MemBright a Family of Fluorescent Membrane Probes for Advanced Cellular Imaging and Neuroscience, bioRxiv, 2018-07-30
AbstractThe proper staining of the plasma membrane (PM) is critical in bioimaging as it delimits the cell. Herein, we developed MemBright a family of six cyanine-based fluorescent turn-on PM probes that emit from orange to near-infrared when reaching the PM, and enable homogeneous and selective PM staining with excellent contrast in mono and two-photon microscopy. These probes are compatible with long-term live cell imaging and immunostaining. Moreover, MemBright label neurons in a brighter manner than surrounding cells allowing identification of neurons in acute brain tissue section and neuromuscular-junctions without any use of transfection or transgenic animals. At last, MemBright were used in super-resolution imaging to unravel the dendritic spines’ neck. 3D multicolor dSTORM in combination with immunostaining revealed en-passant synapse displaying endogenous glutamate receptors clustered at the axonal-dendritic contact site. MemBright probes thus constitute a universal toolkit for cell biology and neuroscience biomembrane imaging with a variety of microscopy techniques.
biorxiv neuroscience 0-100-users 2018Clonal evolution and genome stability in a 2,500-year-old fungal individual, bioRxiv, 2018-07-26
AbstractIn the late 1980s, a genetic individual of the fungus Armillaria gallica that extended over at least 37 hectares of forest floor and encompassed hundreds of tree root systems was discovered on the Upper Peninsula of Michigan. Based on observed growth rates, the individual was estimated to be at least 1500 years old with a mass of more than 105 kg. Nearly three decades on, we returned to the site of individual for new sampling. We report here that the same genetic individual of A. gallica is still alive on its original site, but we estimated that it is older and larger than originally estimated, at least 2,500 years and 4 × 105 kg, respectively. We also show that mutation has occurred within the somatic cells of the individual, reflecting its historical pattern of growth from a single point. The overall rate of mutation, however, was extremely low. The large individual of A. gallica has been remarkably resistant to genomic change as it has persisted in place.
biorxiv evolutionary-biology 0-100-users 2018Moving beyond P values Everyday data analysis with estimation plots, bioRxiv, 2018-07-26
Over the past 75 years, a number of statisticians have advised that the data-analysis method known as null-hypothesis significance testing (NHST) should be deprecated (Berkson, 1942; Halsey et al., 2015; Wasserstein et al., 2019). The limitations of NHST have been extensively discussed, with a broad consensus that current statistical practice in the biological sciences needs reform. However, there is less agreement on reform’s specific nature, with vigorous debate surrounding what would constitute a suitable alternative (Altman et al., 2000; Benjamin et al., 2017; Cumming and Calin-Jageman, 2016). An emerging view is that a more complete analytic technique would use statistical graphics to estimate effect sizes and evaluate their uncertainty (Cohen, 1994; Cumming and Calin-Jageman, 2016). As these estimation methods require only minimal statistical retraining, they have great potential to shift the current data-analysis culture away from dichotomous thinking towards quantitative reasoning (Claridge-Chang and Assam, 2016). The evolution of statistics has been inextricably linked to the development of quantitative displays that support complex visual reasoning (Tufte, 2001). We consider that the graphic we describe here as estimation plot is the most intuitive way to display the complete statistical information about experimental data sets. However, a major obstacle to adopting estimation plots is accessibility to suitable software. To lower this hurdle, we have developed free software that makes high-quality estimation plotting available to all. Here, we explain the rationale for estimation plots by contrasting them with conventional charts used to display data with NHST results, and describe how the use of these graphs affords five major analytical advantages.
biorxiv bioinformatics 500+-users 2018Psychoactive plant- and mushroom-associated alkaloids from two behavior modifying cicada pathogens, bioRxiv, 2018-07-25
AbstractEntomopathogenic fungi routinely kill their hosts before releasing infectious spores, but select species keep insects alive while sporulating, which enhances dispersal. Transcriptomics and metabolomics studies of entomopathogens with post-mortem dissemination from their parasitized hosts have unraveled infection processes and host responses, yet mechanisms underlying active spore transmission by Entomophthoralean fungi in living insects remain elusive. Here we report the discovery, through metabolomics, of the plant-associated amphetamine, cathinone, in four Massospora cicadina-infected periodical cicada populations, and the mushroom-associated tryptamine, psilocybin, in annual cicadas infected with Massospora platypediae or Massospora levispora, which appear to represent a single fungal species. The absence of some fungal enzymes necessary for cathinone and psilocybin biosynthesis along with the inability to detect intermediate metabolites or gene orthologs are consistent with possibly novel biosynthesis pathways in Massospora. The neurogenic activities of these compounds suggest the extended phenotype of Massospora that modifies cicada behavior to maximize dissemination is chemically-induced.
biorxiv ecology 200-500-users 2018Cortical Column and Whole Brain Imaging of Neural Circuits with Molecular Contrast and Nanoscale Resolution, bioRxiv, 2018-07-23
AbstractOptical and electron microscopy have made tremendous inroads in understanding the complexity of the brain, but the former offers insufficient resolution to reveal subcellular details and the latter lacks the throughput and molecular contrast to visualize specific molecular constituents over mm-scale or larger dimensions. We combined expansion microscopy and lattice light sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain, including synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly neuropil domain. The technology should enable statistically rich, large scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.One Sentence SummaryCombined expansion and lattice light sheet microscopy enables high speed, nanoscale molecular imaging of neural circuits over large volumes.
biorxiv neuroscience 200-500-users 2018Panoptic vDISCO imaging reveals neuronal connectivity, remote trauma effects and meningeal vessels in intact transparent mice, bioRxiv, 2018-07-23
Analysis of entire transparent rodent bodies could provide holistic information on biological systems in health and disease. However, it has been challenging to reliably image and quantify signal from endogenously expressed fluorescent proteins in large cleared mouse bodies due to the low signal contrast. Here, we devised a pressure driven, nanobody based whole-body immunolabeling technology to enhance the signal of fluorescent proteins by up to two orders of magnitude. This allowed us to image subcellular details in transparent mouse bodies through bones and highly autofluorescent tissues, and perform quantifications. We visualized for the first-time whole-body neuronal connectivity of an entire adult mouse and discovered that brain trauma induces degeneration of peripheral axons. We also imaged meningeal lymphatic vessels and immune cells through the intact skull and vertebra in naive animals and trauma models. Thus, our new approach can provide an unbiased holistic view of biological events affecting the nervous system and the rest of the body.
biorxiv neuroscience 500+-users 2018