REPSA Directed Assessment of Native Cleavage Resistance of DNA to Type IIS Restriction Endonucleases and Modification of REPSA for High Temperature Application

We have modified the combinatorial selection method Restriction Endonuclease Protection and Selection Assay (REPSA) to work in high temperature conditions for the discovery of new DNA-binding proteins in thermophiles (HT-REPSA). We utilized Thermus thermophilus (HB-8/ATCC 27634/DSM 579) as a test organism due to its amenable nature in a laboratory setting and current status as a model thermophilic organism. We used a TetR Family (TFR) transcription factor SbtR as the model protein for optimization of HT-REPSA protocols, as data had previously been obtained regarding SbtR physical characteristics and DNA-binding properties. REPSA was conducted until a cleavage resistant species arose after 7 rounds. Massively parallel sequencing of the selected DNAs and bioinformatics analysis yielded a consensus binding sequence of 5\u27-GA(t/c)TGACC(c/a)GC(t/g)GGTCA(g/a)TC, a 20base pair palindromic site comparable to that described in the literature. Taken together, our data provide a proof-of-concept that HT-REPSA can be successfully used to identify the preferred DNA-binding sequences of transcription factors from extreme thermophilic organisms

Demonstration of a Thermally Coupled Row-Column SNSPD Imaging Array

While single-pixel superconducting nanowire single photon detectors (SNSPDs) have demonstrated remarkable efficiency and timing performance from the UV to near-IR, scaling these devices to large imaging arrays remains challenging. Here, we propose a new SNSPD multiplexing system using thermal coupling and detection correlations between two photosensitive layers of an array. Using this architecture with the channels of one layer oriented in rows and the second layer in columns, we demonstrate imaging capability in 16-pixel arrays with accurate spot tracking at the few-photon level. We also explore the performance trade-offs of orienting the top layer nanowires parallel and perpendicular to the bottom layer. The thermally coupled row-column scheme is readily able to scale to the kilopixel size with existing readout systems and, when combined with other multiplexing architectures, has the potential to enable megapixel scale SNSPD imaging arrays

Identification of Preferred DNA-Binding Sites for the Thermus thermophilus Transcriptional Regulator SbtR by the Combinatorial Approach REPSA

One of the first steps towards elucidating the biological function of a putative transcriptional regulator is to ascertain its preferred DNA-binding sequences. This may be rapidly and effectively achieved through the application of a combinatorial approach, one involving very large numbers of randomized oligonucleotides and reiterative selection and amplification steps to enrich for high-affinity nucleic acid-binding sequences. Previously, we had developed the novel combinatorial approach Restriction Endonuclease Protection, Selection and Amplification (REPSA), which relies not on the physical separation of ligand-nucleic acid complexes but instead selects on the basis of ligand-dependent inhibition of enzymatic template inactivation, specifically cleavage by type IIS restriction endonucleases. Thus, no prior knowledge of the ligand is required for REPSA, making it more amenable for discovery purposes. Here we describe using REPSA, massively parallel sequencing, and bioinformatics to identify the preferred DNA-binding sites for the transcriptional regulator SbtR, encoded by the TTHA0167 gene from the model extreme thermophile Thermus thermophilus HB8. From the resulting position weight matrix, we can identify multiple operons potentially regulated by SbtR and postulate a biological role for this protein in regulating extracellular transport processes. Our study provides a proof-of-concept for the application of REPSA for the identification of preferred DNA-binding sites for orphan transcriptional regulators and a first step towards determining their possible biological roles

Optically addressing single rare-earth ions in a nanophotonic cavity

We demonstrate optical probing of spectrally resolved single Nd rare-earth ions in yttrium orthovanadate. The ions are coupled to a photonic crystal resonator and show strong enhancement of the optical emission rate via the Purcell effect, resulting in near radiatively limited single photon emission. The measured high coupling cooperativity between a single photon and the ion allows for the observation of coherent optical Rabi oscillations. This could enable optically controlled spin qubits, quantum logic gates, and spin-photon interfaces for future quantum networks

Influence of boundary conditions on the out-of-plane response of brick masonry walls in buildings with RC slabs

In modern unreinforced masonry buildings with stiff RC slabs, walls of the top floor are most susceptible to out-of-plane failure. The out-of-plane response depends not only on the acceleration demand and wall geom-etry but also on the static and kinematic boundary conditions of the walls. This paper discusses the influence of these boundary conditions on the out-of-plane response through evaluation of shake table test results and numerical modelling. As a novum, it shows that the in-plane response of flanking elements, which are or-thogonal to the wall whose out-of-plane response is studied, has a significant influence on the vertical re-straint at the top of the walls. The most critical configuration exists if the flanking elements are unreinforced masonry walls that rock. In this case, the floor slabs can uplift, and the out-of-plane load-bearing walls loose the vertical restraint at the top. Numerical modelling confirms this experimentally observed behaviour and shows that slab uplift and the difference in base and top excitation have a strong influence on the out-of-plane response of the walls analysed

Capacity Design of Coupled RC Walls

Capacity design is an important tool that enables controlled ductile response of structures when subjected to earthquakes. The need for specific capacity design requirements for walls coupled by deep coupling beams, however, is often overlooked and it is assumed in some instances that the same equations used for cantilever walls will give satisfactory results when applied to coupled walls. Furthermore, existing equations for capacity design incorporated into codes and design guides were developed a number of years ago and no longer represent the state-of-the-art. This research investigates the performance of existing capacity design equations for coupled walls and then proposes a new simplified capacity design method based on state-of-the-art-knowledge. The proposed method is then verified through a case study in which a set of 15 coupled walls were analysed using non-linear time-history analyses. Following this, an investigation is made into how the maximum shear force in an individual wall relates to the maximum shear force in the coupled wall system

Comparison of force-based and displacement-based design approaches for RC coupled walls in New Zealand

Reinforced concrete coupled walls are a common lateral load resisting system used in multi-storey buildings. The effect of the coupling beams can improve seismic performance, but at the same time adds complexity to the design procedure. A case study coupled wall building is designed using Force-Based Design (FBD) and Direct Displacement-Based Design (DDBD) and in the case of the latter a step by step design example is provided. Distributed plasticity fibre-section beam element numerical models of the coupled walls are developed in which coupling beams are represented by diagonal truss elements and experimental results are used to confirm that this approach can provide a good representation of hysteretic behaviour. The accuracy of the two different design methods is then assessed by comparing the design predictions to the results of non-linear time-history analyses. It is shown that the DDBD approach gives an accurate prediction of inter-storey drift response. The FBD approach, in accordance with NZS1170.5 and NZS3101, is shown to include an impractical procedure for the assignment of coupling beam strengths and code equations for the calculation of coupling beam characteristics appear to include errors. Finally, the work highlights differences between the P-delta considerations that are made in FBD and DDBD, and shows that the code results are very sensitive to the way in which P-delta effects are accounted for