaithful: The structure of a DNA-dependent DNA polymerase, whose parental ancestor has no significant reverse transcriptase activity, was obtained for the first time incorporating a nucleotide against an RNA template and then structurally characterized. The DNA polymerase variant has significant reverse transcriptase activity that allows its application in PCR-based molecular diagnostics.

In cellular systems environmental and metabolic signals are integrated for the conditional control of gene expression. On the other hand, artificial manipulation of gene expression is of high interest for metabolic and genetic engineering. Especially the reprogramming of gene expression patterns to orchestrate cellular responses in a predictable fashion is considered to be of great importance. Here we introduce a highly modular RNA-based system for performing Boolean logic computation at a post-transcriptional level in Escherichia coli. We have previously shown that artificial riboswitches can be constructed by utilizing ligand-dependent Hammerhead ribozymes (aptazymes). Employing RNA self-cleavage as the expression platform-mechanism of an artificial riboswitch has the advantage that it can be applied to control several classes of RNAs such as mRNAs, tRNAs, and rRNAs. Due to the highly modular and orthogonal nature of these switches it is possible to combine aptazyme regulation of activating a suppressor tRNA with the regulation of mRNA translation initiation. The different RNA classes can be controlled individually by using distinct aptamers for individual RNA switches. Boolean logic devices are assembled by combining such switches in order to act on the expression of a single mRNA. In order to demonstrate the high modularity, a series of two-input Boolean logic operators were constructed. For this purpose, we expanded our aptazyme toolbox with switches comprising novel behaviours with respect to the small molecule triggers thiamine pyrophosphate (TPP) and theophylline. Then, individual switches were combined to yield AND, NOR, and ANDNOT gates. This study demonstrates that post-transcriptional aptazyme-based switches represent versatile tools for engineering advanced genetic devices and circuits without the need for regulatory protein cofactors.

Aptamers binding proteins or small molecules have been shown to be versatile and powerful building blocks for the construction of artificial genetic switches. In this study, we present a novel aptamer-based construct regulating the Tet Off system in a tetracycline-independent manner thus achieving control of transgene expression. For this purpose, a TetR protein-inhibiting aptamer was engineered for use in mammalian cells, enabling the RNA-responsive control of the tetracycline-dependent transactivator (tTA). By rationally attaching the theophylline aptamer as a sensor, the inhibitory TetR aptamer and thus tTA activity became dependent on the ligand of the sensor aptamer. Addition of the small molecule theophylline resulted in enhanced binding to the corresponding protein in vitro and in inhibition of reporter gene expression in mammalian cell lines. By using aptamers as adaptors in order to control protein activity by a predetermined small molecule, we present a simple and straightforward approach for future applications in the field of Chemical Biology. Moreover, aptamer-based control of the widely used Tet system introduces a new layer of regulation thereby facilitating the construction of more complex gene networks., Nucleic Acids Research, 39 (22), ISSN:1362-4962, ISSN:0301-5610

SummaryDeveloping artificial genetic switches in order to control gene expression via an external stimulus is an important aim in chemical and synthetic biology. Here, we expand the application range of RNA switches to the regulation of 16S rRNA function in Escherichia coli. For this purpose, we incorporated hammerhead ribozymes at several positions into orthogonalized 16S rRNA. We observed that ribosomal function is remarkably tolerant toward the incorporation of large additional RNA fragments at certain sites of the 16S rRNA. However, ribozyme-mediated cleavage results in severe reduction of 16S rRNA stability. We carried out an in vivo screen for the identification of sequences acting as ligand-responsive RNA switches, enabling thiamine-dependent switching of 16S rRNA function. In addition to expanding the regulatory toolbox, the presented artificial riboswitches should prove valuable to study aspects of rRNA folding and stability in bacteria.

Toehold-containing DNA strands allow for the assembly of complex nanostructures via kinetically driven hybridization reactions. Here, we have rendered this strategy ligand-dependent, resulting in DNA nanoarchitectures such as four-way junctions inducible via the addition of ATP or arginine amide.

The observation that Cadmium (Cd(2+)) inhibits Msh2-Msh6, which is responsible for identifying base pair mismatches and other discrepancies in DNA, has led to the proposal that selective targeting of this protein and consequent suppression of DNA repair or apoptosis promote the carcinogenic effects of the heavy metal toxin. It has been suggested that Cd(2+) binding to specific sites on Msh2-Msh6 blocks its DNA binding and ATPase activities. To investigate the mechanism of inhibition, we measured Cd(2+) binding to Msh2-Msh6, directly and by monitoring changes in protein structure and enzymatic activity. Global fitting of the data to a multiligand binding model revealed that binding of about 100 Cd(2+) ions per Msh2-Msh6 results in its inactivation. This finding indicates that the inhibitory effect of Cd(2+) occurs via a nonspecific mechanism. Cd(2+) and Msh2-Msh6 interactions involve cysteine sulfhydryl groups, and the high Cd(2+):Msh2-Msh6 ratio implicates other ligands such as histidine, aspartate, glutamate, and the peptide backbone as well. Our study also shows that cadmium inactivates several unrelated enzymes similarly, consistent with a nonspecific mechanism of inhibition. Targeting of a variety of proteins, including Msh2-Msh6, in this generic manner would explain the marked broad-spectrum impact of Cd(2+) on biological processes. We propose that the presence of multiple nonspecific Cd(2+) binding sites on proteins and their propensity to change conformation on interaction with Cd(2+) are critical determinants of the susceptibility of corresponding biological systems to cadmium toxicity.

SummaryRNA-based modules such as riboswitches represent straightforward and simplified approaches for the regulation of gene expression, as no additional proteins are needed. G-rich sequences are known to adopt stable four-stranded structures, and such quadruplexes have been suspected to play important roles in key functions such as the control of gene expression. Here we demonstrate that RNA quadruplexes readily form in vivo. We have constructed mRNA-based G-rich elements that mask the ribosome binding site by folding into four-stranded structures. The suppression of gene expression correlates with the stability of inserted G quadruplexes. Moreover, quadruplexes with moderate stability respond to changes in temperature, thus behaving as artificial RNA thermometers. In conclusion, we introduce tuneable mRNA-based devices that enable modulation of gene expression by a predictable but thus far unknown mechanism.

Fusions of a ribozyme and an aptamer of a natural riboswitch (thiamine pyrophosphate, TPP) are used to construct artificial thiamine-dependent switches of gene expression. As J. S. Hartig et al. describe in their Communication on page 2715 ff., insertion of these RNA elements into bacterial mRNAs allows translation of the message to be switched on or off. TPP triggers changes to the ribozyme-mediated mRNA cleavage, resulting in liberation of the message for translational initiation.