Generic Reporter Sets for Colorimetric Multiplex dPCR Demonstrated with 6-Plex SNP Quantification Panels.

Digital PCR (dPCR) is a powerful method for highly sensitive and precise quantification of nucleic acids. However, designing and optimizing new multiplex dPCR assays using target sequence specific probes remains cumbersome, since fluorescent signals must be optimized for every new target panel. As a solution, we established a generic fluorogenic 6-plex reporter set, based on mediator probe technology, that decouples target detection from signal generation.

Eco-evolutionary experience and behavioral innovation in interactions with non-native species.

Behavioral changes play an important role for animals to cope with human-induced rapid environmental change such as biological invasions. The concept of eco-evolutionary experience (EEE) postulates that native species are more strongly impacted by non-native species the more these differ from species they have coevolved with. Also, EEE could influence the degree of innovation in new behaviors shown by native species.

Phage-assisted evolution of highly active cytosine base editors with enhanced selectivity and minimal sequence context preference.

TadA-derived cytosine base editors (TadCBEs) enable programmable C•G-to-T•A editing while retaining the small size, high on-target activity, and low off-target activity of TadA deaminases. Existing TadCBEs, however, exhibit residual A•T-to-G•C editing at certain positions and lower editing efficiencies at some sequence contexts and with non-SpCas9 targeting domains. To address these limitations, we use phage-assisted evolution to evolve CBE6s from a TadA-mediated dual cytosine and adenine base editor, discovering mutations at N46 and Y73 in TadA that prevent A•T-to-G•C editing and improve C•G-to-T•A editing with expanded sequence-context compatibility, respectively.

Fuzzy logic control for watering system.

A two-dimensional finite element (FEM) model was developed to simulate water propagation in soil during irrigation. The first dimension was water distribution depth in soil, and the second dimension was time. The developed model was tested by analyzing water distribution in a conventional (clock-controlled) irrigation model.

Reporter emission multiplexing in digital PCRs (REM-dPCRs): direct quantification of multiple target sequences per detection channel by population specific reporters.

Digital PCRs (dPCRs) are widely used methods for the detection and quantification of rare abundant sequences relevant to fields such as liquid biopsy or oncology. In order to increase the information content and save valuable sample materials, there is a significant need for digital multiplexing methods that are easy to establish, analyse, and interpret, and ideally allow the usage of existing lab equipment. Herein, we present a novel reporter emission multiplexing approach for the digital PCR method (REM-dPCR), which meets these requirements.

Phage-assisted evolution and protein engineering yield compact, efficient prime editors.

Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size and improved efficiency. Using phage-assisted evolution, we improved editing efficiencies of compact reverse transcriptases by up to 22-fold and generated prime editors that are 516-810 base pairs smaller than the current-generation editor PEmax.

Synergistic Binding of the Halide and Cationic Prime Substrate of l-Lysine 4-Chlorinase, BesD, in Both Ferrous and Ferryl States.

An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl or Br), the halogenation target ("prime substrate"), and dioxygen. In well-studied cases, the three nongaseous substrates must bind to activate the enzyme's Fe(II) cofactor for efficient capture of O. Halide, 2OG, and (lastly) O all coordinate directly to the cofactor to initiate its conversion to a -halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (H) from the non-coordinating prime substrate to enable radicaloid carbon-halogen coupling.

Synergistic Binding of the Halide and Cationic Prime Substrate of the l-Lysine 4-Chlorinase, BesD, in Both Ferrous and Ferryl States.

An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl or Br ), the halogenation target ("prime substrate"), and dioxygen. In well-studied cases, the three non-gaseous substrates must bind to activate the enzyme's Fe(II) cofactor for efficient capture of O . Halide, 2OG, and (lastly) O all coordinate directly to the cofactor to initiate its conversion to a -halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (H•) from the non-coordinating prime substrate to enable radicaloid carbon-halogen coupling.

Biocatalytic control of site-selectivity and chain length-selectivity in radical amino acid halogenases.

Biocatalytic C-H activation has the potential to merge enzymatic and synthetic strategies for bond formation. Fe/αKG-dependent halogenases are particularly distinguished for their ability both to control selective C-H activation as well as to direct group transfer of a bound anion along a reaction axis separate from oxygen rebound, enabling the development of new transformations. In this context, we elucidate the basis for the selectivity of enzymes that perform selective halogenation to yield 4-Cl-lysine (BesD), 5-Cl-lysine (HalB), and 4-Cl-ornithine (HalD), allowing us to probe how site-selectivity and chain length selectivity are achieved.

Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.

Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination.

Analyzing siRNA Concentration, Complexation and Stability in Cationic Dendriplexes by Stem-Loop Reverse Transcription-qPCR.

RNA interference (RNAi) is a powerful therapeutic approach for messenger RNA (mRNA) level regulation in human cells. RNAi can be triggered by small interfering RNAs (siRNAs) which are delivered by non-viral carriers, e.g.

Higher education non-completion, employers, and labor market integration: Experimental evidence.

Theories of job assignment suggest that employers give a lot of weight to achieved educational credentials. But what if credentials are missing? We theorize how college non-completion affects the hiring chances, identify its causal effect in different labor market segments, and assess which factors facilitate labor market entry for dropouts. Based on a simulated hiring process with N = 1382 German employers who rated more than 10,000 fictitious CVs, we show that college non-completion is not a scar per se, but rather depends on the educational attainment of the competitors who constitute the labor queue, and on the degree of occupational closure which varies on a granular level between firms that hire for the same occupations.

Substrate-Triggered μ-Peroxodiiron(III) Intermediate in the 4-Chloro-l-Lysine-Fragmenting Heme-Oxygenase-like Diiron Oxidase (HDO) BesC: Substrate Dissociation from, and C4 Targeting by, the Intermediate.

The enzyme BesC from the -thynyl-l-erine biosynthetic pathway in fragments 4-chloro-l-lysine (produced from l-Lysine by BesD) to ammonia, formaldehyde, and 4-chloro-l-allylglycine and can analogously fragment l-Lys itself. BesC belongs to the emerging family of O-activating non-heme-diiron enzymes with the "heme-oxygenase-like" protein fold (HDOs). Here, we show that the binding of l-Lys or an analogue triggers capture of O by the protein's diiron(II) cofactor to form a blue μ-peroxodiiron(III) intermediate analogous to those previously characterized in two other HDOs, the olefin-installing fatty acid decarboxylase, UndA, and the guanidino--oxygenase domain of SznF.

Reaction pathway engineering converts a radical hydroxylase into a halogenase.

Fe/α-ketoglutarate (Fe/αKG)-dependent enzymes offer a promising biocatalytic platform for halogenation chemistry owing to their ability to functionalize unactivated C-H bonds. However, relatively few radical halogenases have been identified to date, limiting their synthetic utility. Here, we report a strategy to expand the palette of enzymatic halogenation by engineering a reaction pathway rather than substrate selectivity.

Absolute characterization of high numerical aperture microscope objectives utilizing a dipole scatterer.

Measuring the aberrations of optical systems is an essential step in the fabrication of high precision optical components. Such a characterization is usually based on comparing the device under investigation with a calibrated reference object. However, when working at the cutting-edge of technology, it is increasingly difficult to provide an even better or well-known reference device.

Microsphere kinematics from the polarization of tightly focused nonseparable light.

Recently, it was shown that vector beams can be utilized for fast kinematic sensing via measurements of their global polarization state [Optica2, 864 (2015)10.1364/OPTICA.2.

Cribellate thread production as model for spider's spinneret kinematics.

Spider silk attracts researchers from the most diverse fields, such as material science or medicine. However, still little is known about silk aside from its molecular structure and material strength. Spiders produce many different silks and even join several silk types to one functional unit.

[Evaluation of the new German measles protection act: An online survey].

Introduction: The current COVID-19 pandemic reveals the dangerousness of infectious diseases and the threats we face. Often however, effective vaccinations are carried out insufficiently. In March 2020, the German measles protection law was introduced to raise the level of population (herd) immunity to over 95 %.

Interactive editing of virtual chordae tendineae for the simulation of the mitral valve in a decision support system.

Purpose: Decision support systems for mitral valve disease are an important step toward personalized surgery planning. A simulation of the mitral valve apparatus is required for decision support. Building a model of the chordae tendineae is an essential component of a mitral valve simulation.

Towards fully integrated photonic displacement sensors.

The field of optical metrology with its high precision position, rotation and wavefront sensors represents the basis for lithography and high resolution microscopy. However, the on-chip integration-a task highly relevant for future nanotechnological devices-necessitates the reduction of the spatial footprint of sensing schemes by the deployment of novel concepts. A promising route towards this goal is predicated on the controllable directional emission of the fundamentally smallest emitters of light, i.

A clinical decision support system improves antibiotic therapy for upper urinary tract infection in a randomized single-blinded study.

Background: Due to increasing bacterial resistance rates choosing a correct empiric antibiotic therapy is getting more and more complex. Often medical doctors use information tools to make the right treatment choice.

Methods: One hundred sixty six participants (77 medical doctors and 89 medical students) were asked to provide a diagnosis and antibiotic therapy in a simple fictive paper case of upper urinary tract infection (UTI) in a randomized single-blinded study.

A family of radical halogenases for the engineering of amino-acid-based products.

The integration of synthetic and biological catalysis enables new approaches to the synthesis of small molecules by combining the high selectivity of enzymes with the reaction diversity offered by synthetic chemistry. While organohalogens are valued for their bioactivity and utility as synthetic building blocks, only a handful of enzymes that carry out the regioselective halogenation of unactivated [Formula: see text] bonds have previously been identified. In this context, we report the structural characterization of BesD, a recently discovered radical halogenase from the Fe/α-ketogluturate-dependent family that chlorinates the free amino acid lysine.

[Lack of information and provision of information at the workplace as potential reasons for inappropriate antibiotic therapy in Germany].

Background: The increased use of antibiotics leads to a rise in drug-resistant bacteria. It is critical to reduce inadequate prescribing of antibiotics in order to prevent a post-antibiotic era.

Aim Of The Study: To explore if healthcare providers in Germany have access to current treatment guidelines, information about antibiotics and local resistance data at their workplace and how they access the information in their daily routine.

Emission of circularly polarized light by a linear dipole.

Controlling the polarization state and the propagation direction of photons is a fundamental prerequisite for many nanophotonic devices and a precursor for future on-chip communication, where the emission properties of individual emitters are particularly relevant. Here, we report on the emission of partially circularly polarized photons by a linear dipole. The underlying effect is linked to the near-field part of the angular spectrum of the dipole, and it occurs in any type of linear dipole emitter, ranging from atoms and quantum dots to molecules and dipole-like antennas.

Experimental demonstration of linear and spinning Janus dipoles for polarisation- and wavelength-selective near-field coupling.

The electromagnetic field scattered by nano-objects contains a broad range of wavevectors and can be efficiently coupled to waveguided modes. The dominant contribution to scattering from subwavelength dielectric and plasmonic nanoparticles is determined by electric and magnetic dipolar responses. Here, we experimentally demonstrate spectral and phase selective excitation of Janus dipoles, sources with electric and magnetic dipoles oscillating out of phase, in order to control near-field interference and directional coupling to waveguides.