title: Structural anther mimics improve reproductive success through dishonest signalling that enhances both attraction and the morphological fit of pollinators with flowers

abstract:
O_LINumerous studies have identified traits associated with pollen mimicry, however, the processes underlying floral deception remains poorly documented for these structures. We studied the importance of attraction and mechanical fit of anther mimics in Tritonia laxifolia (Iridaceae) and their relative contributions to reproductive success.
C_LIO_LITo determine anther mimics role in pollinator attraction, we offered bees binary preferences to flowers painted with UV absorbent and reflecting paint. We also conducted preference experiments between flowers with excised anther mimics and unmanipulated controls, from which mechanical fit was assessed using single visits. Anther mimics effects on female reproductive success was determined using similar treatments, but on rooted plants.
C_LIO_LIBees preferred UV absorbent over UV reflecting anther mimics. Preference for flowers with and without the three-dimensional structures was equal. Single visits resulted in more pollen deposition on unmanipulated controls over flowers with their anther mimics excised, which was directly linked to pollen-collecting behaviour. Controls with unmanipulated anther mimics experienced more seed set than those with their anther mimics excised.
C_LIO_LIThis study provides insights into pollinator-mediated selection on deceptive floral signals and shows that three-dimensional anther mimics increases reproductive success through both attraction and pollen collecting behaviours that improves the fit between flowers and pollinators.
C_LI


title: Systematic analysis of intrinsic enhancer-promoter compatibility in the mouse genome.

abstract: Gene expression is in part controlled by cis-regulatory elements (CREs) such as enhancers and repressive elements. Anecdotal evidence has indicated that a CRE and a promoter need to be biochemically compatible for promoter regulation to occur, but this compatibility has remained poorly characterised in mammalian cells. We used high-throughput combinatorial reporter assays to test thousands of CRE - promoter pairs from three Mb-sized genomic regions in mouse cells. This revealed that CREs vary substantially in their promoter compatibility, ranging from striking specificity for a single promoter to quantitative differences in activation across a broad set of promoters. More than half of the tested CREs exhibit significant promoter selectivity. Housekeeping promoters tend to have similar CRE preferences, but other promoters exhibit a wide diversity of compatibilities. Higher-order TF motif combinations may account for compatibility. CRE-promoter selectivity does not correlate with looping interactions in the native genomic context, suggesting that chromatin folding and compatibility are two orthogonal mechanisms that confer specificity to gene regulation.

category: genetics


title: Colour Vision in Stomatopod Crustaceans: more questions than answers

abstract: Stomatopod crustaceans, or mantis shrimps, are known for their extensive range of spectral sensitivities but relatively poor spectral discrimination. Instead of the colour-opponent mechanism of other colour vision systems, the 12 narrow-band colour channels they possess may underlie a different method of colour processing. We investigated one hypothesis, in which the photoreceptors are proposed to act as individual wave-band detectors, interpreting colour as a parallel pattern of photoreceptor activation, rather than a ratiometric comparison of individual signals. This different form of colour detection has been used to explain previous behavioural tests in which low saturation blue was not discriminated from grey potentially because of similar activation patterns. Results here, however, indicate that the stomatopod, Haptosquilla trispinosa was able to easily distinguish several colours, including blue of both high and low saturation, from greys. The animals did show a decrease in performance over time in an artificially lit environment, indicating plasticity in colour discrimination ability. This rapid plasticity, most likely the result of a change in opsin (visual pigment) expression, has now been noted in several animal lineages (both invertebrate and vertebrate) and is a factor we suggest needing care and potential re-examination in any colour-based behavioural tests. As for stomatopods, it remains unclear why they achieve poor colour discrimination using the most comprehensive set of spectral sensitivities in the animal kingdom and also what form of colour processing they may utilise.

category: animal behavior and cognition


title: 3D visualisation of the cardiac ryanodine receptor clusters and the molecular-scale fraying of dyads

abstract: Clusters of ryanodine receptor calcium channels (RyRs) form the primary molecular machinery in cardiomyocytes. Various adaptations of super-resolution microscopy have revealed intricate details of the structure, molecular composition and locations of these couplons. However, most optical super-resolution techniques lack the capacity for three-dimensional (3D) visualisation. Enhanced Expansion Microscopy (EExM) offers resolution (in-plane and axially) sufficient to spatially resolve individual proteins within peripheral couplons and within dyads located in the interior. We have combined immunocytochemistry and immunohistochemistry variations of EExM with 3D visualisation to examine the complex topologies, geometries and molecular sub-domains within RyR clusters. We observed that peripheral couplons exhibit variable co-clustering ratios and patterns between RyR and the structural protein, junctophilin-2 (JPH2). Dyads possessed sub-domains of JPH2 which occupied the central regions of the RyR cluster, whilst the poles were typically devoid of JPH2 and broader, and likely specialise in turnover and remodelling of the cluster. In right ventricular myocytes from rats with monocrotaline-induced right ventricular failure, we observed hallmarks of RyR cluster fragmentation accompanied by similar fragmentations of the JPH2 sub-domains. We hypothesise that the frayed morphology of RyRs in close proximity to fragmented JPH2 structural sub-domains may form the primordial foci of RyR mobilisation and dyad remodelling.

category: biophysics


title: Spatially-resolved transcriptomics analyses of invasive fronts in solid tumors

abstract: Solid tumors are complex ecosystems, and heterogeneity is the major challenge for overcoming tumor relapse and metastasis. Uncovering the spatial heterogeneity of cell types and functional states in tumors is essential for developing effective treatment, especially in invasive fronts of tumor, the most active region for tumor cells infiltration and invasion. We firstly used SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq) with a nanoscale resolution to characterize the tumor microenvironment of intrahepatic cholangiocarcinoma (ICC). Enrichment of distinctive immune cells, suppressive immune microenvironment and metabolic reprogramming of tumor cells were identified in the 500{micro}m-wide zone centered bilaterally on the tumor boundary, namely invasive fronts of tumor. Furthermore, we found the damaged states of hepatocytes with overexpression of Serum Amyloid A (SAA) in invasive fronts, recruiting macrophages for facilitating further tumor invasion, and thus resulting in a worse prognosis. We also confirmed these findings in hepatocellular carcinoma and other liver metastatic cancers. Our work highlights the remarkable potential of high-resolution-spatially resolved transcriptomic approaches to provide meaningful biological insights for comprehensively dissecting the tumor ecosystem and guiding the development of novel therapeutic strategies for solid tumors.

category: cancer biology


title: Automated and parallelized spike collision tests to identify spike signal projections

abstract: The spike collision test is a highly reliable technique to identify axonal projection of a neuron recorded electrophysiologically for investigating functional spike information among brain areas. It is potentially applicable to more neuronal projections by combining multi-channel recording with optogenetic stimulation. Yet, it remains inefficient and laborious because an experimenter must visually select spikes in every channel and manually repeat spike collision tests for each neuron serially. Here, we established a novel technique to automatically perform spike collision tests for all channels in parallel (Multi-Linc analysis), employing two distinct protocols implemented in a multi-channel real-time processing system. The rat cortical neurons identified with this technique displayed physiological spike features consistent with excitatory projection neurons. Their antidromic spikes were similar in shape but slightly larger in amplitude compared with spontaneous spikes. In addition, we demonstrated simultaneous identification of reciprocal or bifurcating projections among cortical areas. Thus, our Multi-Linc analysis will be a powerful research approach to elucidate interareal spike communication.

category: neuroscience


title: Aurora B/C-dependent phosphorylation promotesRec8 cleavage in mammalian oocytes

abstract: To generate haploid gametes, cohesin is removed in a step-wise manner from chromosome arms in meiosis I and the centromere region in meiosis II, to segregate chromosomes and sister chromatids, respectively. Meiotic cohesin removal requires cleavage of the meiosis-specific kleisin subunit Rec8 by the protease Separase[1, 2]. In yeast, Rec8 is kept in a non-phosphorylated state by the action of PP2A-B56, which is localised to the centromere region, thereby preventing cohesin removal from this region in meiosis I[3-5]. However, it is unknown whether Rec8 has to be equally phosphorylated for cleavage, and whether centromeric cohesin protection is indeed brought about by dephosphorylation of Rec8 preventing cleavage, in mammalian meiosis. The identity of one or several potential Rec8-specific kinase(s) is also unknown. This is due to technical challenges, as Rec8 is poorly conserved preventing a direct translation of the knowledge gained from model systems such as yeast and C. elegans to mammals, and additionally, there is no turn-over of Rec8 after cohesion establishment, preventing phosphomutant analysis of functional Rec8. To address how Rec8 cleavage is brought about in mammals, we adapted a biosensor for Separase to study Rec8 cleavage in single mouse oocytes by live imaging, and identified phosphorylation sites promoting cleavage. We found that Rec8 cleavage by Separase depends on Aurora B/C kinase activity, and identified a residue promoting cleavage and being phosphorylated in an Aurora B/C kinase-dependent manner. Accordingly, inhibition of Aurora B/C kinase during meiotic maturation impairs endogenous Rec8 phosphorylation and chromosome segregation.

category: cell biology


title: Myosin-X-dependent assembly of the extracellular matrix limits breast cancer invasion

abstract:
Ductal carcinoma in situ (DCIS) is a pre-invasive stage of breast cancer, where the tumor is encapsulated by a basement membrane (BM). At the invasive phase, the BM barrier is compromised enabling tumor cells to escape into the surrounding stroma. The molecular mechanisms that establish and maintain an epithelial BM barrier in vivo are poorly understood. Myosin-X (MYO10) is a filopodia-inducing motor protein implicated in metastasis and poor clinical outcome in patients with invasive breast cancer (IBC). We compared MYO10 expression in patient-matched normal breast tissue and DCIS lesions and found elevated MYO10 expression in DCIS samples, suggesting that MYO10 might facilitate the transition from DCIS to IBC. Indeed, MYO10 promoted the formation of filopodia and cell invasion in vitro and positively regulated the dissemination of individual cancer cells from IBC lesions in vivo. However, MYO10-depleted DCIS xenografts were, unexpectedly, more invasive. In these xenografts, MYO10 depletion compromised BM formation around the lesions resulting in poorly defined tumor borders and increased cancer cell dispersal into the surrounding stroma. Moreover, MYO10-depleted tumors showed increased EMT-marker-positive cells, specifically at the tumor periphery. We also observed cancer spheroids undergoing rotational motion and recruiting BM components in a filopodia-dependent manner to generate a near-continuous extracellular matrix boundary. Taken together, our data identify a protective role for MYO10 in early-stage breast cancer, where MYO10-dependent tumor cell protrusions support BM assembly at the tumor-stroma interface to limit cancer progression, and a pro-invasive role that facilitates cancer cell dissemination at later stages.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/464987v2_ufig1.gif" ALT="Figure 1">
View larger version (28K):
org.highwire.dtl.DTLVardef@d90a4eorg.highwire.dtl.DTLVardef@133ba9borg.highwire.dtl.DTLVardef@1204a05org.highwire.dtl.DTLVardef@dba254_HPS_FORMAT_FIGEXP  M_FIG C_FIG Highlights- Filopodia sculpt the tumor-proximal stroma in pre-invasive ductal carcinoma in situ (DCIS).
- Filopodia-dependent basement membrane (BM) assembly limits invasive transition of DCIS-like tumors in vivo.
- Loss of MYO10-dependent filopodia impairs BM assembly and induces an EMT-like phenotype at the tumor-stroma interface in vivo.
- MYO10 filopodia are anti-invasive in DCIS but facilitate dissemination in invasive breast cancer.


title: Filopodia-mediated basement membrane assembly at pre-invasive tumor boundaries


title: Spatiotemporal mapping of gene expression landscapes and developmental trajectories during zebrafish embryogenesis

abstract: Vertebrate embryogenesis is a remarkably dynamic process during which numerous cell types of different lineages generate, change, or disappear within a short period of time. A major challenge in understanding this process is the lack of topographical transcriptomic information that can help correlate microenvironmental cues within the hierarchy of cell fate decisions. Here, we employed Stereo-seq, a high-definition spatially resolved transcriptomic technology, to dissect the spatiotemporal dynamics of gene expression and regulatory networks in the developing zebrafish embryos. We profiled 91 embryo sections covering six critical time points during the first 24 hours of development, obtaining a total of 139,391 spots at cellular size ([~]100 m2) with spatial coordinates. Meanwhile, we identified spatial modules and co-varying genes for specific tissue organizations. By performing the integrative analysis of the Stereo-seq and scRNA-seq data from each time point, we reconstructed the spatially resolved developmental trajectories of cell fate transitions and molecular changes during zebrafish embryogenesis. We further investigated the spatial distribution of ligand-receptor pairs for major signaling pathways and identified novel interactions that potentially crosstalk with the Notch signaling pathway during zebrafish development. Our study constitutes a fundamental reference for further studies aiming to understand vertebrate development.

category: developmental biology


title: High-resolution spatiotemporal transcriptomic maps of developing Drosophila embryos and larvae

abstract: Drosophila has long been a successful model organism in multiple fields such as genetics and developmental biology. Drosophila genome is relatively smaller and less redundant, yet largely conserved with mammals, making it a productive model in studies of embryogenesis, cell signaling, disease mechanisms, etc. Spatial gene expression pattern is critical for understanding of complex signaling pathways and cell-cell interactions, whereas temporal gene expression changes need to be tracked during highly dynamic activities such as tissue development and disease progression. Systematic studies in Drosophila as a whole are still impeded by lack of these spatiotemporal transcriptomic information. Drosophila embryos and tissues are of relatively small size, limiting the application of current technologies to comprehensively resolve their spatiotemporal gene expression patterns. Here, utilizing SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq), we dissected the spatiotemporal transcriptomic changes of developing Drosophila with high resolution and sensitivity. Our data recapitulated the spatial transcriptomes of embryonic and larval development in Drosophila. With these data, we identified known and previously undetected subregions in several tissues during development, and revealed known and potential gene regulatory networks of transcription factors within their topographic background. We further demonstrated that Stereo-seq data can be used for 3D reconstruction of Drosophila embryo spatial transcriptomes. Our data provides Drosophila research community with useful resources of spatiotemporally resolved transcriptomic information across developmental stages.


title: Habitat loss exacerbates pathogen spread: An Agent-based model of avian influenza infection in migratory waterfowl

abstract:
Habitat availability determines the distribution of migratory waterfowl along their flyway, which further influences the transmission and spatial spread of avian influenza viruses (AIVs). The extensive habitat loss in the East Asian-Australasian Flyway (EAAF) may have potentially altered the virus transmission and spread, but those consequences are rarely studied. We constructed 6 fall migration networks that differed in their level of habitat loss, wherein an increase in habitat loss resulted in smaller networks with fewer sites. The networks were integrated with an agent-based model and a susceptible-infected-recovered model to simulate waterfowl migration and AIV transmission. We found that extensive habitat loss in the EAAF can 1) relocate the outbreaks northwards responding to the distribution changes of wintering waterfowl geese, 2) increase the outbreak risk in remaining sites due to larger bird congregations, and 3) facilitate AIV transmission among wintering geese. Our modelling output suggested that there was a certain system resilience of migration network to confront the site removal. In addition, the outputs were in line with the predictions from the concept of "migratory escape", affecting the pattern of infection prevalence in the waterfowl population. Our modelling shed light on the potential consequences of habitat loss in transmitting and spreading AIV at the flyway scale, and suggested the driving mechanisms behind these effects, advocating the importance of nature conservation in changing spatial and temporal patterns of AIV outbreak.

Author summaryWhat are the possible consequences of extensive habitat loss on the transmission and spread of avian influenza viruses (AIVs)? We used a logistic regression model to select the suitable habitats of Greater white-fronted goose in the East Asian-Australasian Flyway and treated these habitats as sites to construct 6 fall migration networks that differed in their level of habitat loss (i.e., site removal). We then simulate geese migration in these networks, and explore the impacts of habitat loss on habitat connectivity and AIV transmission. We found the extensive habitat loss can cause relocation of the outbreaks and increase the outbreak risk and AIV transmission. Our modelling outputs suggested a certain network resilience to confront the site loss, and a "migratory escape" to change the spatial and temporal pattern of infection prevalence in the population. Overall, our study showed that land use changes and habitat loss can affect disease distribution and prevalence, suggested the importance of habitat conservation in changing the spatial and temporal pattern of AIVs transmission and spread.

category: ecology


title: Non-monotonic fibril surface occlusion by GFP tags from coarse-grained molecular simulations

abstract: The pathological growth of amyloid fibrils in neurons underlies the progression of neurodegenerative diseases including Alzheimers and Parkinsons disease. Fibrils form when soluble monomers oligomerise in the cytoplasm. Their subsequent growth occurs via nucleated polymerization mechanisms involving the free ends of the fibrils augmented by secondary nucleation of new oligomers at their surface. Amyloid fibrils possess a complex interactome with diffusing cytoplasmic proteins that regulates many aspects of their growth, seeding capacity, biochemical activity and transition to pathological inclusions in diseased brains. Changes to their surface are also expected to modify their interactome, pathogenicity and spreading in the brain. Many assays visualise fibril formation, growth and inclusion formation by decorating monomeric proteins with fluorescent tags such as GFP. Recent studies from our group suggest that tags with sizes comparable to the fibril radius may modify the fibril surface accessibility and thus their PTM pattern, interactome and ability to form inclusions. Using coarse-grained molecular simulations of a single alpha synuclein fibril tagged with GFP we find that thermal fluctuations of the tags create a non-monotonic, size-dependent sieve around the fibril that perturbs its interactome with diffusing species. Our results indicate that experiments using tagged and untagged monomers to study the growth and interactome of fibrils should be compared with caution, and the confounding effects of the tags are more complex than a reduction in surface accessibility. The prevalence of fluorescent tags in amyloid fibril growth experiments suggests this has implications beyond the specific alpha synuclein fibrils we model here.


title: Implementation of residue-level coarse-grained models in GENESIS for large-scale molecular dynamics simulations

abstract:
Residue-level coarse-grained (CG) models have become one of the most popular tools in biomolecular simulations in the trade-off between modeling accuracy and computational efficiency. To investigate large-scale biological phenomena in molecular dynamics (MD) simulations with CG models, unified treatments of proteins and nucleic acids, as well as efficient parallel computations, are indispensable. In the GENESIS MD software, we implement several residue-level CG models, covering structure-based and context-based potentials for both well-folded biomolecules and intrinsically disordered regions. An amino acid residue in protein is represented as a single CG particle centered at the C atom position, while a nucleotide in RNA or DNA is modeled with three beads. Then, a single CG particle represents around ten heavy atoms in both proteins and nucleic acids. The input data in CG MD simulations are treated as GROMACS-style input files generated from a newly developed toolbox, GENESIS-CG-tool. To optimize the performance in CG MD simulations, we utilize multiple neighbor lists, each of which is attached to a different nonbonded interaction potential in the cell-linked list method. We found that random number generations for Gaussian distributions in the Langevin thermostat are one of the bottlenecks in CG MD simulations. Therefore, we parallelize the computations with message-passing-interface (MPI) to improve the performance on PC clusters or supercomputers. We simulate Herpes simplex virus (HSV) type 2 B-capsid and chromatin models containing more than 1,000 nucleosomes in GENESIS as examples of large-scale biomolecular simulations with residue-level CG models. This framework extends accessible spatial and temporal scales by multi-scale simulations to study biologically relevant phenomena, such as genome-scale chromatin folding or phase-separated membrane-less condensations.

Author summaryMolecular dynamics (MD) simulations have been widely used to investigate biological phenomena that are difficult to study only with experiments. Since all-atom MD simulations of large biomolecular complexes are computationally expensive, coarse-grained (CG) models based on different approximations and interaction potentials have been developed so far. There are two practical issues in biological MD simulations with CG models. The first issue is the input file generations of highly heterogeneous systems. In contrast to well-established all-atom models, specific features are introduced in each CG model, making it difficult to generate input data for the systems containing different types of biomolecules. The second issue is how to improve the computational performance in CG MD simulations of heterogeneous biological systems. Here, we introduce a user-friendly toolbox to generate input files of residue-level CG models containing folded and disordered proteins, RNAs, and DNAs using a unified format and optimize the performance of CG MD simulations via efficient parallelization in GENESIS software. Our implementation will serve as a framework to develop novel CG models and investigate various biological phenomena in the cell.


title: Effect of reversible osmotic stress on live cell plasma membranes, probed via Laurdan general polarization measurements

abstract:
Here we seek to gain insight into changes in the plasma membrane of live cells upon the application of osmotic stress using Laurdan, a fluorescent probe that reports on membrane organization, hydration, and dynamics. It is known that the application of osmotic stress to lipid vesicles causes a decrease in Laurdans generalized polarization (GP), which has been interpreted as an indication of membrane stretching. In cells, we see the opposite effects, as GP increases when the osmolarity of the solution is decreased. This increase in GP is associated with the presence of caveolae, which are known to disassemble and flatten in response to osmotic stress, in a process that supplies extra plasma membrane in physiological processes.

SignificanceCells can experience multiple stresses in vivo. Furthermore, the application of osmotic stress is used as a biophysical tool to interrogate membrane proocesses in vitro. We sought to investigate the consequences of osmotic stress on the plasma membrane properties using the fluorescent probe Laurdan. Unexpectedly, we find that osmotic stress leads to an increase in GP in live cells. The opposite change in GP has been observed in model lipid bilayers, reminding us there are limitations to the utility of model systems in understanding cell membrane behavior. Despite years of research, the cell membrane still has ways to surprise us.


title: Incorporating Target-Specific Pharmacophoric Information Into Deep Generative Models For Fragment Elaboration

abstract: Despite recent interest in deep generative models for scaffold elaboration, their applicability to fragment-to-lead campaigns has so far been limited. This is primarily due to their inability to account for local protein structure or a users design hypothesis. We propose a novel method for fragment elaboration, STRIFE that overcomes these issues. STRIFE takes as input Fragment Hotspot Maps (FHMs) extracted from a protein target, and processes them to provide meaningful and interpretable structural information to its generative model, which in turn is able to rapidly generate elaborations with complementary pharmacophores to the protein. In a large-scale evaluation, STRIFE outperforms existing, structure-unaware, fragment elaboration methods in proposing highly ligand efficient elaborations. In addition to automatically extracting pharmacophoric information from a protein targets FHM, STRIFE optionally allows the user to specify their own design hypotheses.

category: systems biology


title: ChemChaste: Simulating spatially inhomogenous biochemical reaction-diffusion systems for modelling cell-environment feedbacks

abstract:
MotivationSpatial organisation plays an important role in the function of many biological systems, from cell fate specification in animal development to multi-step metabolic conversions in microbial communities. The study of such systems benefits from the use of spatially explicit computational models that combine a discrete description of cells with a continuum description of one or more chemicals diffusing within a surrounding bulk medium. These models allow the in silico testing and refinement of mechanistic hypotheses. However, most existing models of this type do not account for concurrent bulk and intracellular biochemical reactions and their possible coupling.

ResultsHere, we describe ChemChaste, an extension for the open-source C++ computational biology library Chaste. ChemChaste enables the spatial simulation of both multicellular and bulk biochemistry by expanding on Chastes existing capabilities. In particular, ChemChaste enables: (i) simulation of an arbitrary number of spatially diffusing chemicals; (ii) spatially heterogeneous chemical diffusion coefficients; and (iii) inclusion of both bulk and intracellular biochemical reactions and their coupling. ChemChaste also introduces a file-based interface that allows users to define the parameters relating to these functional features without the need to interact directly with Chastes core C++ code. We describe ChemChaste and demonstrate its functionality using a selection of chemical and biochemical exemplars, with a focus on demonstrating increased ability in modelling bulk chemical reactions and their coupling with intracellular reactions.

Availability and implementationChemChaste is a free, open-source C++ library, available via GitHub at https://github.com/OSS-Lab/ChemChaste under the BSD license.

ContactO.Soyer@warwick.ac.uk or a.g.fletcher@sheffield.ac.uk


title: Roosting ecology and the evolution of bat landing maneuvers

abstract: Biomechanics is poised at the intersection of organismal form, function, and ecology, and forms a practical lens through which to investigate evolutionary linkages among these factors. We conducted the first evolutionary analysis of bat flight dynamics by examining the phylogenetic patterning of landing mechanics. We discovered that bats perform stereotyped maneuvers that are correlated with landing performance quantified as impact force, and that these are linked with roosting ecology, a critical aspect of bat biology. Our findings suggest that bat ancestors performed simple, four-limbed landings, similar to those performed by gliding mammals, and that more complex landings evolved in association with novel roost types. This explicit connection between ecology and biomechanics presents the opportunity to identify traits that are associated with a locomotor behavior of known ecological relevance, thus laying the foundation for a broader understanding of the evolution of flight and wing architecture in this extraordinarily successful mammalian lineage.

category: zoology


title: Learning the statistics of pain: computational and neural mechanisms

abstract: Pain invariably changes over time, and these temporal fluctuations are riddled with uncertainty about body safety. In theory, statistical regularities of pain through time contain useful information that can be learned, allowing the brain to generate expectations and inform behaviour. To investigate this, we exposed healthy participants to probabilistic sequences of low and high-intensity electrical stimuli to the left hand, containing sudden changes in stimulus frequencies. We demonstrate that humans can learn to extract these regularities, and explicitly predict the likelihood of forthcoming pain intensities in a manner consistent with optimal Bayesian models with dynamic update of beliefs. We studied brain activity using functional MRI whilst subjects performed the task, which allowed us to dissect the underlying neural correlates of these statistical inferences from their uncertainty and update. We found that the inferred frequency (posterior probability) of high intensity pain correlated with activity in bilateral sensorimotor cortex, secondary somatosensory cortex and right caudate. The uncertainty of statistical inferences of pain was encoded in the right superior parietal cortex. An intrinsic part of this hierarchical Bayesian model is the way that unexpected changes in frequency lead to shift beliefs and update the internal model. This is reflected by the KL divergence between consecutive posterior distributions and associated with brain responses in the premotor cortex, dorsolateral prefrontal cortex, and posterior parietal cortex. In conclusion, this study extends what is conventionally considered a sensory pain pathway dedicated to process pain intensity, to include the generation of Bayesian internal models of temporal statistics of pain intensity levels in sensorimotor regions, which are updated dynamically through the engagement of premotor, prefrontal and parietal regions.


title: The 2D Ising model, criticality and AIT

abstract: In this short note we study the 2D Ising model, a universal computational model which reflects phase transitions and critical phenomena, as a framework for establishing links between systems that exhibit criticality with the notions of complexity. This is motivated in the context of neuroscience applications stemming from algorithmic information theory (AIT). Starting with the original 2D Ising model, we show that -- together with correlation length of the spin lattice, susceptibility to a uniform external field -- the correlation time of the magnetization time series, the compression ratio of the spin lattice, the complexity of the magnetization time series -- as derived from Lempel-Ziv-Welch compression--, and the rate of information transmission in the lattice, all reflect the effects of the phase transition, which results in spacetime pockets of uniform magnetization at all scales. We also show that in the Ising model the insertion of sparse long-range couplings has a direct effect on the critical temperature and other parameters. The addition of positive links extends the ordered regime to higher critical temperatures, while negative links have a stronger, disordering influence at the global scale. We discuss some implications for the study of long-range (e.g., ephaptic) interactions in the human brain and the effects of weak perturbations in neural dynamics.


title: Visualization of Sympathetic Neural Innervation in Human White Adipose Tissue

abstract: Obesity, defined as an excess of adipose tissue that adversely affects health, is a major cause of morbidity and mortality. However, understanding the structure and function of human adipose tissue has been limited by the inability to visualize cellular components due to the innate structure of adipocytes, which are characterized by large lipid droplets. Combining the iDISCO and the CUBIC protocols for whole tissue staining and optical clearing, we developed a protocol to enable immunostaining and clearing of human subcutaneous white adipose tissue (WAT) obtained from individuals with severe obesity. We were able to perform immunolabeling of sympathetic nerve terminals in whole white adipose tissue and subsequent optical clearing by eliminating lipids to render the opaque tissue completely transparent. We then used light sheet confocal microscopy to visualize innervation of human WAT from obese individuals in a 3D manner.We demonstrate the visualization of sympathetic nerve terminals in human WAT. This protocol can be modified to visualize other structures such as blood vessels involved in the development, maintenance and function of human adipose tissue in health and disease.


title: An in-vivo digital atlas of the spatially reliable cerebral vasculature in mice

abstract: Cerebral vascular abnormalities can have a large impact on brain function and have been frequently detected as a comorbidity in various neuropathologies. The mouse is the most common pre-clinical animal model used to investigate neuropathologies and thus cerebral vascular atlases of this species are indispensable. In particular, an atlas derived from multiple animals and that can address inter-subject variability is still absent. The current study presents a mouse cerebral vascular atlas developed from MR 2D-TOF angiograms acquired from a relatively high number (N=21) of 12 weeks old male C57BL/6 mice. The vascular atlas we present depicts large arteries, veins and sinuses that are more spatially reliable across animals than smaller vessels. The atlas is available for download and contains multiple datasets: i) a cerebral vascular atlas; ii) a digital segmentation of large vessels (arteries and veins); iii) a high resolution T2 anatomical background aligned to the atlas; iv) a skeletonized vascular atlas; and v) a vascular graph model. We suggest that these components provide a very potent analysis tool for mouse fMRI data and that the atlas can be used as a template for multimodal brain imaging studies. To this end, we also demonstrate an explicit application of the atlas for the investigation of vascular influences in resting-state fMRI.


title: Readers move their eyes mindlessly using midbrain visuo-motor principles

abstract: Saccadic eye movements rapidly shift our gaze over 100,000 times daily, enabling countless tasks ranging from driving to reading. Long regarded as a window to the mind1 and human information processing2, they are thought to be cortically/cognitively controlled movements aimed at objects/words of interest3-10. Saccades however involve a complex cerebral network11-13 wherein the contribution of phylogenetically older sensory-motor pathways14-15 remains unclear. Here we show using a neuro-computational approach16 that mindless visuo-motor computations, akin to reflexive orienting responses17 in neonates18-19 and vertebrates with little neocortex15,20, guide humans eye movements in a quintessentially cognitive task, reading. These computations occur in the superior colliculus, an ancestral midbrain structure15, that integrates retinal and (sub)cortical afferent signals13 over retinotopically organized, and size-invariant, neuronal populations21. Simply considering retinal and primary-visual-cortex afferents, which convey the distribution of luminance contrast over sentences (visual-saliency map22), we find that collicular population-averaging principles capture readers prototypical word-based oculomotor behavior2, leaving essentially rereading behavior unexplained. These principles reveal that inter-word spacing is unnecessary23-24, explaining metadata across languages and writing systems using only print size as a predictor25-26. Our findings demonstrate that saccades, rather than being a window into cognitive/linguistic processes, primarily reflect rudimentary visuo-motor mechanisms in the midbrain that survived brain-evolution pressure27.


title: Systematic reduction of the dimensionality of natural scenes allows accurate predictions of retinal ganglion cell spike outputs.

abstract: The mammalian retina engages a broad array of linear and nonlinear circuit mechanisms to convert natural scenes into retinal ganglion cell (RGC) spike outputs. Although many individual integration mechanisms are well understood, predictive models of natural scene encoding perform poorly, likely due to interactions among the active mechanisms. Here, we identified spatial integration mechanisms relevant for natural scene encoding and used computational modeling to predict spike outputs in primate parasol RGCs. Our approach was to simplify the structure of natural scenes and empirically ask whether these changes were reflected in RGC spike responses. We observed that reducing natural movies to 16 linearly integrated regions described [~]80% of the structure of parasol RGC spike responses. We then used simplified stimuli to create high-dimensional metamers that recapitulated the spike response of full-field naturalistic movies. Finally, we identified the retinal computations that convert natural images in 16-dimensional space into 1-dimensional spike outputs.


title: A comprehensive benchmark for structural assessment in bipartite networks

abstract: The use of null models has been a cornerstone to assess the emergence of many network properties at different levels of organization (micro-, meso- and macroscale). Notwithstanding, the debate around which is the most appropriate randomization procedure for a given problem is far from being over. Within the ecological community, for example, the discussion around whether nestedness is -or is not- a frequent pattern in natural systems, and under which assumptions, remains open. For this particular problem, efforts have been devoted to exploring to what extent current models are vulnerable to statistical errors, or to introduce new models that employ different randomization procedures. However, few or no attention has been devoted to the performance of those null models against other architectures. Here, we show that assessing alternative structures under a single null model may produce ambiguous results, which difficult the comparison regarding the joint emergence of different arrangements within a single network. To this aim, we analyze the statistical significance -in terms of z-scores- of nestedness, modularity, and in-block nestedness scores, employing five different null models on a benchmark of [~] 2.5 x 104 synthetic bipartite networks with prescribed levels of the mentioned patterns. We show that some null models systematically over- or underestimate the presence of one or another structural pattern. In light of these ambiguities, we introduce an alternative model (termed Corrected Probabilistic model) that reduces the observed biases towards under- and overestimation, and highlight the need for the development of new frameworks that take into account those biases.


title: Mimicked synthetic ribosomal protein complex for benchmarking cross-linking mass spectrometry workflows

abstract:
The field of cross-linking mass spectrometry has matured to a frequently used tool for the investigation of protein structures as well as interactome studies up to a system wide level. The growing community generated a broad spectrum of applications, linker types, acquisition strategies and specialized data analysis tools, which makes it challenging, especially for newcomers, to decide for an appropriate analysis workflow. Therefore, we here present a large and flexible synthetic peptide library as reliable instrument to benchmark crosslinkers with different reactive sites as well as acquisition techniques and data analysis algorithms. Additionally, we provide a tool, IMP-X-FDR, that calculates the real, experimentally validated, FDR, compares results across search engine platforms and analyses crosslink properties in an automated manner. The library was used with the reagents DSSO, DSBU, CDI, ADH, DHSO and azide-a-DSBSO and data were analysed using the algorithms MeroX, MS Annika, XlinkX, pLink 2, MaxLynx and xiSearch. We thereby show that the correct algorithm and search setting choice is highly important to improve ID rate and FDR in combination with software and sample-complexity specific score cut-offs. When analysing DSSO data with MS Annika, we reach high identification rates of up to [~]70 % of the theoretical maximum (i.e. 700 unique lysine-lysine cross-links) while maintaining a low real FDR of < 3 % at cross-link level and with high reproducibility, representatively showing that our test system delivers valuable and statistically solid results.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/465295v2_ufig1.gif" ALT="Figure 1">
View larger version (30K):
org.highwire.dtl.DTLVardef@146c751org.highwire.dtl.DTLVardef@1c7a6e1org.highwire.dtl.DTLVardef@9d2ee7org.highwire.dtl.DTLVardef@1725693_HPS_FORMAT_FIGEXP  M_FIG C_FIG

category: bioinformatics


title: Neuropeptide Neuromedin B does not alter body weight and glucose homeostasis nor does it act as an insulin-releasing peptide

abstract: Neuromedin B (NMB) is a member of the neuromedin family of neuropeptides with a high level of region-specific expression in the brain. Several GWAS studies on non-obese and obese patients suggested that polymorphisms in NMB predispose to obesity by affecting appetite control and feeding preference. Furthermore, several studies proposed that NMB can act as an insulin releasing peptide. Since the functional study has never been done, the in vivo role of NMB as modulator of weight gain or glucose metabolism remains unclear. Here, we generated Nmb conditional mice and nervous system deficient NmB mice. We then performed olfactory and food preference analysis, as well as metabolic analysis under standard and high fat diet. Additionally, in direct islet studies we evaluated the role of NMB on basal and glucose-stimulated insulin secretion in mouse and humans.

category: physiology


title: Individual-based modelling of shelled pteropods

abstract: Shelled pteropods are cosmopolitan, free-swimming organisms of biogeochemical and commercial importance. They are widely used as sentinel species for the overall response of marine ecosystems to environmental stressors associated with climate change and changes in ocean chemistry. However, currently we are unable to project the effects of climate change on shelled pteropods at the population level, due to the missing spatio-temporal characterization of the response of pteropods to environmental stressors, and the limited information on the pteropod life history and life cycle. In this study, we implement a shelled pteropod Individual-Based Model (IBM), i.e. we simulate a pteropod population as a set of discrete individuals over several generations, life stages (eggs, larvae, juveniles and adults) and as a function of temperature, food availability and aragonite saturation state. The model is able to provide an abundance signal that is consistent with the abundance signal measured in the temperate region. In addition, the modeled life stage progression matches the reported size spectrum across the year, with two major spawning periods in spring and fall, and maturation in March and September. Furthermore, our IBM correctly predicts the abundance maxima of younger, smaller and potentially more susceptible life stages in spring and winter. Thus, our model provides a tool for advancing our understanding of the response of pteropod populations to future environmental changes.


title: Early detection of cerebrovascular pathology and protective antiviral immunity by MRI

abstract: Central nervous system (CNS) infections are a major cause of human morbidity and mortality worldwide. Even patients that survive CNS infections can have lasting neurological dysfunction resulting from immune and pathogen induced pathology. Developing approaches to noninvasively track pathology and immunity in the infected CNS is crucial for patient management and development of new therapeutics. Here, we develop novel MRI-based approaches to monitor virus-specific CD8+ T cells and their relationship to cerebrovascular pathology in the living brain. We studied a relevant murine model in which a neurotropic virus (vesicular stomatitis virus) was introduced intranasally and then entered the brain via olfactory sensory neurons - a route exploited by many pathogens in humans. Using T2*-weighted high-resolution MRI, we identified small cerebral microbleeds as the earliest form of pathology associated with viral entry into the brain. Mechanistically, these microbleeds occurred in the absence of peripheral immune cells and were associated with infection of vascular endothelial cells. We monitored the adaptive response to this infection by developing methods to iron label and track individual virus specific CD8+ T cells by MRI. Transferred antiviral T cells were detected in the brain within a day of infection and were able to reduce cerebral microbleeds. These data demonstrate the utility of MRI in detecting the earliest pathological events in the virally infected CNS as well as the therapeutic potential of antiviral T cells in mitigating this pathology.


title: The human frontal operculum is involved in visuomotor performance monitoring

abstract: For adaptive goal-directed action, the brain needs to monitor action performance and detect errors. The corresponding information may be conveyed via different sensory modalities; for instance, visual and proprioceptive body position cues may inform about current manual action performance. Thereby, contextual factors such as the current task set may also determine the relative importance of each sensory modality for action guidance. Here, we analysed human behavioral, fMRI, and MEG data from two VR-based hand-target phase matching studies to identify the neuronal correlates of performance monitoring and error processing under instructed visual or proprioceptive task sets. Our main result was a general, modality-independent response of the bilateral FO to poor phase matching accuracy, as evident from increased BOLD signal and increased gamma power. Furthermore, functional connectivity of the bilateral FO to the right PPC increased under a visual vs proprioceptive task set. These findings suggest that the bilateral FO generally monitors manual action performance; and, moreover, that when visual action feedback is used to guide action, the FO may signal an increased need for control to visuomotor regions in the right PPC following errors.


title: Use-dependent biases primarily originate from a contaminated motor plan

abstract: Repetition of a specific movement biases subsequent actions towards the recently practiced movement, a phenomenon referred to as use-dependent learning (UDL). UDL has been attributed to shifts in the tuning of neurons in the motor cortex. However, recent studies employing a forced reaction time task, including the eLife article by Marinovic et al (2017), indicate that these biases may also arise from a contaminated motor plan, one that is biased towards the practiced direction. We advanced this line of inquiry, seeking to establish the relative contribution of execution and planning processes to UDL in a center-out reaching task in which participants were able to initiate movements of their own volition. On most trials, the target appeared at a designated "frequent" location; on other trials, the target appeared at one of six "rare" locations. In Experiment 1, participants exhibited a robust movement bias towards the frequent target when movements were self-initiated quickly, but a small movement bias when movements were self-initiated slowly - the signature of a contaminated motor plan. Strikingly, the heading angles were bimodally distributed, with one peak at the frequent target location and the other at the rare target location - a finding reinforced by a re-analysis of two widely cited studies on UDL. Notably, the latter peak was shifted in the frequently practiced direction, a signature of a motor execution bias. To eliminate the contribution of planning-related UDL, we imposed a delay between target onset and movement initiation in Experiment 2. As predicted, the heading angles became unimodally distributed around the rare target. The peak of this distribution was again shifted towards the location of the frequent target, indicative of a persistent bias in motor execution. Taken together, these results highlight two distinct components of UDL even when movements are self-initiated: First, the temporal dynamics underlying movement planning, in which a default plan is progressively overridden by a new plan, produces a pronounced motor planning bias. Second, there is a small, temporally stable bias that may reflect shifts in motor unit tuning.


title: Pupillometry and electroencephalography in the digit span task

abstract: This dataset consists of raw 64-channel EEG, cardiovascular (electrocardiography and photoplethysmography), and pupillometry data from 86 human participants recorded during 4 minutes of eyes-closed resting and during performance of a classic working memory task - digit span task with serial recall. The participants either memorized or just listened to sequences of 5, 9, or 13 digits presented auditorily every 2 seconds. The dataset can be used for (1) developing algorithms for cognitive load discrimination and detection of cognitive overload; (2) studying neural (event-related potentials and brain oscillations) and peripheral (electrocardiography, photoplethysmography, and pupillometry) physiological signals during encoding and maintenance of each sequentially presented memory item; (3) correlating cognitive load and individual differences in working memory to neural and peripheral physiology, and studying the relationship between the physiological signals; (4) integration of the physiological findings with the vast knowledge coming from behavioral studies of verbal working memory in simple span paradigms. The data are shared in Brain Imaging Data Structure (BIDS) format and freely available on OpenNeuro (https://openneuro.org/datasets/ds003838).


title: Sex differences in the strength of selection under facultative sex

abstract: Facultative sex, the ability to reproduce both sexually and asexually, is widespread across the tree of life. In anisogamous species, the frequency of sex modulates selection on traits with sex-specific expression. Current theory on conditional gene expression posits that the strength of selection on loci only expressed by a subset of individuals, and/or in a subset of environments, is proportional to the frequency of expressers in the population. We show here that this assumption does not hold when the subsets in question are males or females (because of the Fisher condition) and is most important in facultatively sexual populations. In this case, the proportion of sexually produced offspring is not determined by male frequency (sex ratio), but on relative female investment in sexual versus asexual reproduction. This breaks the link between the frequency of expressers and selection for loci with sex-specific expression. However, certain conditions can re-establish this link, for example male traits that predict mating success better under strong male-male competition, and sex ratio affecting the relative fecundity of sexual versus asexual females. Our work highlights the importance of the Fisher condition for understanding the efficiency of selection and has implications for differences in the genetic load among sexes.

category: evolutionary biology


title: The natural history of the black soldier fly, Hermetia illucens: insights from complete mitochondrial genome sequences

abstract: The Black Soldier Fly (BSF) Hermetia illucens is a cosmopolitan fly heavily used by industrial companies to reduce biowaste and produce protein and fat for poultry and aquaculture feed. However, the natural history and the genetic diversity of the BSF is poorly known. In this study, we analysed 677 CO1 sequences derived from samples found all over the five continents leading us to the discovery of 52 haplotypes including 10 major haplotypes. We refined the definition of these haplotypes by sequencing 59 mitochondrial genomes. We could derive an estimate of the separation events of the different haplotypes at more than two million years for the oldest branches. This worldwide cryptic genetic and genomic diversity is mirrored at local scale in France in which we found five major haplotypes sometimes in sympatry. Our data resolve the phylogenetic relationships between the major lineages and give insights into the dispersal and the numbers of BSF neo-introduction at global and local scales. Our results indicate that the genetic and genomic diversity of commercial BSF stock is very low and these brood stock participated in the dissemination of the BSF in the wild. Taken together these results call for a better understanding of the genomic diversity of the BSF to unravel possible specific adaptations of the different lineages for industrial needs and to initiate the selection process.


title: Hybridisation and chloroplast capture between ancient Themeda triandra ecotypes in Australia

abstract: Ecotypes are distinct populations within a species which are adapted to specific environmental conditions. Understanding how these ecotypes become established, and how they interact when reunited, is fundamental to elucidating how ecological adaptations are maintained. This study focuses on Themeda triandra, a dominant grassland species across Asia, Africa and Australia. It is the most widespread plant in Australia, where it has distinct ecotypes that are usually restricted to either wetter and cooler coastal regions or the drier and hotter interior. We use whole genome sequencing for over 80 Themeda accessions to reconstruct the evolutionary history of T. triandra and related taxa. A chloroplast phylogeny confirms that Australia was colonised by T. triandra twice, with the division between ecotypes predating their arrival in Australia. The nuclear genome provides evidence of gene-flow among the ecotypes, largely restricted to two geographic areas. In northern Queensland there appears to be a hybrid zone with admixed nuclear genomes and shared plastid haplotypes. Conversely, in the cracking claypans of Western Australia, there is cytonuclear discordance with individuals possessing the coastal plastid and interior clade nuclear genomes. This chloroplast capture is potentially a result of adaptive introgression, with selection detected in the rpoC2 gene which is associated with water use efficiency. A stable hybrid zone in the east, and the displacement of one ecotype in the west, highlights the unpredictable nature of hybrid zones, with repeated contacts between the same ecotypes producing different outcomes.


title: A molecular switch between mammalian MLL complexes dictates response to Menin-MLL inhibition

abstract: The chromatin adaptor Menin interacts with oncogenic fusion proteins encoded by MLL1-rearrangements (MLL1-r), and small molecules that disrupt these associations are currently in clinical trials for the treatment of leukemia. By integrating chromatin-focused and genome-wide CRISPR screens with genetic, pharmacological, and biochemical approaches in mouse and human systems, we discovered a molecular switch between the MLL1-Menin and MLL3/4-UTX chromatin modifying complexes that dictates response to Menin-MLL inhibitors. We show that MLL1-Menin safeguards leukemia survival by impeding binding of the MLL3/4-UTX complex at a subset of target gene promoters. Disrupting the interaction between Menin and MLL1 leads to UTX-dependent transcriptional activation of a tumor suppressor gene-program that is crucial for a therapeutic response in murine and human leukemia. We establish the therapeutic relevance of this mechanism by showing that CDK4/6 inhibitors allow re-activation of this tumor-suppressor program in Menin-inhibitor insensitive leukemia cells, mitigating treatment resistance. The discovery of a molecular switch between MLL1-Menin and MLL3/4-UTX complexes on chromatin sheds light on novel functions of these evolutionary conserved epigenetic mediators and is particularly relevant to understand and target molecular pathways determining response and resistance in ongoing phase 1/2 clinical trials.


title: Molecular switch between mammalian MLL complexes dictates response to Menin-MLL inhibition


title: Within-generation and transgenerational social plasticity interact during rapid adaptive evolution

abstract:
O_LIThe role of within-generation phenotypic plasticity (WGP) versus transgenerational plasticity (TGP) during evolutionary adaptation are not well understood, particularly for socially-cued TGP.
C_LIO_LIWe tested how genetics, WGP, and TGP jointly influence expression of fitness traits facilitating adaptive evolution in the field cricket Teleogryllus oceanicus. A male-silencing mutation ("flatwing") spread to fixation in ca. 50 generations in a Hawaiian cricket population attacked by acoustically-orienting parasitoids. This rapid loss of song caused the social environment to dramatically change.
C_LIO_LIJuveniles carrying the flatwing (fw) genotype exhibited greater locomotive activity than those carrying the normal-wing (nw) allele, consistent with genetic coupling of increased locomotion with fw.
C_LIO_LIConsistent with adaptive WGP, homozygous fw females developing in the absence of song showed reduced body condition and reproductive investment at adulthood.
C_LIO_LIAdult but not juvenile offspring exhibited TGP in response to maternal social environment for structural size, somatic condition, and reproductive investment, whereas adult locomotion and flight was only influenced by WGP. WGP and TGP interacted to shape multiple traits at adulthood, though effect sizes were modest.
C_LIO_LIInteractions between genetic effects and social plasticity within and across generations are likely to have influenced the evolutionary spread of flatwing crickets. However, interactions among these effects can be complex, and it is notable that TGP manifested most strongly later in development. Our findings stress the importance of evaluating trait plasticity at different developmental stages and across generations when studying phenotypic plasticitys role in evolution.
C_LI


title: Novel algorithms deriving clinical performance measures from smartphone sensor data collected under a walking test

abstract: The modern ubiquity of smartphones which incorporate sensors such as inertial measurement units (IMU) and global positioning system (GPS) receivers, raises the possibility that they might be used as inexpensive devices for clinical performance testing. Here, we report on the development and testing of an app and associated statistical signal processing algorithms, to measure the basic gait properties of step rate, walking speed and distance under an outside short walk test experimental protocol, from digital accelerometry and GPS. We provide extensive details on the assumptions underlying the development of the algorithms. A small set of experiments across a range of conditions and individuals, demonstrate the reliability of the combined app, protocol and algorithms. With reasonable adherence to the test protocol, these experiments show 2% error across all metrics. The emphasis in this paper is on illustrating the design principles which all such systems, aiming to re-purpose consumer smartphones as clinical gait measurement devices, must address.

category: bioengineering


title: Morphology and gene expression profiling provide complementary information for mapping cell state

abstract: Deep profiling of cell states can provide a broad picture of biological changes that occur in disease, mutation, or in response to drug or chemical treatments. Morphological and gene expression profiling, for example, can cost-effectively capture thousands of features in thousands of samples across perturbations, but it is unclear to what extent the two modalities capture overlapping versus complementary mechanistic information. Here, using both the L1000 and Cell Painting assays to profile gene expression and cell morphology, respectively, we perturb A549 lung cancer cells with 1,327 small molecules from the Drug Repurposing Hub across six doses. We determine that the two assays capture some shared and some complementary information in mapping cell state. We find that as compared to L1000, Cell Painting captures a higher proportion of reproducible compounds and has more diverse samples, but measures fewer distinct groups of features. In an unsupervised analysis, Cell Painting grouped more compound mechanisms of action (MOA) whereas in a supervised deep learning analysis, L1000 predicted more MOAs. In general, the two assays together provide a complementary view of drug mechanisms for follow up analyses. Our analyses answer fundamental biological questions comparing the two biological modalities and, given the numerous applications of profiling in biology, provide guidance for planning experiments that profile cells for detecting distinct cell types, disease phenotypes, and response to chemical or genetic perturbations.


title: The SAMBA tool uses long reads to improve the contiguity of genome assemblies

abstract: Third-generation sequencing technologies can generate very long reads with relatively high error rates. The lengths of the reads, which sometimes exceed one million bases, make them invaluable for resolving complex repeats that cannot be assembled using shorter reads. Many high-quality genome assemblies have already been produced, curated, and annotated using the previous generation of sequencing data, and full re-assembly of these genomes with long reads is not always practical or cost-effective. One strategy to upgrade existing assemblies is to generate additional coverage using long-read data, and add that to the previously assembled contigs. SAMBA is a tool that is designed to scaffold and gap-fill existing genome assemblies with additional long-read data, resulting in substantially greater contiguity. SAMBA is the only tool of its kind that also computes and fills in the sequence for all spanned gaps in the scaffolds, yielding much longer contigs. Here we compare SAMBA to several similar tools capable of re-scaffolding assemblies using long-read data, and we show that SAMBA yields better contiguity and introduces fewer errors than competing methods. SAMBA is open-source software that is distributed at https://github.com/alekseyzimin/masurca.


title: Formulation of Dry Powders of Vaccines Containing MF59 or AddaVax by Thin-Film Freeze-Drying

abstract: Oil-in-water (O/W) nanoemulsion-based vaccine adjuvants such as MF59(R) are often used in seasonal and pandemic influenza vaccines. However, vaccines containing nanoemulsions require cold chain for storage and are sensitive to accidental freezing. We explored the feasibility of developing dry powders of vaccines adjuvanted with MF59 or AddaVax, a preclinical grade nanoemulsion that has the same composition and droplet size as MF59, by thin-film freeze-drying (TFFD). AddaVax alone was successfully converted from a liquid to dry powders by TFFD using trehalose as a stabilizing agent while maintaining the droplet size distribution of the AddaVax when reconstituted, whereas subjecting the same AddaVax composition to conventional shelf freeze-drying led to significant aggregation or fusion. TFFD was then applied to convert liquid AddaVax-adjuvanted vaccines containing either model antigens such as ovalbumin and lysozyme, mono-, bi-, and tri-valent recombinant hemagglutinin (rHA) protein-based H1 and/or H3 (universal) influenza vaccine candidates, as well as the MF59-containing Fluad(R) Quadrivalent influenza vaccine to dry powders. Antigens, stabilizing agents, and buffer showed different effects on the physical properties of the vaccines (e.g., mean particle size and particle size distribution) after subjected to TFFD, but the integrity and hemagglutination activity of the rHA antigens did not significantly change and the immunogenicity of reconstituted influenza vaccine candidates was preserved when evaluated in BALB/c mice. The vaccine dry powder was not sensititve to repeated freezing-and-thawing, in contrast to its liquid counterpart. It is concluded that TFFD can be applied to convert vaccines containing MF59 or an nanoemulsion with the same composition and droplet size as MF59 from liquid to dry powders while maintaining the immunogencity of the vaccines, and it may be used to prepare dry powders of multivalent universal influenza vaccines.

category: pharmacology and toxicology


title: Proteomic analysis of mouse kidney tissue associates peroxisomal dysfunction with early diabetic kidney disease

abstract:
BackgroundThe absence of efficient inhibitors for DKD progression reflects the gaps in our understanding of DKD molecular pathogenesis. A comprehensive proteomic analysis was performed on glomeruli and kidney cortex of diabetic mice with subsequent validation of findings in human biopsies and - omics datasets aiming to better understand the underlying molecular biology of early DKD development and progression.

MethodsLC-MS/MS was employed to analyze the kidney proteome of DKD mouse models: Glomeruli of Ins2Akita mice 2 month and 4 month old, and cortex of db/db mice 6 month old. Following label-free quantification, the abundance of detected proteins were correlated with existing kidney datasets and functionally annotated. Tissue sections from 16 DKD patients were analyzed by IHC.

ResultsPathway analysis of differentially expressed proteins in the early and late DKD versus controls predicted dysregulation in DKD hallmarks (such as peroxisomal lipid metabolism, {beta}-oxidation and TCA cycle) supporting the functional relevance of the findings. Comparing the observed protein changes in early and late DKD, consistent upregulation of 21 and downregulation of 18 proteins was detected. Among these were downregulated peroxisomal proteins such as NUDT19, ACOX1, and AMACR and upregulated mitochondrial proteins related to aminoacid metabolism including GLS, GLDC, and GCAT. Several of these changes were also observed in the kidney cortex proteome of db/db mice. IHC of human kidney further confirmed the differential expression of NUDT19, AGPS, AMACR and CAT proteins in DKD.

ConclusionsOur study shows an extensive differential expression of peroxisomal proteins in the early stages of DKD that persists regardless of the disease severity. These proteins therefore represent potential markers of early DKD pathogenesis. Collectively, essential pathways associated with peroxisomes such as lipid {beta}-oxidation, plasmalogen synthesis, aminoacid metabolism and response to oxidative stress are downregulated in early DKD, providing new perspectives and potential markers of diabetic kidney dysfunction.


title: High-frequency hearing is required for generating a topographic map of auditory space in the mouse superior colliculus

abstract:
A topographic map of auditory space is a feature found in the superior colliculus (SC) of many species, including CBA/CaJ mice. In this genetic background, high-frequency monaural spectral cues and interaural level differences are used to generate spatial receptive fields (RFs) that form a topographic map along the azimuth. Unfortunately, C57BL/6 mice, a strain widely used for transgenic manipulation, display age-related hearing loss (AHL) due to an inbred mutation in the Cadherin 23 gene (Cdh23) that affects hair cell mechanotransduction. To overcome this problem, researchers have used young C57BL/6 mice in their studies, as they have been shown to have normal hearing thresholds. However, important details of the auditory response characteristics of the SC such as spectral responses and spatial localization, have not been characterized in young C57BL/6 mice.

Here we show that 2-4-month C57BL/6 mice lack neurons with frontal auditory RFs and therefore lack a topographic representation of auditory space in the SC. Analysis of the spectrotemporal receptive fields (STRFs) of the SC auditory neurons shows that C57BL/6 mouse SC neurons lack the ability to detect the high-frequency (>40kHz) spectral cues that are needed to compute frontal RFs. We also show that crossing C57BL/6 mice with CBA/CaJ mice or introducing one copy of the wild-type Cdh23 to C57BL/6 mice rescues the high-frequency hearing deficit and improves the topographic map of auditory space. Taken together, these results demonstrate the importance of high-frequency hearing in computing a topographic map of auditory space.

Significance StatementDespite the strains age-dependent hearing loss, C57BL/6 mice are widely used in auditory studies because of the development of transgenic reporter and Cre lines in this genetic background. Here we examined the topographic map of auditory space and spectrotemporal properties of neurons in the SC of C57BL/6 mice. We found an early-onset high-frequency hearing deficit that results in the loss of SC neurons with frontal RFs and, consequently, an absence of a topographic map of auditory space. These findings stress the importance of high-frequency hearing in generating spatially restricted receptive fields and serve as a caution to researchers that doing auditory-related research using the C57BL/6 genetic background may not be representative of true wild-type mice.


title: De novo motor learning of a bimanual control task over multiple days of practice

abstract: Although much research on motor learning has focused on how we adapt our movements to maintain performance in the face of imposed perturbations, in many cases we must learn new skills from scratch, or de novo. In comparison to adaptation, relatively little is known about de novo learning. In part, this is because learning a new skill can involve many challenges, including learning to recognize new patterns of sensory input and generate new patterns of motor output. However, even with familiar sensory cues and well-practiced movements, the problem of quickly selecting the appropriate actions in response to the current state is challenging. Here, we devised a bimanual hand-to-cursor mapping which isolates this control problem. We find that participants initially struggled to control the cursor under this bimanual mapping, despite explicit knowledge of the mapping. Performance improved steadily over multiple days of practice, however. Participants exhibited no aftereffects when reverting to a veridical cursor, confirming that participants learned the new task de novo, rather than through adaptation. Corrective responses to mid-movement perturbations of the target were initially weak, but, with practice participants gradually became able to respond rapidly and robustly to these perturbations. After four days of practice, participants behavior under the bimanual mapping almost matched performance with a veridically mapped cursor. However, there remained a small but persistent difference in performance level. Our findings illustrate the dynamics and limitations of learning a novel controller and introduce a promising paradigm for tractably investigating this aspect of motor skill learning.


title: Nasal prevention of SARS-CoV-2 infection by intranasal influenza-based boost vaccination

abstract:
BackgroundVaccines in emergency use are efficacious against COVID-19, yet vaccine-induced prevention against nasal SARS-CoV-2 infection remains suboptimal.

MethodsSince mucosal immunity is critical for nasal prevention, we investigated an intramuscular PD1-based receptor-binding domain (RBD) DNA vaccine (PD1-RBD-DNA) and intranasal live attenuated influenza-based vaccines (LAIV-CA4-RBD and LAIV-HK68-RBD) against SARS-CoV-2.

FindingsSubstantially higher systemic and mucosal immune responses, including bronchoalveolar lavage IgA/IgG and lung polyfunctional memory CD8 T cells, were induced by the heterologous PD1-RBD-DNA/LAIV-HK68-RBD as compared with other regimens. When vaccinated animals were challenged at the memory phase, prevention of robust SARS-CoV-2 infection in nasal turbinate was achieved primarily by the heterologous regimen besides consistent protection in lungs. The regimen-induced antibodies cross-neutralized variants of concerns. Furthermore, LAIV-CA4-RBD could boost the BioNTech vaccine for improved mucosal immunity.

InterpretationOur results demonstrated that intranasal influenza-based boost vaccination is required for inducing mucosal and systemic immunity for effective SARS-CoV-2 prevention in both upper and lower respiratory systems.

FundingThis study was supported by the Research Grants Council Collaborative Research Fund (C7156-20G, C1134-20G and C5110-20G), General Research Fund (17107019) and Health and Medical Research Fund (19181052 and 19181012) in Hong Kong; Outbreak Response to Novel Coronavirus (COVID-19) by the Coalition for Epidemic Preparedness Innovations; Shenzhen Science and Technology Program (JSGG20200225151410198); the Health@InnoHK, Innovation and Technology Commission of Hong Kong; and National Program on Key Research Project of China (2020YFC0860600, 2020YFA0707500 and 2020YFA0707504); and donations from the Friends of Hope Education Fund. Z.C.s team was also partly supported by the Theme-Based Research Scheme (T11-706/18-N).

category: immunology


title: Gramicidin S and Melittin - Potential anti-viral therapeutic peptides to treat SARS-CoV-2 infection

abstract: The COVID19 pandemic has resulted in multipronged approaches for treatment of the disease. Since de novo discovery of drugs is time consuming, repurposing of molecules is now considered as one of the alternative strategies to treat COVID19. Antibacterial peptides are being recognized as attractive candidates for repurposing to treat viral infections. In this study, we describe the anti-SARS-CoV-2 activity of gramicidin S and melittin peptides obtained from Bacillus brevis and bee venom respectively. Our in vitro antiviral assay results showed significant decrease in the viral load compared to the untreated group with no/very less cytotoxicity. The EC50 values for gramicidin S and melittin are calculated as 1.571g and 0.656g respectively. Both the peptides treated to the SARS-CoV-2 infected Vero cells showed viral clearance from 12 hours onwards with a maximal clearance after 24 hours post infection. Based on proteome analysis it was found that more than 250 proteins were found to be differentially regulated in the gramicidin S and melittin treated SARS-CoV-2 infected Vero cells against control SARS-CoV-2 infected Vero cells after 24 and 48 hours post infection. The identified proteins were found to be associated in the metabolic and mRNA processing of the Vero cells post-treatment and infection. Both these peptides could be attractive candidates for repurposing to treat SARS-CoV-2 infection.


title: Autopolyploid establishment depends on life history strategy and the mating outcomes of clonal architecture

abstract: Polyploidy is a significant component in the evolution of many taxa, particularly plant groups. However, the mechanisms promoting or preventing initial polyploid establishment in natural populations are often unclear. We develop spatially explicit agent-based simulation models to explore how perennial life history and clonal propagation influence the early stages of polyploid establishment. Our models show that polyploid establishment is unlikely among short-lived plants. Polyploids have increased establishment probability when both diploid and polyploid lifespans are long, especially when unreduced gamete production is non-zero. Further, polyploids that combine sexual and clonal reproduction can establish across a wide range of life history strategies. Polyploid genets containing many, far spreading ramets are most successful, but genets with tightly clumped ramets have similar establishment probability when pollen dispersal is local and rates of self-fertilization are high. Clonal architecture has a substantial impact on the spatial structure of the mixed cytotype population during establishment; altering patterns of mating within or between cytotypes, the mechanisms through which polyploid establishment proceeds, and the final composition of the polyploid population after successful establishment. Overall, our findings provide insight into the complex relationship between polyploidy, perenniality, and clonal reproduction, and offer testable predictions for future empirical work.


title: Total evidence tip-dating phylogeny of platyrrhine primates and 27 well-justified fossil calibrations for primate divergences

abstract: Phylogenies with estimates of divergence times are essential for investigating many evolutionary questions. In principle, "tip-dating" is arguably the most appropriate approach, with fossil and extant taxa analyzed together in a single analysis, and topology and divergence times estimated simultaneously. However, "node-dating" (as used in many molecular clock analyses), in which fossil evidence is used to calibrate the age of particular nodes a priori, will probably remain the dominant approach, due to various issues with analyzing morphological and molecular data together. Tip-dating may nevertheless play a key role in robustly identifying fossil taxa that can be used to inform node-dating calibrations. Here, we present tip-dating analyses of platyrrhine primates (so-called "New World monkeys") based on a total evidence dataset of 418 morphological characters and 10.2 kb of DNA sequence data from 17 nuclear genes, combined from previous studies. The resultant analyses support a late Oligocene or early Miocene age for crown Platyrrhini (composite age estimate: 20.7-28.2 Ma). Other key findings include placement of the early Miocene putative cebid Panamacebus outside crown Platyrrhini, equivocal support for Proteropithecia being a pitheciine, and support for a clade comprising three subfossil platyrrhines from the Caribbean (Xenothrix, Antillothrix and Paralouatta), related to Callicebinae. Based on these results and the available literature, we provide a list of 27 well-justified node calibrations for primate divergences, following best practices: 17 within Haplorhini, five within Strepsirrhini, one for crown Primates, and four for deeper divergences within Euarchontoglires. In each case, we provide a hard minimum bound, and for 23 of these we also provide a soft maximum bound and a suggested prior distribution. For each calibrated node, we provide the age of the oldest fossil of each daughter lineage that descends from it, which allows use of the "CladeAge" method for specifying priors on node ages.


title: Underlying selection for the diversity of Spike protein sequences of SARS-CoV-2

abstract: The global spread of SARS-CoV-2 is fast moving and has caused a worldwide public health crisis. In the present manuscript we analyzed spike protein sequences of SARS-CoV-2 genomes to assess the impact of mutational diversity. We observed from amino acid usage patterns that spike proteins are associated with a diversity of mutational changes and most important underlying cause of variation of amino acid usage is the changes in hydrophobicity of spike proteins. The changing patterns of hydrophobicity of spike proteins over time and its influence on the receptor binding affinity provides crucial information on the SARS-CoV-2 interaction with human receptor. Our results also show that spike proteins have evolved to prefer more hydrophobic residues over time. The present study provides a comprehensive analysis of molecular sequence data to consider that mutational variants might play a crucial role in modulating the virulence and spread of the virus and has immediate implications for therapeutic strategies.


title: Molecular Insights into the Differential Dynamics of SARS-CoV-2 Variants of Concern (VOCs)

abstract: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected the lives and livelihood of millions of individuals around the world. It has mutated several times after its first inception, with an estimated two mutations occurring every month. Although we have been successful in developing vaccines against the virus, emergence of variants has enabled it to escape therapy. Few of the generated variants are also reported to be more infectious than the wild-type (WT). In this study, we analyze the attributes of all RBD/ACE2 complexes for the reported VOCs, namely, Alpha, Beta, Gamma, and Delta through computer simulations. Results indicate differences in orientation and binding energies of the VOCs from the WT. Overall, it was observed that electrostatic interactions play a major role in the binding of the complexes. Detailed residue level energetics revealed that the most prominent changes in interaction energies were seen particularly at the mutated residues which were present at RBD/ACE2 interface. We found that the Delta variant is one of the most tightly bound variants of SARS-CoV-2 with dynamics similar to WT. High binding affinity of RBD towards ACE2 is indicative of an increase in the viral transmission and infectivity. The details presented in our study would prove extremely useful for the design and development of effective therapeutic strategies for the emerging variants of the virus.


title: A model that predicts resistant starch in a dog kibble using a small-scale twin-screw extruder

abstract: The objective of this work was to modify extrusion parameters to yield greater resistant starch (RS) in a kibble and create a model to predict its concentration. A dog food was extruded through a small-scale twin-screw extruder as a central composite design with 6 central points (replicates) and 14 single replicates. There were three factors tested at three levels: corn particle size, extruder shaft speed, and in-barrel moisture (IBM). The remaining processing inputs were kept constant. Chemical and physical starch analyses were performed. A model to predict RS was created using the REG procedure from SAS. Pearson correlations between extrusion parameters and starch analyses were conducted. A model to predict RS was created (R2adj= 0.834; P < .0001). Both SME and extrudate temperature had a high negative correlation with RS and RVA raw starch. Results suggest that low mechanical energy and high IBM increase kibble RS.


title: Non-glycosylated Seipin to Cause a Motor Neuron Disease Induces ER stress and Apoptosis by Inactivating the ER Calcium Pump SERCA2b

abstract: A causal relationship between endoplasmic reticulum (ER) stress and the development of neurodegenerative diseases remains controversial. Here, we focused on Seipinopathy, a dominant motor neuron disease, based on the finding that its causal gene product, Seipin, is a hairpin-like transmembrane protein in the ER. Gain-of-function mutations of Seipin produce non-glycosylated Seipin (ngSeipin), which was previously shown to induce ER stress and apoptosis at both cell and mouse levels albeit with no clarified mechanism. We found that aggregation-prone ngSeipin dominantly inactivated SERCA2b, the major calcium pump in the ER, and decreased the calcium concentration in the ER, leading to ER stress and apoptosis. This inactivation required oligomerization of ngSeipin and direct interaction of the ngSeipin C-terminus with SERCA2b, and was observed in Seipin-deficient human colorectal carcinoma-derived cells (HCT116) expressing ngSeipin at a level comparable with that in neuroblastoma cells (SH-SY5T). Our results thus provide a new direction to controversy noted above.


title: Hingepoint emergence in mammalian spinal neurulation

abstract: Neurulation is the process in early vertebrate embryonic development during which the neural plate folds to form the neural tube. Spinal neural tube folding in the posterior neuropore changes over time, first showing a median hingepoint, then both the median hingepoint and dorsolateral hingepoints, followed by dorsolateral hingepoints only. The biomechanical mechanism of hingepoint formation in the mammalian neural tube is poorly understood. Here, we employ a mechanical finite element model to study neural tube formation. The computational model mimics the mammalian neural tube using microscopy data from mouse and human embryos. While intrinsic curvature at the neural plate midline has been hypothesized to drive neural tube folding, intrinsic curvature was not sufficient for tube closure in our simulations. We achieved neural tube closure with an alternative model combining mesoderm expansion, non-neural ectoderm expansion and neural plate adhesion to the notochord. Dorsolateral hingepoints emerged in simulations with low mesoderm expansion and zippering. We propose that zippering provides the biomechanical force for dorsolateral hingepoint formation in settings where the neural plate lateral sides extend above the mesoderm. Together, these results provide a new perspective on the biomechanical and molecular mechanism of mammalian spinal neurulation.


title: Building new compartments for unconventional protein secretion from the early and late Golgi membranes

abstract: CUPS, a compartment for unconventional secretion of signal sequence lacking proteins, is built during starvation. CUPS, lacking the Golgi specific glycosyltransferases, form by COPI independent extraction of membranes from the early Golgi cisterna, require PI4P for their biogenesis and PI3P for stability. We now show that a PI4P effector Drs2 of the trans-Golgi network, relocates to a new compartment monikered TCUPS because it touches CUPS. Although localized to TCUPS, Drs2 is required for CUPS formation specifically by interacting with Rcy1, and this process is essential for unconventional secretion. Visualizing cells by 4D SCLIM technology revealed that tubules emanating from TCUPS are often collared by CUPS and severed. Incidentally, while CUPS are stable, TCUPS are vesiculated at late stages of starvation. This mirrors the dynamics of the early and late Golgi during conventional protein secretion. TCUPS and CUPS thus emerge as the functional equivalent of early and late Golgi of the conventional secretory pathway, thus representing key compartments in unconventional secretion.


title: Single-particle tracking of dynein identifies PP2A B55/SUR-6 as a cell cycle regulator of cortical force generation

abstract: Convergence and positioning of the pronuclei and mitotic spindle of many zygotes aids efficient division and is essential for early embryonic patterning. In the C. elegans zygote, interactions between microtubules and cortically anchored dynein are key to early development. However, how cortical microtubule pulling forces are controlled through the cell cycle is less well understood. We used single-molecule imaging and a windowed mean squared displacement analysis to uncover the behavior of dynein during cortical force generation, and provide a regulatory role for protein phosphatase PP2A-B55/SUR6 via NuMA-like protein LIN-5 in this process. Previous findings and our results suggest that PP2A regulates cortical microtubule pulling forces by increasing dynein binding and unbinding to the cortical force generation complex. Our data also suggests that cortical occupancy of dynein is abrogated to vary force generation. Our approach will be broadly applicable to classify the force generation behavior of single molecules in living organisms.


title: The Rpd3-complex regulates expression of multiple cell surface recycling factors in yeast

abstract: Intracellular trafficking pathways control residency and bioactivity of integral membrane proteins at the cell surface. Upon internalisation, surface cargo proteins can be delivered back to the plasma membrane via endosomal recycling pathways. Recycling is thought to be controlled at the metabolic and transcriptional level, but such mechanisms are not fully understood. In yeast, recycling of surface proteins can be triggered by cargo deubiquitination and a series of molecular factors have been implicated in this trafficking. In this study, we follow up on the observation that many subunits of the Rpd3 lysine deacetylase complex are required for recycling. We validate ten Rpd3-complex subunits in recycling using two distinct assays and developed tools to quantify both. Fluorescently labelled Rpd3 localises to the nucleus and complements recycling defects, which we hypothesised were mediated by modulated expression of Rpd3 target gene(s). Bioinformatics implicated 32 candidates that function downstream of Rpd3, which were over-expressed and assessed for capacity to suppress recycling defects of rpd3{Delta} cells. This effort yielded 3 hits: Sit4, Dit1 and Ldb7, which were validated with a lipid dye recycling assay. Additionally, the essential phosphatidylinositol-4-kinase Pik1 was shown to have a role in recycling. We propose recycling is governed by Rpd3 at the transcriptional level via multiple downstream target genes.


title: Trap-TRAP, a versatile tool for tissue-specific translatomics in zebrafish

abstract: Developmental and physiological processes depend on the transcriptional and translational activity of heterogeneous cell populations. A main challenge in gene expression studies is dealing with this intrinsic complexity while keeping sequencing efficiency. Translating ribosome affinity purification (TRAP) methods have allowed cell-specific recovery of polyribosome-associated RNAs by genetic tagging of ribosomes in selected cell populations. Here we combined the TRAP approach with adapted enhancer trap methods (trap-TRAP) to systematically generate zebrafish transgenic lines suitable for tissue-specific translatome interrogation. Through the random integration of the eGFP:rpl10a cassette, we have generated stable lines driving expression in a variety of tissues, including the retina, skeletal muscle, lateral line primordia, rhombomeres, or jaws. To increase the range of applications, a UAS:TRAP transgenic line compatible with available Gal4 lines was also generated and tested. The resulting collection of lines and applications constitutes a resource for the zebrafish community in developmental genetics, organ physiology and disease modelling.


title: The post-transcriptional regulation of TFs in immature motoneurons shapes the axon-muscle connectome

abstract: Temporal factors expressed sequentially in neural stem cells, such as RNA binding proteins (RBPs) or transcription factors (TFs), are key elements in the generation of neuronal diversity. The molecular mechanism underlying how the temporal identity of stem cells is decoded into their progeny to generate neuronal diversity is largely unknown. Here, we used genetic and new computational tools to study with precision the unique fates of the progeny of a stem cell producing 29 morphologically distinct leg motoneurons (MNs) in Drosophila. We identified 40 TFs expressed in this MN lineage, 15 of which are expressed in a combinatorial manner in immature MNs just before their morphological differentiation. By following TF expression patterns at an earlier developmental stages, we discovered 19 combinatorial codes of TFs that were progressively established in immature MNs as a function of their birth order. The comparison of the RNA and protein expression profiles of 6 TFs revealed that post-transcriptional regulation plays an essential role in shaping these TF codes. We found that the two known RBPs, Imp and Syp, expressed sequentially in neuronal stem cells, are upstream regulators of the TF codes. Both RBPs are key players in the construction of axon-muscle connectome through the post-transcriptional regulation of 5 of the 6 TFs examined. By deciphering the function of Imp in the immature MNs with respect to the stem cell of the same lineage, we propose a model where RBPs shape the morphological fates of MNs through post-transcriptional regulation of TF codes in immature MNs. Taken together, our study reveals that immature MNs are plastic cells that have the potential to acquire many morphological fates. The molecular basis of MN plasticity originates in the broad expression of different TF mRNA, that are post-transcriptionally shaped into TF codes by Imp and Syp, and potentially by other RBPs that remain to be discovered, to determine their morphological fates.


title: Innexin function dictates the spatial relationship between distal somatic cells in the Caenorhabditis elegans gonad without impacting the germline stem cell pool

abstract: Gap-junctional signaling mediates myriad cellular interactions in metazoans. Yet, how gap junctions control the positioning of cells in organs is not well understood. Innexins compose gap junctions in invertebrates and affect organ architecture. Here, we investigate the roles of gap-junctions in controlling distal somatic gonad architecture and its relationship to underlying germline stem cells in Caenorhabditis elegans. We show that a reduction of soma-germline gap-junctional activity causes displacement of distal sheath cells (Sh1) towards the distal end of the gonad. We confirm, by live imaging, transmission electron microscopy, and antibody staining, that bare regions--lacking somatic gonadal cell coverage of germ cells--are present between the distal tip cell (DTC) and Sh1, and we show that an innexin fusion protein used in a prior study encodes an antimorphic gap junction subunit that mispositions Sh1. We determine that, contrary to the model put forth in the prior study based on this fusion protein, Sh1 mispositioning does not markedly alter the position of the borders of the stem cell pool nor of the progenitor cell pool. Together, these results demonstrate that gap junctions can control the position of Sh1, but that Sh1 position is neither relevant for GLP-1/Notch signaling nor for the exit of germ cells from the stem cell pool.


title: Coordinated DNA and Histone Dynamics Drive Accurate Histone H2A.Z Exchange

abstract:
Nucleosomal histone H2A is exchanged for its variant H2A.Z by the SWR1 chromatin remodeler, but the mechanism and timing of histone exchange remain unclear. Here, we quantify DNA and histone dynamics during histone exchange in real-time using a three-color single-molecule FRET assay. We show that SWR1 operates with timed precision to unwrap DNA with large displacement from one face of the nucleosome, remove H2A-H2B from the same face, and rewrap DNA, all within 2.3 seconds. Such productive DNA unwrapping requires full SWR1 activation and differs from unproductive, smaller-scale DNA unwrapping caused by SWR1 binding alone. On an asymmetrically positioned nucleosome, SWR1 intrinsically senses long-linker DNA to preferentially exchange H2A.Z on the distal face as observed in vivo. The displaced H2A-H2B dimer remains briefly associated with the SWR1-nucleosome complex and is dissociated by histone chaperones. These findings reveal how SWR1 coordinates DNA unwrapping with histone dynamics to rapidly and accurately place H2A.Z at physiological sites on chromatin.

One-Sentence SummaryMulticolor single-molecule FRET reveals how SWR1 unwraps DNA to exchange nucleosomal H2A-H2B for H2A.Z-H2B.


title: MutS and DNA Function as a Clamp Loader for the MutL Sliding Clamp During Mismatch Repair

abstract: DNA mismatch repair (MMR) is accomplished by highly conserved MutS and MutL homologs. MutS proteins recognize mismatch nucleotides and in the presence of ATP form a stable sliding clamp on the DNA. The MutS sliding clamp then promotes the cascade assembly of a MutL sliding clamp, which ultimately coordinates downstream mismatch excision. The MutS clamp-loader mechanics are unknown. Here we have examined a conserved positively charged cleft (PCC) located on the MutL N-terminal domain (NTD) proposed to mediate stable DNA binding events in several MMR models. We show that MutL does not bind DNA in physiological ionic conditions. Instead, the MutS sliding clamps and DNA together exploit the PCC to position the MutL NTD for clamp loading. Once in a sliding clamp form, the MutL PCC aids in UvrD helicase capture but not interactions with MutH during mismatch excision. The MutS-DNA clamp-loader progressions are significantly different from the replication clamp-loaders that attach polymerase processivity factors such as {beta}-clamp and PCNA to the DNA. These studies underlining the breadth of mechanisms for stably linking crucial genome maintenance proteins to the DNA.