Positive and Negative Regulation of Gli Activity by Kif7 in the Zebrafish Embryo

Loss of function mutations of Kif7, the vertebrate orthologue of the Drosophila Hh pathway component Costal2, cause defects in the limbs and neural tubes of mice, attributable to ectopic expression of Hh target genes. While this implies a functional conservation of Cos2 and Kif7 between flies and vertebrates, the association of Kif7 with the primary cilium, an organelle absent from most Drosophila cells, suggests their mechanisms of action may have diverged. Here, using mutant alleles induced by Zinc Finger Nuclease-mediated targeted mutagenesis, we show that in zebrafish, Kif7 acts principally to suppress the activity of the Gli1 transcription factor. Notably, we find that endogenous Kif7 protein accumulates not only in the primary cilium, as previously observed in mammalian cells, but also in cytoplasmic puncta that disperse in response to Hh pathway activation. Moreover, we show that Drosophila Costal2 can substitute for Kif7, suggesting a conserved mode of action of the two proteins. We show that Kif7 interacts with both Gli1 and Gli2a and suggest that it functions to sequester Gli proteins in the cytoplasm, in a manner analogous to the regulation of Ci by Cos2 in Drosophila. We also show that zebrafish Kif7 potentiates Gli2a activity by promoting its dissociation from the Suppressor of Fused (Sufu) protein and present evidence that it mediates a Smo dependent modification of the full length form of Gli2a. Surprisingly, the function of Kif7 in the zebrafish embryo appears restricted principally to mesodermal derivatives, its inactivation having little effect on neural tube patterning, even when Sufu protein levels are depleted. Remarkably, zebrafish lacking all Kif7 function are viable, in contrast to the peri-natal lethality of mouse kif7 mutants but similar to some Acrocallosal or Joubert syndrome patients who are homozygous for loss of function KIF7 alleles

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Effect of Mn on hardenability of 25CrMo axle steel by an improved end-quench test

With the sixth large-scale railway speed-up, the quality of the axles is essential to the safety of the locomotive. According to the high-speed axle technical standard for the control of alloy elements in axle steel, optimization experiments of 25CrMo steel composition were performed by vacuum inductive melting. In order to study the hardenability of high-speed rail axles, an improved end-quench test was put forward. The advantage is that it enables the heat to transfer along the axial direction, thus avoiding edge effects. The hardenability of 25CrMo axle steels with Mn content of 0.60wt.% and 0.80wt.% was investigated mainly by means of optical microscopy and hardness tests. The experimental results indicate that the Mn has a pronounced effect on the hardenability of the steel. With an increase in Mn content from 0.60wt.% and 0.80wt.%, the hardenability of 25CrMo axle steel increases and the hard microstructure is maintained at an increasing distance from the quenched end. From the surface of the water quenched end to the center of the sample, the microstructure is martensite, martensite with bainite, and bainite

Manufacturing of AcMNPV baculovirus vectors to enable gene therapy trials

Over the past two decades, baculoviruses have become workhorse research tools for transient transgene expression. Although they have not yet been used directly as a gene therapy vector in the clinical setting, numerous preclinical studies have suggested the highly promising potential of baculovirus as a delivery vector for a variety of therapeutic applications including vaccination, tissue engineering, and cancer treatment. As such, there is growing interest in using baculoviruses as human gene therapy vectors, which has led to advances in baculovirus bioprocessing methods. This review provides an overview of the current approaches for scaled-up amplification, concentration, purification, and formulation of AcMNPV baculoviruses, and highlights the key regulatory requirements that must be met before gene therapy clinical trials can be initiated

A NOVEL RHODAMINE-BASED FLUORESCENCE CHEMOSENSOR CONTAINING POLYETHER FOR MERCURY (II) IONS IN AQUEOUS SOLUTION

A novel rhodamine-based Hg2+ chemosensor P2 containing polyether was readily synthesized and investigated, which displayed high selectivity and sensitivity for Hg2+. Because of good water-solubility of polyther, the rhodamine-based chemosensor containing polyether can be used in aqueous solution. The sensor responded rapidly to Hg2+ in pure water solutions with a 1:1 stoichiometry. Meanwhile, it indicated excellent adaptability and also the responsiveness

Pt Deposites on TiO2 for Photocatalytic H2 Evolution: Pt Is Not Only the Cocatalyst, but Also the Defect Repair Agent

Pt, as a common cocatalyst, has been widely used in photocatalytic H2 evolution. However, the specific role of Pt in photocatalytic H2 evolution has not been thoroughly studied. In this paper, by employing three Pt sources with different charges (positive, negative and neutral), we systematically studied the charge effect of Pt sources on photocatalytic H2 evolution via TiO2 catalyst. According to the results of Raman, X-ray photoelectron spectroscopy (XPS), recycle experiments and photocurrent characterizations, it was found that TiO2 would produce electropositive defects during photocatalytic H2 evolution, inevitably leading to the decline of H2 production activity. Thanks to the electrostatic interaction, the electronegative Pt source not only promoted charge separation, but preferential deposited on electropositive defects, which acted as the defect repair agent, and thus resulted in the increased photocatalytic stability. This work may provide a new perspective for enhancing photocatalytic stability of hydrogen production

Mapping RNA–chromatin interactions by sequencing with iMARGI

RNA-chromatin interactions represent an important aspect of the transcriptional regulation of genes and transposable elements. However, analyses of chromatin-associated RNAs (caRNAs) are often limited to one caRNA at a time. Here, we describe the iMARGI (in situ mapping of RNA-genome interactome) technique, which is used to discover caRNAs and reveal their respective genomic interaction loci. iMARGI starts with in situ crosslinking and genome fragmentation, followed by converting each proximal RNA-DNA pair into an RNA-linker-DNA chimeric sequence. These chimeric sequences are subsequently converted into a sequencing library suitable for paired-end sequencing. A standardized bioinformatic software package, iMARGI-Docker, is provided to decode the paired-end sequencing data into caRNA-DNA interactions. Compared to its predecessor MARGI (mapping RNA-genome interactions), the number of input cells for iMARGI is 3-5 million (a 100-fold reduction), experimental time is reduced, and clear checkpoints have been established. It takes a few hours a day and a total of 8 d to complete the construction of an iMARGI sequencing library and 1 d to carry out data processing with iMARGI-Docker

Visual-Based Multi-Section Welding Path Generation Algorithm

As an important form of additive manufacturing, welding is widely used in steel components welding work of construction, shipbuilding and other fields. In this study, an intelligent welding path generation algorithm based on multi-section interpolation is proposed in order to deal with non-standard multi-pass welding grooves which are difficult to be handled by automatic welding equipment in the construction site. Firstly, the non-standard grooves are classified and the reasons for their occurrence are discussed. Secondly, an automatic welding additive manufacturing system framework is discussed and an appropriate detection method is selected. Then, combining with the welding standard of non-standard grooves and the characteristics of the welding process, a multi-section interpolation-based welding path generation algorithm is proposed. Finally, a visual experiment platform was built to detection the typical non-standard groove and the welding experiment is implemented to verify the feasibility of the algorithm. According to the path generated by the algorithm, the welded steel components test plate meets the actual engineering standard after quality inspection. The experimental results and simulation results conclude the algorithm can be used to generate the welding path of the non-standard groove