An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR mediated homologous recombination

Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination (Lee et al., 2018). These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps) (Kanca et al., 2019a). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 50% of Drosophila genes lack suitable coding introns for integration of artificial exons. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.

An efficient semi-analytical static and free vibration analysis of laminated and sandwich beams based on linear elasticity theory

Based on the two-dimensional (2D) elastic theory without enforcing any beam assumption, an efficient semi-analytical scaled boundary finite element method (SBFEM) is proposed to solve the bending and free vibration responses of composite laminated and sandwich beams under the mechanical load. The scaled center is placed at infinity, which produces the accurate result by discretizing only the longitudinal direction of the beam structure treated as a one-dimensional (1D) discretization problem. A new kind of 1D high-order spectral element shape functions with the advantages of high accuracy and superior convergence is introduced in SBFEM coordinate system to approximate the geometric model and corresponding variables. The principle of weighted residual in conjunction with the Green’s theorem are applied to obtain the SBFEM governing equation of each layer with respect to radial displacement fields. The solution of equation is indicated analytically by a matrix exponential function, which can be accurately solved by using the precise integration technique (PIT). Finally, an effective and simple stiffness matrix is obtained. By comparing two examples with the solutions based on the finite element method (FEM), the results show that the proposed method has good accuracy and rapid convergence with only a few meshes. The numerical examples are given to investigate the parametric effects of the stacking sequence, thickness ratio, boundary condition, and load form on the variation of the displacement, stress and natural frequency. The results validate that the present technique is also applicable to the complex beam structure with softcore layer inside.

Precise-Integration Time-Domain Formulation for Optical Periodic Media

A numerical formulation based on the precise-integration time-domain (PITD) method for simulating periodic media is extended for overcoming the Courant-Friedrich-Levy (CFL) limit on the time-step size in a finite-difference time-domain (FDTD) simulation. In this new method, the periodic boundary conditions are implemented, permitting the simulation of a wide range of periodic optical media, i.e., gratings, or thin-film filters. Furthermore, the complete tensorial derivation for the permittivity also allows simulating anisotropic periodic media. Numerical results demonstrate that PITD is reliable and even considering anisotropic media can be competitive compared to traditional FDTD solutions. Furthermore, the maximum allowable time-step size has been demonstrated to be much larger than that of the CFL limit of the FDTD method, being a valuable tool in cases in which the steady-state requires a large number of time-steps.

Autopolyploidization affects transcript patterns and gene targeting frequencies in Physcomitrella

Key message In Physcomitrella, whole-genome duplications affected the expression of about 3.7% of the protein-encoding genes, some of them relevant for DNA repair, resulting in a massively reduced gene-targeting frequency. Abstract Qualitative changes in gene expression after an autopolyploidization event, a pure duplication of the whole genome (WGD), might be relevant for a different regulation of molecular mechanisms between angiosperms growing in a life cycle with a dominant diploid sporophytic stage and the haploid-dominant mosses. Whereas angiosperms repair DNA double-strand breaks (DSB) preferentially via non-homologous end joining (NHEJ), in the moss Physcomitrella homologous recombination (HR) is the main DNA–DSB repair pathway. HR facilitates the precise integration of foreign DNA into the genome via gene targeting (GT). Here, we studied the influence of ploidy on gene expression patterns and GT efficiency in Physcomitrella using haploid plants and autodiploid plants, generated via an artificial WGD. Single cells (protoplasts) were transfected with a GT construct and material from different time-points after transfection was analysed by microarrays and SuperSAGE sequencing. In the SuperSAGE data, we detected 3.7% of the Physcomitrella genes as differentially expressed in response to the WGD event. Among the differentially expressed genes involved in DNA–DSB repair was an upregulated gene encoding the X-ray repair cross-complementing protein 4 (XRCC4), a key player in NHEJ. Analysing the GT efficiency, we observed that autodiploid plants were significantly GT suppressed (p < 0.001) attaining only one third of the expected GT rates. Hence, an alteration of global transcript patterns, including genes related to DNA repair, in autodiploid Physcomitrella plants correlated with a drastic suppression of HR.

Self-assembled multifunctional neural probes for precise integration of optogenetics and electrophysiology

Optogenetics combined with electrical recording has emerged as a powerful tool for investigating causal relationships between neural circuit activity and function. However, the size of optogenetically manipulated tissue is typically 1-2 orders of magnitude larger than that can be electrically recorded, rendering difficulty for assigning functional roles of recorded neurons. Here we report a viral vector-delivery optrode (VVD-optrode) system for precise integration of optogenetics and electrophysiology in the brain. Our system consists of flexible microelectrode filaments and fiber optics that are simultaneously self-assembled in a nanoliter-scale, viral vector-delivery polymer carrier. The highly localized delivery and neuronal expression of opsin genes at microelectrode-tissue interfaces ensure high spatial congruence between optogenetically manipulated and electrically recorded neuronal populations. We demonstrate that this multifunctional system is capable of optogenetic manipulation and electrical recording of spatially defined neuronal populations for three months, allowing precise and long-term studies of neural circuit functions.

A Block Matrix Based Precise Integration Algorithm for Solving Non-Homogeneous Dynamic Response

This paper puts forward a computationally-efficient parallel precise integration algorithm for solving vibration response subjected to time-variable excitation and nonlinearity, especially for non-homogenous dynamic response solution with large-scale degree of freedom. In detail, both of the nonlinear parts and time-varying inputs of the dynamic system are separated from the original dynamic equations and then simulated by employing a piecewise interpolation function within a computing time-step. A novel closed-form iteration formula is presented in conjunction with the block matrix strategy and modified increment-dimensional precise integration technique. Interestingly, the presented approach is essentially a high-accuracy and parallel algorithm, which exhibits a high prediction accuracy without the limitation of matrix inversion, higher-order derivative, periodicity requirement nor cycle oscillation and instability of high-order interpolation. At last, the feasibility and advantage of the proposed method is verified with two numerical examples.

Raising climate resilient crops: Journey from the conventional breeding to new breeding approaches

Background: In order to meet the demands of ever-increasing human population, it has become necessary to raise climate-resilient crops. Plant breeding which involves crossing and selecting superior gene pools has contributed tremendously towards achieving this goal during the past few decades. The relatively newer methods of crop improvement based on genetic engineering are relatively simple and targets can be achieved in an expeditious manner. More recently emerged genome editing technique using CRISPR has raised strong hopes among plant scientists for precise integration of valuable traits and removal of undesirable ones. Conclusion: Genome editing using Site Specific Nucleases (SSNs) is a good alternative to the plant breeding and genetic engineering approaches as it can modify the genomes specifically and precisely at the target site in the host genome. Another added advantage of the genome editing approach is the simpler biosafety regulations that have been adopted by many countries for commercialization of the products thus generated. This review provides a critical assessment of the available methods for improving the stress tolerance in crop plants. Special emphasis has been given on genome editing approach in light of the diversity of tools which are being discovered on everyday basis and the practical applications of the same. This information will serve a beginner’s guide to initiate the crop improvement programs as well as giving technical insight to the expert to plan the research strategically to tackle even multigenic traits in crop plants.

Effect of delayed resonator on the vibration reduction performance of vehicle active seat suspension

The combination of dynamic vibration absorber and partial state feedback with time-delay is called delayed resonator. In order to suppress the seat vibration caused by uneven road surface and improve ride comfort, the delayed resonator is applied to the seat suspension to realize active control of the seat suspension system. The dynamic model of the half-vehicle suspension system is established, and the time-delay differential equation of the system under external excitation is solved by the precise integration method. The root mean square of the time-domain vibration response of seat displacement, seat acceleration and vehicle acceleration are selected as the objective function. Then, the optimal time-delay control parameters are obtained by particle swarm optimization algorithm. The frequency sweeping method is used to obtain the critical time-delay value and time-delay stable interval of the system. Finally, an active seat suspension model with delayed resonator is established for numerical simulation. The results show that the delayed resonator can greatly suppress the seat vibration response regardless of the road simple harmonic excitation or random excitation. Compared with dynamic vibration absorber, it has a better vibration absorption effect and a wider vibration reduction frequency band.

A non-iterative inverse method for the identification of time and space-dependent heat sources of multi-dimensional FGMs by boundary temperature data

Background: Detection of heat sources is frequently encountered in many fields of science and engineering and plays a significant role in monitoring and control of many engineering thermal systems. Objective: The objective of this paper is to estimate the space and time-dependent heat sources in multi-dimensional functionally graded materials using boundary temperature data. Methods: First, the dimensionless temperature at the measurement points on the boundary is obtained by a direct process. Then, the objective function is obtained by a series of matrix operations, and the relationship between the temperature at the measurement points and the unknown parameters is established. After that, the strength of the heat sources can be inversed directly by the least-square error method, then the location and number of the heat sources can be obtained directly from the strength distribution of heat sources. The coefficient expansion method and truncated singular value decomposition method are applied to reduce the ill-posed degree of the inverse process. Results: Through the analysis of typical 2-dimensional and 3-dimensional examples, the influence of various factors such as the type of basis functions, the circular supported radius and the measurement noise on the inversion results is discussed, which shows that this method can identify the strength, location and number of heat sources of FGMs well. Conclusion: A non-iterative inverse method based on precise integration finite element method and least-square error method is established to estimate the strength, location and number of the unknown heat sources of functionally graded materials using boundary temperature data.

Earthquake Response Spectra Analysis of Bridges considering Pounding at Bilateral Beam Ends Based on an Improved Precise Pounding Algorithm

Asynchronous vibration was generated between the main bridge and approach spans or abutments due to differences in stiffness and mass during an earthquake, thus further leading to pounding at the bilateral beam ends. By taking a T-shaped rigid frame bridge as an example, the bilateral pounding model was abstracted, and the earthquake response spectra considering pounding at the bilateral beam ends were studied, including the maximum displacement spectrum, the acceleration dynamic coefficient spectrum, the pounding force response spectrum, and the response spectrum for the number of pounding events. An improved precise pounding algorithm was proposed to solve the dynamic equation of the bilateral pounding model. This algorithm is based on the precise integration method for solving the second-order dynamic differential equation and reduces the order thereof by introducing a new velocity vector and uses the series method to find the nonhomogeneous term. The system matrix is simpler, and the inversion of the system matrix can be avoided. On this basis, a multipoint earthquake-induced pounding response spectrum program was developed. A total of 18 seismic waves from Class II sites were selected, and the response spectra of 18 waves were analyzed using this new program. Furthermore, the effects of structural stiffness, mass, stiffness of contact element, pounding recovery coefficient, and peak ground acceleration (PGA) on the earthquake response spectrum were studied. Through the analysis of earthquake response spectra and a parametric study, the phenomenon of earthquake-induced pounding of bridges was clarified to the benefit of the analysis and engineering control of earthquake-induced pounding of bridges.