Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues, bioRxiv, 2019-02-13
Recent advances in optical clearing and light-sheet microscopy have provided unprecedented access to structural and molecular information from intact tissues. However, current light-sheet microscopes have imposed constraints on the size, shape, number of specimens, and compatibility with various clearing protocols. Here we present a multi-immersion open-top light-sheet microscope that enables simple mounting of multiple specimens processed with a variety of protocols, which will facilitate wider adoption by preclinical researchers and clinical laboratories.
biorxiv bioengineering 0-100-users 2019Resting-state cross-frequency coupling networks in human electrophysiological recordings, bioRxiv, 2019-02-13
Neuronal oscillations underlie temporal coordination of neuronal processing and their synchronization enables neuronal communication across distributed brain areas to serve a variety of sensory, motor, and cognitive functions. The regulation and integration of neuronal processing between oscillating assemblies at distinct frequencies, and thereby the coordination of distinct computational functions, is thought to be achieved via cross-frequency coupling (CFC). Although many studies have observed CFC locally within a brain region during cognitive processing, the large-scale networks of CFC have remained largely uncharted. Critically, also the validity of prior CFC observations and the presence of true neuronal CFC has been recently questioned because non-sinusoidal or non-zero-mean waveforms that are commonplace in electrophysiological data cause filtering artefacts that lead to false positive CFC findings. We used a unique dataset of stereo-electroencephalography (SEEG) and source-reconstructed magnetoencephalography (MEG) data to chart whole-brain CFC networks from human resting-state brain dynamics. Using a novel graph theoretical method to distinguish true inter-areal CFC from potentially false positive CFC, we show that the resting state is characterized by two separable forms of true inter-areal CFC phase-amplitude coupling (PAC) and nm-cross-frequency phase synchrony (CFS). PAC and CFS large-scale networks coupled prefrontal, visual and sensorimotor cortices, but with opposing anatomical architectures. Crucially also directionalities between low- and high-frequency oscillations were opposite between CFS and PAC. We also found CFC to decay as a function of distance and to be stronger in the superficial than deep layers of the cortex. In conclusion, these results provide conclusive evidence for the presence of two forms of genuine inter-areal CFC and elucidate the large-scale organization of CFC resting-state networks.
biorxiv neuroscience 0-100-users 2019Bacterial Phage Tail-like Structure Kills Eukaryotic Cells by Injecting a Nuclease Effector, bioRxiv, 2019-02-12
Many bacteria interact with target organisms using syringe-like structures called Contractile Injection Systems (CIS). CIS structurally resemble headless bacteriophages and share evolutionarily related proteins such as the tail tube, sheath, and baseplate complex. Recent evidence shows that CIS are specialized to puncture membranes and often deliver effectors to target cells. In many cases, CIS mediate trans-kingdom interactions between bacteria and eukaryotes, however the effectors delivered to target cells and their mode of action are often unknown. In this work, we establish an in vitro model to study a CIS called Metamorphosis Associated Contractile structures (MACs) that target eukaryotic cells. We show that MACs kill two eukaryotic cell lines, Fall Armyworm Sf9 cells and J774A.1 murine macrophage cells through the action of a newly identified MAC effector, termed Pne1. To our knowledge, Pne1 is the first CIS effector exhibiting nuclease activity against eukaryotic cells. Our results define a new mechanism of CIS-mediated bacteria-eukaryote interaction and are a first step toward understanding structures with the potential to be developed as novel delivery systems for eukaryotic hosts.
biorxiv microbiology 0-100-users 2019Extensive loss of cell cycle and DNA repair genes in an ancient lineage of bipolar budding yeasts, bioRxiv, 2019-02-12
Cell cycle checkpoints and DNA repair processes protect organisms from potentially lethal mutational damage. Compared to other budding yeasts in the subphylum Saccharomycotina, we noticed that a lineage in the genus Hanseniaspora exhibited very high evolutionary rates, low GC content, small genome sizes, and lower gene numbers. To better understand Hanseniaspora evolution, we analyzed 25 genomes, including 11 newly sequenced, representing 18 21 known species in the genus. Our phylogenomic analyses identify two Hanseniaspora lineages, the fast-evolving lineage (FEL), which began diversifying ~87 million years ago (mya), and the slow-evolving lineage (SEL), which began diversifying ~54 mya. Remarkably, both lineages lost genes associated with the cell cycle and genome integrity, but these losses were greater in the FEL. For example, all species lost the cell cycle regulator WHI5, and the FEL lost components of the spindle checkpoint pathway (e.g., MAD1, MAD2) and DNA damage checkpoint pathway (e.g., MEC3, RAD9). Similarly, both lineages lost genes involved in DNA repair pathways, including the DNA glycosylase gene MAG1, which is part of the base excision repair pathway, and the DNA photolyase gene PHR1, which is involved in pyrimidine dimer repair. Strikingly, the FEL lost 33 additional genes, including polymerases (i.e., POL4 and POL32) and telomere-associated genes (e.g., RIF1, RFA3, CDC13, PBP2). Echoing these losses, molecular evolutionary analyses reveal that, compared to the SEL, the FEL stem lineage underwent a burst of accelerated evolution, which resulted in greater mutational loads, homopolymer instabilities, and higher fractions of mutations associated with the common endogenously damaged base, 8-oxoguanine. We conclude that Hanseniaspora is an ancient lineage that has diversified and thrived, despite lacking many otherwise highly conserved cell cycle and genome integrity genes and pathways, and may represent a novel system for studying cellular life without them.
biorxiv evolutionary-biology 0-100-users 2019Predictive neural processing in adult zebrafish depends on shank3b, bioRxiv, 2019-02-12
Intelligent behavior requires a comparison between the predicted and the actual consequences of behavioral actions. According to the theory of predictive processing, this comparison relies on a neuronal error signal that reflects the mismatch between an internal prediction and sensory input. Inappropriate error signals may generate pathological experiences in neuropsychiatric conditions. To examine the processing of sensorimotor prediction errors across different telencephalic brain areas we optically measured neuronal activity in head-fixed, adult zebrafish in a virtual reality. Brief perturbations of visuomotor feedback triggered distinct changes in swimming behavior and different neuronal responses. Neuronal activity reflecting sensorimotor mismatch, rather than sensory input or motor output alone, was prominent throughout multiple forebrain areas. This activity preceded and predicted the transition in motor behavior. Error signals were altered in specific forebrain regions by a mutation in the autism-related gene shank3b. Predictive processing is therefore a widespread phenomenon that may contribute to disease phenotypes.
biorxiv neuroscience 0-100-users 2019Environmental DNA (eDNA) metabarcoding of pond water as a tool to survey conservation and management priority mammals, bioRxiv, 2019-02-11
Abstract<jatslist list-type=order><jatslist-item>Environmental DNA (eDNA) metabarcoding is largely used to survey aquatic communities, but can also provide data on terrestrial taxa utilising aquatic habitats. However, the entry, dispersal, and detection of terrestrial species’ DNA within waterbodies is understudied.<jatslist-item><jatslist-item>We evaluated eDNA metabarcoding of pond water for monitoring semi-aquatic, ground-dwelling, and arboreal mammals, and examined spatiotemporal variation in mammal eDNA signals using experiments in captive and wild conditions.<jatslist-item><jatslist-item>We selected nine focal species of conservation and management concern European water vole, European otter, Eurasian beaver, European hedgehog, European badger, red deer, Eurasian lynx, red squirrel, and European pine marten. We hypothesised that eDNA signals (i.e. proportional read counts) would be stronger for semi-aquatic than terrestrial species, and at sites where mammals exhibited behaviours (e.g. swimming, urination). We tested this by sampling waterbodies in enclosures of captive focal species at specific sites where behaviours had been observed (‘directed’ sampling) and at equidistant intervals along the shoreline (‘stratified’ sampling). We then surveyed natural ponds (N = 6) where focal species were present using stratified water sampling, camera traps, and field signs. eDNA samples were metabarcoded using vertebrate-specific primers.<jatslist-item><jatslist-item>All focal species were detected in captivity. eDNA signal strength did not differ between directed and stratified samples across or within species, between species lifestyles (i.e. semi-aquatic, ground-dwelling, arboreal), or according to behaviours. Therefore, eDNA was evenly distributed within artificial waterbodies. Conversely, eDNA was unevenly distributed in natural ponds. eDNA metabarcoding, camera trapping, and field signs detected beaver, red deer, and roe deer. Badger and red fox were recorded with cameras and field signs, but not eDNA metabarcoding. However, eDNA metabarcoding detected small mammals missed by cameras and field signs, e.g. water vole. Terrestrial mammal eDNA signals were weaker and detected in fewer samples than semi-aquatic mammal eDNA signals.<jatslist-item><jatslist-item>eDNA metabarcoding has potential for inclusion in mammal monitoring schemes by enabling large-scale, multi-species distribution assessment for priority and difficult to survey species, and could provide early indication of range expansions or contractions. However, eDNA surveys need high spatiotemporal resolution and metabarcoding biases require further investigation before this tool is routinely implemented.<jatslist-item>
biorxiv molecular-biology 0-100-users 2019