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The community-driven initiative Quality Assessment and Reproducibility for Instruments & Images in Light Microscopy (QUAREP-LiMi) wants to improve reproducibility for light microscopy image data through quality control (QC) management of instruments and images. It aims for a common set of QC guidelines for hardware calibration and image acquisition, management and analysis.

A deeper epigenomic understanding of spatial organization of cells in human tissues is an important challenge. Here we report the first combined positional analysis of transcriptomes and methylomes across three micro-dissected zones (pericentral, intermediate and periportal) of human liver. We identify pronounced anti-correlated transcriptional and methylation gradients including a core of 271 genes controlling zonated metabolic and morphogen networks and observe a prominent porto-central gradient of DNA methylation at binding sites of 46 transcription factors. The gradient includes an epigenetic and transcriptional Wnt signature supporting the concept of a pericentral hepatocyte regeneration pathway under steady-state conditions. While donors with non-alcoholic fatty liver disease show consistent gene expression differences corresponding to the severity of the disease across all zones, the relative zonated gene expression and DNA methylation patterns remain unchanged. Overall our data provide a wealth of new positional insights into zonal networks controlled by epigenetic and transcriptional gradients in human liver. Spatial mapping of genomic programs in tissue cells is an important step in the understanding of organ function and disease. Here, the authors provide a spatially resolved epigenomic and transcriptomic map of human liver and show porto-central gradients in metabolic and morphogen networks and transcription factor binding sites as a basis to better understand liver regeneration and function.

Previous studies have shown that human topoisomerase I interacts directly with the tumor-suppressor protein p53. In the past few years it has repeatedly been suggested that topoisomerase I and p53 may play a joint role in the response to genotoxic stress. This led to the suggestion that p53 and human topoisomerase I may cooperate in the process of DNA repair and/or apoptosis. Recently we have demonstrated that a human topoisomerase I cleavage complex can be recognized by an additional topoisomerase I molecule and thereby form a so-called double cleavage complex. The double cleavage complex creates an about 13 nucleotides long single-stranded gap that may provide an entry site for recombinational repair events. Here we demonstrate that p53 stimulates both the DNA relaxation activity as well as the formation of the human topoisomerase I double cleavage complex by at least a factor of six. Stimulation of topoisomerase I activity by p53 is mediated via the central part of topoisomerase I. We also show that human, bovine, and murine p53 stimulate human topoisomerase I relaxation activity equally well. From these results it is conceivable that p53's stimulatory activity on topoisomerase I may play a role in DNA recombination and repair as well as in apoptosis.

Unlike humans, teleosts like zebrafish exhibit robust retinal regeneration after injury from endogenous stem cells. However, understanding the functional recovery of the regenerated retina remains a challenge. In particular, it is unclear if regenerating cone-photoreceptors can regain physiological function and integrate correctly into post-synaptic circuits. To bridge this gap, we employed two-photon calcium imaging of living retina, focusing on photoreceptor responses before and after an intense light-induced lesion in the adult zebrafish retina. To assess the functional recovery of cones and their downstream circuits, we exploited the colour opponency in adult zebrafish cones. We find that UV cones exhibit an intrinsic Off-response to short-wavelength blue light, but an On-response to longer-wavelength green light, which depends on feedback signals from outer retinal circuits. Accordingly, we examined the presence and quality of Off- versus On-responses, to assess the functional recovery of cones and their correct integration into outer retinal circuits. We find that regenerated UV cones regain both Off-responses to short-wavelength and On-responses to long-wavelength within 3 months after light lesion. Hence, physiological circuit functionality is restored in regenerated cone photoreceptors, suggesting that inducing endogenous regeneration is a promising strategy for human retinal repair.Competing Interest StatementThe authors have declared no competing interest.

Fluorescence lifetime imaging microscopy (FLIM) translates the duration of excited states of fluorophores into lifetime information as additional source of contrast in images of biological samples. This offers the possibility to separate fluorophores particularly beneficial in case of similar excitation spectra. Here, we demonstrate the distinction of fluorescent molecules based on FLIM phasor analysis, called lifetime unmixing, in live-cell imaging using open-source software for analysis. We showcase two applications using Caenorhabditis elegans as a model system. First, we unmixed the highly spectrally overlapping fluorophores mCherry and mKate2 to distinctively track tagged proteins in six-dimensional datasets to investigate cell division in the developing early embryo. Second, we unmixed fluorescence of tagged proteins of interest from masking natural autofluorescence in adult hermaphrodites. For FLIM data handling and workflow implementation, we developed the open-source plugin napari-FLIM-phasor-plotter to implement conversion, visualization, analysis and reuse of FLIM data of different formats. Our work thus advances technical applications and bioimage data management and analysis in FLIM microscopy for life science research.Competing Interest StatementThe authors have declared no competing interest.Footnotes* https://omero.med.tu-dresden.de/webclient/?show=project-1401/* http://omero.med.tu-dresden.de/figure/file/5748/* http://omero.med.tu-dresden.de/figure/file/5749/* http://omero.med.tu-dresden.de/figure/file/5819/* http://omero.med.tu-dresden.de/figure/file/5820/* https://github.com/zoccoler/napari-flim-phasor-plotter/* https://github.com/zoccoler/Lifetime-Unmixing/

Morphogen gradients impart positional information to cells in a homogenous tissue field. Fgf8a, a highly conserved growth factor, has been proposed to act as a morphogen during zebrafish gastrulation. However, technical limitations have so far prevented direct visualization of the endogenous Fgf8a gradient and confirmation of its morphogenic activity. Here, we monitored Fgf8a propagation in the developing neural plate using a CRISPR/Cas9-mediated EGFP knock-in at the endogenous fgf8a locus. By combining sensitive imaging platforms with single-molecule Fluorescence Correlation Spectroscopy (FCS), we demonstrate that Fgf8a, produced at the embryonic margin, propagates by free diffusion through the extracellular space and forms a graded distribution towards the animal pole. Overlaying the Fgf8a gradient curve with expression profiles of its downstream targets determines the precise input-output relationship of Fgf8a mediated patterning. Manipulation of the Fgf8a input alters the signaling outcome, thereby establishing Fgf8a as a bona fide morphogen during zebrafish gastrulation. Furthermore, using diffusion-hindered versions of Fgf8a, we demonstrate that extracellular diffusion of the protein from the source is critical for it to achieve its morphogenic potential.

An angular analysis of the decay $B^0 \\to \\phi K^*(892)^0$ is reported based on a $pp$ collision data sample, corresponding to an integrated luminosity of 1.0 fb$^{-1}$, collected at a centre-of-mass energy of $\\sqrt{s} = 7$ TeV with the LHCb detector. The P-wave amplitudes and phases are measured with a greater precision than by previous experiments, and confirm about equal amounts of longitudinal and transverse polarization. The S-wave $K^+ \\pi^-$ and $K^+K^-$ contributions are taken into account and found to be significant. A comparison of the $B^0 \\to \\phi K^*(892)^0$ and $\\bar{B}^0 \\to \\phi \\bar{K}^*(892)^0$ results shows no evidence for direct CP violation in the rate asymmetry, in the triple-product asymmetries or in the polarization amplitudes and phases.

Measurements of $b$-hadron lifetimes are reported using $pp$ collision data, corresponding to an integrated luminosity of 1.0fb$^{-1}$, collected by the LHCb detector at a centre-of-mass energy of $7$Tev. Using the exclusive decays $B^+\\to J/\\psi K^+$, $B^0\\to J/\\psi K^*(892)^0$, $B^0\\to J/\\psi K^0_{\\rm S}$, $\\Lambda_b^0\\to J/\\psi \\Lambda$ and $B^0_s\\to J/\\psi \\phi$ the average decay times in these modes are measured to be $\\tau_{B^+\\to J/\\psi K^+}$ = $1.637 \\pm$ 0.004 $\\pm$ 0.003 ps, $\\tau_{B^0\\to J/\\psi K^*(892)^0}$ = $1.524 \\pm$ 0.006 $\\pm$ 0.004 ps, $\\tau_{B^0\\to J/\\psi K^0_{\\rm S}}$ = $1.499 \\pm$ 0.013 $\\pm$ 0.005 ps, $\\tau_{\\Lambda_b^0\\to J/\\psi \\Lambda}$ = $1.415 \\pm$ 0.027 $\\pm$ 0.006 ps and $\\tau_{B^0_s\\to J/\\psi \\phi}$ = $1.480 \\pm$ 0.011 $\\pm$ 0.005 ps, where the first uncertainty is statistical and the second is systematic. These represent the most precise lifetime measurements in these decay modes. In addition, ratios of these lifetimes, and the ratio of the decay-width difference, $\\Delta\\Gamma_d$, to the average width, $\\Gamma_d$, in the $B^0$ system, $\\Delta \\Gamma_d/\\Gamma_d = -0.044 \\pm 0.025 \\pm 0.011$, are reported. All quantities are found to be consistent with Standard Model expectations.

A search for previously unobserved decays of beauty baryons to the final states $K^0_{\\rm\\scriptscriptstyle S} p \\pi^{-}$ and $K^0_{\\rm\\scriptscriptstyle S}p K^{-}$ is reported. The analysis is based on a data sample corresponding to an integrated luminosity of $1.0\\,$fb$^{-1}$ of $pp$ collisions. The $\\Lambda^0_{b} \\rightarrow \\overline{\\kern -0.2em K}^0_{\\rm\\scriptscriptstyle S}p \\pi^{-}$ decay is observed with a significance of $8.6\\,\\sigma$, with branching fraction \\begin{eqnarray*} {\\cal{B}}(\\Lambda^0_{b} \\rightarrow \\overline{\\kern -0.2em K}^0 p \\pi^{-}) & = & \\left( 1.26 \\pm 0.19 \\pm 0.09 \\pm 0.34 \\pm 0.05 \\right) \\times 10^{-5} \\,, \\end{eqnarray*} where the uncertainties are statistical, systematic, from the ratio of fragmentation fractions $f_{\\Lambda}/f_{d}$, and from the branching fraction of the $B^0 \\rightarrow K^0_{\\rm\\scriptscriptstyle S}\\pi^{+}\\pi^{-}$ normalisation channel, respectively. A first measurement is made of the $CP$ asymmetry, giving \\begin{eqnarray*} A_{C\\!P} (\\Lambda^0_{b} \\rightarrow \\overline{\\kern -0.2em K}^0 p \\pi^{-}) & = & 0.22 \\pm 0.13\\mathrm{\\,(stat)} \\pm 0.03\\mathrm{\\,(syst)} \\, . \\end{eqnarray*} No significant signals are seen for $\\Lambda^0_{b} \\rightarrow K^0_{\\rm\\scriptscriptstyle S}p K^{-}$ decays, $\\Xi^{0}_{b}$ decays to both the $K^0_{\\rm\\scriptscriptstyle S}p \\pi^{-}$ and $K^0_{\\rm\\scriptscriptstyle S}p K^{-}$ final states, and the $\\Lambda^0_{b} \\rightarrow D^{-}_{s} (\\rightarrow K^0_{\\rm\\scriptscriptstyle S} K^{-}) p$ decay, and upper limits on their branching fractions are reported.