title: Differential adhesion regulates neurite placement via a retrograde zippering mechanism

abstract: During development, neurites and synapses segregate into specific neighborhoods or layers within nerve bundles. The developmental programs guiding placement of neurites in specific layers, and hence their incorporation into specific circuits, are not well understood. We implement novel imaging methods and quantitative models to document the embryonic development of the C. elegans brain neuropil, and discover that differential adhesion mechanisms control precise placement of single neurites onto specific layers. Differential adhesion is orchestrated via developmentally-regulated expression of the IgCAM SYG-1, and its partner ligand SYG-2. Changes in SYG-1 expression across neuropil layers result in changes in adhesive forces, which sort SYG-2-expressing neurons. Sorting to layers occurs, not via outgrowth from the neurite tip, but via an alternate mechanism of retrograde zippering, involving interactions between neurite shafts. Our study indicates that biophysical principles from differential adhesion govern neurite placement and synaptic specificity in vivo in developing neuropil bundles.


title: ORP1L regulates dynein clustering on endolysosmal membranes in response to cholesterol levels

abstract: The sub-cellular positioning of endolysosomes is crucial for regulating their function. Particularly, the positioning of endolysosomes between the cell periphery versus the peri-nuclear region impacts autophagy, mTOR (mechanistic target of rapamycin) signaling and other processes. The mechanisms that regulate the positioning of endolysosomes at these two locations are still being uncovered. Here, using super-resolution microscopy, we show that the retrograde motor dynein forms nano-clusters on endolysosomal membranes containing 1-2 dyneins, with an average of ~3 nanoclusters per endolysosome. These data suggest that a very small number of dynein motors (1-6) drive endolysosome motility. Surprisingly, dynein nano-clusters are slightly larger on peripheral endolysosomes having higher cholesterol levels compared to peri-nuclear ones. By perturbing endolysosomal membrane cholesterol levels, we show that dynein copy number within nano-clusters is influenced by the amount of endolysosomal cholesterol while the total number of nano-clusters per endolysosome is independent of cholesterol. Finally, we show that the dynein adapter protein ORP1L (Oxysterol Binding Protein Homologue) regulates the number of dynein motors within nano-clusters in response to cholesterol levels. We propose a new model by which endolysosomal transport and positioning is influenced by the cholesterol sensing adapter protein ORP1L, which influences dyneins copy number within nano-clusters.

category: cell biology


title: Quantifying the dynamics of viral recombination during free virus and cell-to-cell transmission in HIV-1 infection

abstract: Recombination has been shown to contribute to HIV-1 evolution in vivo, but the underlying dynamics are extremely complex, depending on the nature of the fitness landscapes and of epistatic interactions. A less well-studied determinant of recombinant evolution is the mode of virus transmission in the cell population. HIV-1 can spread by free virus transmission, resulting largely in singly infected cells, and also by direct cell-to-cell transmission, resulting in the simultaneous infection of cells with multiple viruses. We investigate the contribution of these two transmission pathways to recombinant evolution, by applying mathematical models to in vitro experimental data on the growth of fluorescent reporter viruses under static conditions (where both transmission pathways operate), and under gentle shaking conditions, where cell-to-cell transmission is largely inhibited. The parameterized mathematical models are then used to extrapolate the viral evolutionary dynamics beyond the experimental settings. Assuming a fixed basic reproductive ratio of the virus (independent of transmission pathway), we find that recombinant evolution is fastest if virus spread is driven only by cell-to-cell transmission, and slows down if both transmission pathways operate. Recombinant evolution is slowest if all virus spread occurs through free virus transmission. This is due to cell-to-cell transmission (i) increasing infection multiplicity, (ii) promoting the co-transmission of different virus strains from cell to cell, and (iii) increasing the rate at which point mutations are generated as a result of more reverse transcription events. This work further resulted in the estimation of various parameters that characterize these evolutionary processes. For example, we estimate that during cell-to-cell transmission, an average of 3 viruses successfully integrated into the target cell, which can significantly raise the infection multiplicity compared to free virus transmission. In general, our study points towards the importance of infection multiplicity and cell-to-cell transmission for HIV-evolution.

category: evolutionary biology


title: Blocking the Rac1-TRPC5 pathway protects human podocytes

abstract: Podocyte injury and the appearance of proteinuria are key features of several progressive kidney diseases. Genetic deletion or selective inhibition of TRPC5 channels with small-molecule inhibitors protects podocytes in rodent models of disease, but less is known about the human relevance and translatability of TRPC5 inhibition. Here, we investigate the effect of TRPC5 inhibition in puromycin aminonucleoside (PAN)-treated human iPSC-derived podocytes and kidney organoids. We first established that systemic administration of the TRPC5-specific blocker AC1903 was sufficient to protect podocyte cytoskeletal proteins and suppress proteinuria in PAN-induced nephrosis in rats, an established model of podocyte injury and progressive kidney disease. PAN treatment also triggered the Rac1-TRPC5 injury pathway in human iPSC-derived podocytes and kidney organoids. TRPC5 current was recorded in human iPSC-derived podocytes, and was blocked by AC1903. The TRPC5 blocker also reversed the effects of PAN-induced injury in human podocytes in both 2D and 3D culture systems. Taken together, these results revealed the relevance of the TRPC5-Rac1 pathway in human kidney tissue highlighting the potential of this therapeutic strategy for patients.


title: Different combinations of insect Na,K-ATPase α- and β-subunit paralogs enable fine tuning of toxin resistance and enzyme kinetics

abstract:
BackgroundCardiac glycosides are known to fatally inhibit the Na,K-ATPase throughout the animal kingdom. Several animals, however, evolved target-site insensitivity by substitutions in the otherwise highly conserved cardiac glycoside binding pocket located on the Na,K-ATPase -subunit. The minimal functional enzyme consist of an - and a {beta}-subunit, the latter considered mainly as a chaperone responsible for correct folding and membrane integration. We here analyze resistance to cardiac glycosides and kinetic properties of different Na,K-ATPase /{beta}-combinations of the large milkweed bug, Oncopeltus fasciatus. These insects have adapted to high concentrations of cardiac glycosides in their food plants via several rounds of Na,K-ATPase gene duplications followed by differential resistance conferring substitutions and subfunctionalization of the enzymes.

ResultsTo investigate their characteristics we expressed nine combinations of O. fasciatus Na,K-ATPase /{beta}-sunbunits (three each) in Sf9 cells and tested them with two structurally distinct cardiac glycosides, calotropin, a host plant compound, and ouabain, a commonly used toxin. Differences in the number and identity of amino acid substitutions in the cardiac glycoside binding site resulted in large differences in activity and toxin resistance of the three -subunits. The enzymes kinetics were also influenced by the {beta}-subunits leading to increased activities (C{beta}3) or altered resistances. The host plant toxin calotropin proved to be a much more potent inhibitor than ouabain for the phylogenetically oldest C based enzymes. This effect was compensated for in the B and A based enzymes with A{beta}1 having higher resistance against calotropin than against ouabain.

ConclusionThe originally higher inhibitory potency of the host compound calotropin supports a coevolutionary escalation of plant defenses and herbivore tolerance mechanisms. For the bugs the possession of multiple paralogs improved adaptation to plant toxins in a stepwise manner and mitigates pleiotropic effects by a compromise between ion pumping activity and resistance.


title: Genetics of phosphorus use efficiency in a MAGIC wheat population grown in the field

abstract:
Phosphorus (P) is an essential plant nutrient and regular applications are essential in most farming systems to maintain high yields. Yet the P fertilizers applied to crops and pastures are derived from non-renewable resources. It is therefore important to find agronomic and genetic strategies for using this resource efficiently, especially since only a proportion of the applied P is absorbed by crops. The aim of this study was to identify Quantitative Trait Loci (QTL) for P use efficiency (PUE) in wheat using a Multiparent Advanced Generation InterCross (MAGIC) population grown in the field. The 357 genotypes were arranged in paired plots with and without P fertilization. Yield and biomass were measured and PUE was calculated as either the performance of the genotype relative to the average response to fertilization, or the performance of the genotype relative to the average resilience in the absence of fertilization. Five trials were conducted over three years in Australia at three sites with contrasting clay and sandy soil types.

Genotypic variation for response and resilience were identified in all trials with moderate to strong correlation with productivity with and without P between trials. Multiparent Whole Genome Average Interval Mapping (WGAIM) QTL analyses were conducted on the four traits (Biomass / Yield x P Response / Resilience) across the five trials and identified 130 QTL in total. QTL within 10 cM of each other were clustered into 56 groups that were likely to represent identical or linked loci. Of the clusters 27 (48%) contained only a single QTL but 17 (31%) contained 3 or more in different trials or traits. This suggests multiple biological mechanisms driving PUE in different environments. Eight of the 56 groups collocated with QTL for seedling root hair length identified in the same MAGIC population in an earlier study.

HighlightIdentification of genetic loci for phosphorus use efficiency in a multigenic population of Australian wheats grown on contrasting soils.

category: plant biology


title: Neural vulnerability and hurricane-related media are associated with post-traumatic stress in youth

abstract: As natural disasters increase in frequency and severity1, 2, mounting evidence reveals that their human toll extends beyond death, injury, and loss. Posttraumatic stress (PTS) can be common among directly exposed individuals, and children are particularly vulnerable3, 4. Curiously, PTS can even be found among youth far removed from harms way, and media-based exposure may partially account for this phenomenon5-8. Unfortunately, susceptibility to media effects has been difficult to characterize because most research is initiated post-event, precluding examination of pre-disaster factors. In this study, we mitigate this issue with data from nearly 400 9-to 11-year-old children collected prior to and after Hurricane Irma. We evaluate whether preexisting neural patterns moderate associations between hurricane experiences and later Irma-related PTS. We show that "dose" of both objective exposure and Irma-related media exposure predicted Irma-related PTS, the latter even among children dwelling thousands of kilometers away from the hurricane. Furthermore, we show, using pre-hurricane functional magnetic resonance imaging data, that neural responses in brain regions associated with anxiety and stress confer particular vulnerability to the psychological effects of hurricane exposure among certain children. Surprisingly, this was even the case for media exposure- we found that that right amygdala reactivity to fearful stimuli moderated the association between Irma-related media exposure and PTS symptoms, with the media-PTS association strongest for children showing pre-hurricane heightened amygdala reactivity to Fear vs Neutral Faces. In contrast, in bilateral orbitofrontal cortex and left parahippocampal gyrus, children showing a weak response to the Fear condition relative to the Neutral condition were especially susceptible to PTS as a result of Irma-related media exposure. Collectively, these findings run counter to outdated "bullseye" models of disaster exposure that assume negative effects are narrowly circumscribed around a disasters geographic epicenter9. In contrast, for some youth with measurable preexisting vulnerability, consumption of extensive disaster-related media appears to offer an alternative pathway to disaster exposure that transcends geography and objective risk. This preventable exposure should be considered in disaster-related mental health efforts.

category: neuroscience


title: Evolution of Single Gyroid Photonic Crystals in Bird Feathers

abstract: Vivid, saturated structural colors are conspicuous and important features of many animals. A rich diversity of three-dimensional periodic photonic nanostructures is found in the chitinaceous exoskeletons of invertebrates. Three-dimensional photonic nanostructures have been described in bird feathers, but they are typically quasi-ordered. Here, we report bi-continuous single gyroid {beta}-keratin and air photonic crystal networks in the feather barbs of blue-winged leafbirds (Chloropsis cochinchinensis sensu lato), which have evolved from ancestral quasi-ordered channel-type nanostructures. Self-assembled avian photonic crystals may serve as inspiration for multi-functional applications, as they suggest efficient, alternative routes to single gyroid synthesis at optical length-scales, which has been experimentally elusive.


title: Learning Distance-Dependent Motif Interactions: An Explicitly Interpretable Neural Model of Genomic Events

abstract: In many biological studies, prediction is used primarily to validate the model; the real quest is to understand the underlying phenomenon. Therefore, interpretable deep models for biological studies are required. Here, we propose the Hyper-parameter eXplainable Motif Pair framework (HyperXPair) to model biological motifs and their distance-dependent context through explicitly interpretable parameters. This makes HyperXPair more than a decision-support tool; it is also a hypothesis-generating tool designed to advance knowledge in the field. We demonstrate the utility of our model by learning distance-dependent motif interactions for two biological problems: transcription initiation and RNA splicing.

category: bioinformatics


title: Chance, contingency, and necessity in the experimental evolution of ancestral proteins

abstract: To understand why evolution produced the biological systems that exist today, we must know how important chance, contingency, and necessity were during history. Previous observations suggest that each of these modes of causality affects evolution in various settings, but their relative roles and interactions are not well characterized because they have never been systematically assessed in a single system or on a timescale relevant to evolutionary history. To this end, we reconstructed ancestral B-cell-lymphoma-2-family proteins and developed a continuous evolution method to select for defined protein-protein interaction specificities. By repeatedly evolving a series of ancestral proteins to acquire specificities that occurred during history, we show that contingency steadily overwhelms chance and erases necessity as the primary cause of sequence variation in proteins over long phylogenetic timescales. As a result, evolutionary trajectories launched from distant starting points are essentially unpredictable, even under strong and identical selection pressures. Genetic dissection of the outcomes shows that chance arises because numerous sets of mutations can alter specificity at any point in time, while contingency arises because historical substitutions change these sets. Patterns of variation in extant protein sequences are therefore largely the idiosyncratic product of a particular course of unpredictable historical events.


title: Contingency and chance erase necessity in the experimental evolution of ancestral proteins


title: Protein Phosphatase 1 in association with Bud14 inhibits mitotic exit in Saccharomyces cerevisiae

abstract: Mitotic exit in budding yeast is dependent on correct orientation of the mitotic spindle along the cell polarity axis. When accurate positioning of the spindle fails, a surveillance mechanism named the Spindle Position Checkpoint (SPOC) prevents cells from exiting mitosis. Mutants with a defective SPOC become multinucleated and lose their genomic integrity. Yet, a comprehensive understanding of the SPOC mechanism is missing. In this study, we identified the type 1 protein phosphatase, Glc7, in association with its regulatory protein Bud14 as a novel checkpoint component. We further showed that Glc7-Bud14 promotes dephosphorylation of the SPOC effector protein Bfa1. Our results suggest a model in which two mechanisms act in parallel for a robust checkpoint response: first, the SPOC kinase Kin4 isolates Bfa1 away from the inhibitory kinase Cdc5 and second, Glc7-Bud14 dephosphorylates Bfa1 to fully activate the checkpoint effector.


title: Rest is Required to Learn an Appetitively-Reinforced Operant Task in Drosophila

abstract: Maladaptive operant conditioning contributes to development of neuropsychiatric disorders. Candidate genes have been identified that contribute to this maladaptive plasticity, but the neural basis of operant conditioning in genetic model organisms remains poorly understood. The fruit fly Drosophila melanogaster is a versatile genetic model organism that readily forms operant associations with punishment stimuli. However, operant conditioning with a food reward has not been demonstrated in flies, limiting the types of neural circuits that can be studied. Here we present the first sucrose-reinforced operant conditioning paradigm for flies. In the paradigm, flies walk along a Y-shaped track with reward locations at the terminus of each hallway. When flies turn in the reinforced direction at the center of the track, sucrose is presented at the end of the hallway. Only flies that rest early in training learn the reward contingency normally. Flies rewarded independently of their behavior do not form a learned association but have the same amount of rest as trained flies, showing that rest is not driven by learning. Optogenetically-induced sleep does not promote learning, indicating that sleep itself is not sufficient for learning the operant task. We validated the sensitivity of this assay to detect the effect of genetic manipulations by testing the classic learning mutant dunce. Dunce flies are learning impaired in the Y-Track task, indicating a likely role for cAMP in the operant coincidence detector. This novel training paradigm will provide valuable insight into the molecular mechanisms of disease and the link between sleep and learning.


title: Organisation of afferents along the anterior-posterior and medial-lateral axes of the rat OFC

abstract: The orbitofrontal cortex (OFC) has been anatomically divided into a number of subregions along its medial-lateral axis, which behavioural research suggests have distinct functions. Recently, evidence has emerged suggesting functional diversity is also present along the anterior-posterior axis of the rodent OFC. However, the patterns of anatomical connections that underlie these differences have not been well characterised. Here, we use the retrograde tracer cholera toxin subunit B to simultaneously label the projections into the anterior lateral (ALO), posterior lateral (PLO), and posterior ventral (PVO) portions of the rat OFC. Our methodological approach allowed us to simultaneously compare the density and input patterns into these OFC subdivisions. We observed distinct and topographically organised projection patterns into ALO, PLO, and PVO from the mediodorsal and the submedius nuclei of the thalamus. We also observed different levels of connectivity strength into these OFC subdivisions from the amygdala, motor cortex, sensory cortices and medial prefrontal cortical structures, including medial OFC, infralimbic and prelimbic cortices. Interestingly, while labelling in some of these input regions revealed only a gradient in connectivity strength, other regions seem to project almost exclusively to specific OFC subdivisions. Moreover, differences in input patterns between ALO and PLO were as pronounced as those between PLO and PVO. Together, our results support the existence of distinct anatomical circuits within lateral OFC along its anterior-posterior axis.


title: The subcellular architecture of the xyl gene expression flow of the TOL catabolic plasmid of Pseudomonas putida mt-2

abstract:
Despite intensive research on the biochemical and regulatory features of the archetypal catabolic TOL system borne by pWW0 of Pseudomonas putida mt-2, the physical arrangement and tridimensional logic of the xyl gene expression flow remains unknown. In this work, the spatial distribution of specific xyl mRNAs with respect to the host nucleoid, the TOL plasmid and the ribosomal pool has been investigated. In situ hybridization of target transcripts with fluorescent oligonucleotide probes revealed that xyl mRNAs cluster in discrete foci, adjacent but clearly separated from the TOL plasmid and the cell nucleoid. Also, they co-localize with ribosome-rich domains of the intracellular milieu. This arrangement was kept even when the xyl genes were artificially relocated at different chromosomal locations. The same happened when genes were expressed through a heterologous T7 polymerase-based system, which originated mRNA foci outside the DNA. In contrast, rifampicin treatment, known to ease crowding, blurred the confinement of xyl transcripts. This suggested that xyl mRNAs intrinsically run away from their initiation sites to ribosome-rich points for translation--rather than being translated coupled to transcription. Moreover, the results suggest that the distinct subcellular motion of xyl mRNAs results both from innate properties of the sequence at stake and the physical forces that keep the ribosomal pool away from the nucleoid in P. putida. This scenario is discussed on the background of current knowledge on the 3D organization of the gene expression flow in other bacteria and the environmental lifestyle of this soil microorganism.

IMPORTANCEThe transfer of information between DNA, RNA and proteins in a bacterium is often compared to the decoding of a piece of software in a computer. However, the tridimensional layout and the relational logic of the cognate biological hardware i.e. the nucleoid, the RNA polymerase and the ribosomes, are habitually taken for granted. In this work we inspected the localization and fate of the transcripts that stem from the archetypal biodegradative plasmid pWW0 of soil bacterium Pseudomonas putida KT2440 through the non-homogenous milieu of the bacterial cytoplasm. The results expose that-- similarly to computers also--the material components that enable the expression flow are well separated physically and they decipher the sequences through a distinct tridimensional arrangement with no indication of transcription/translation coupling. We argue that the resulting subcellular architecture enters an extra regulatory layer that obeys a species-specific positional code that accompanies the environmental lifestyle of this bacterium.

category: microbiology


title: Natural selection and the advantage of recombination

abstract: The ubiquity of sex and recombination in nature is widely viewed as enigmatic, despite an abundance of limited-scope explanations. Natural selection, it seems, should amplify well-matched combinations of genes. Recombination would break up these well-matched combinations and should thus be suppressed. We show, to the contrary, that natural selection amplifies poorly-matched gene combinations and creates negative associations in the process. Recombination breaks up these poorly-matched combinations, neutralizes the negative associations, and should thus be passively and universally favored.


title: Full-length de novo protein structure determination from cryo-EM maps using deep learning

abstract:
Advances in microscopy instruments and image processing algorithms have led to an increasing number of cryo-EM maps. However, building accurate models for the EM maps at 3-5 [A] resolution remains a challenging and time-consuming process. With the rapid growth of deposited EM maps, there is an increasing gap between the maps and reconstructed/modeled 3-dimensional (3D) structures. Therefore, automatic reconstruction of atomic-accuracy full-atom structures from EM maps is pressingly needed. Here, we present a semi-automatic de novo structure determination method using a deep learning-based framework, named as DeepMM, which builds atomic-accuracy all-atom models from cryo-EM maps at near-atomic resolution. In our method, the main-chain and C positions as well as their amino acid and secondary structure types are predicted in the EM map using Densely Connected Convolutional Networks. DeepMM was extensively validated on 40 simulated maps at 5 [A] resolution and 30 experimental maps at 2.6-4.8 [A] resolution as well as an EMDB-wide data set of 2931 experimental maps at 2.6-4.9 [A] resolution, and compared with state-of-the-art algorithms including RosettaES, MAINMAST, and Phenix. Overall, our DeepMM algorithm obtained a significant improvement over existing methods in terms of both accuracy and coverage in building full-length protein structures on all test sets, demonstrating the efficacy and general applicability of DeepMM.

Availabilityhttps://github.com/JiahuaHe/DeepMM

Supplementary informationSupplementary data are available.


title: Using Neutralization Landscapes to enumerate Antibody Behavior and Decompose Antibody Mixtures

abstract:
Antibodies constitute a key line of defense against the diverse pathogens we encounter in our lives. While the interactions between a single antibody and a single virus are routinely characterized in exquisite detail, the inherent tradeoffs between attributes such as potency and breadth remain unclear. In addition, there is a wide gap between the discrete interactions of single antibodies and how antibody mixtures collectively repel multiple viruses. Here, we develop a new form of antigenic cartography called a Neutralization Landscape that enumerates the full space of antibody-virus interactions for antibodies targeting the influenza hemagglutinin stem. This reference set of antibody behaviors transforms the potency-breadth tradeoff into a readily solvable geometry problem. Using the Neutralization Landscape, we decompose the collective neutralization from multiple antibodies to characterize the composition and functional properties of the stem antibodies within. Looking forward, this framework can leverage the serological assays routinely performed for influenza surveillance to analyze how an individuals antibody repertoire evolves over time in response to vaccination or infection.

One-Sentence SummaryWe describe the full range of behaviors for antibodies targeting the stem of influenza hemagglutinin, enabling us to decompose the collective behavior of antibody mixtures and characterize the individual antibodies within.

category: immunology


title: Natural enemies have inconsistent impacts on the coexistence of competing species

abstract:
O_LIThe role of natural enemies in promoting coexistence of competing species has generated substantial debate. Modern coexistence theory provides a detailed framework to investigate this topic, but there have been remarkably few empirical applications to the impact of natural enemies.
C_LIO_LIWe tested experimentally the capacity for a generalist enemy to promote coexistence of competing insect species, and the extent to which any impact can be predicted by trade-offs between reproductive rate and susceptibility to natural enemies.
C_LIO_LIWe used experimental mesocosms to conduct a fully-factorial pairwise competition experiment for six rainforest Drosophila species, with and without a generalist pupal parasitoid. We then parameterised models of competition and examined the coexistence of each pair of Drosophila species within the framework of modern coexistence theory.
C_LIO_LIWe found idiosyncratic impacts of parasitism on pairwise coexistence, mediated through changes in fitness differences, not niche differences. There was no evidence of an overall reproductive rate - susceptibility trade-off. Pairwise reproductive rate - susceptibility relationships were not useful shortcuts for predicting the impact of parasitism on coexistence.
C_LIO_LIOur results exemplify the value of modern coexistence theory in multi-trophic contexts and the importance of contextualising the impact of natural enemies. In the set of species investigated, competition was affected by the higher trophic level, but the overall impact on coexistence cannot be easily predicted just from knowledge of relative susceptibility. Methodologically, our Bayesian approach highlights issues with the separability of model parameters within modern coexistence theory and shows how using the full posterior parameter distribution improves inferences. This method should be widely applicable for understanding species coexistence in a range of systems.
C_LI

category: ecology


title: Lattice micropatterning for cryo-electron tomography studies of cell-cell contacts

abstract: Cryo-electron tomography is the highest resolution tool available for structural analysis of macromolecular complexes within their native cellular environment. At present, data acquisition suffers from low throughput, in part due to the low probability of positioning a cell such that the subcellular structure of interest is on a region of the electron microscopy (EM) grid that is suitable for imaging. Here, we photo-micropatterned EM grids to optimally position endothelial cells so as to enable high-throughput imaging of cell-cell contacts. Lattice micropatterned grids increased the average distance between intercellular contacts and the thicker cell nuclei such that the regions of interest were sufficiently thin for direct imaging. We observed a diverse array of membranous and cytoskeletal structures at intercellular contacts, demonstrating the utility of this technique in enhancing the rate of data acquisition for cellular cryo-electron tomography studies.

category: bioengineering


title: Identifying seaweeds species of Chlorophyta, Phaeophyta and Rhodophyta using DNA barcodes

abstract: Strengthening the DNA barcode database is important for a species level identification, which was lacking for seaweeds. We made an effort to collect and barcode seaweeds occurring along Southeast coast of India. We barcoded 31 seaweeds species belonging to 21 genera, 14 family, 12 order of 3 phyla (viz., Chlorophyta, Phaeophyta and Rhodophyta). We found 10 species in 3 phyla and 2 genera (Anthophycus and Chnoospora) of Phaeophyta were barcoded for the first time. Uncorrected p-distance calculated using K2P, nucleotide diversity and Tajimas test statistics reveals highest values among the species of Chlorophyta. Over all K2P distance was 0.36. The present study revealed the potentiality of rbcL gene sequences in identification of all 3 phyla of seaweeds. We also found that the present barcode reference libraries (GenBank and BOLD) were insufficient in seaweeds identification and more efforts were needed for strengthening local seaweed barcode library to benefit rapids developing field such as environmental DNA barcoding. We also show that the constructed barcode library could aid various industrial experts involved in seaweed bio-resource exploration and taxonomy/non-taxonomic researches involved in climate, agriculture and epigenetics research in precise seaweed identification. Since the rise of modern high-throughput sequencing technologies is significantly altering bio-monitoring applications and surveys, reference datasets such as ours will become essential in ecosystems health assessment and monitoring.


title: Cellxgene VIP unleashes full power of interactive visualization and integrative analysis of scRNA-seq, spatial transcriptomics, and multiome data

abstract: To meet the growing demands from scientists to effectively extract deep insights from single cell RNA sequencing, spatial transcriptomics, and emerging multiome datasets, we developed cellxgene VIP (Visualization In Plugin), a frontend interactive visualization plugin of cellxgene framework, which greatly expanded capabilities of the base tool in the following aspects. First, it generates a comprehensive set of over eighteen commonly used quality control and analytical plots in high resolution with highly customizable settings in real time. Second, it provides more advanced analytical functions to gain insights on cellular compositions and deep biology, such as marker gene identification, differential gene expression analysis, and gene set enrichment analysis. Third, it empowers advanced users to perform analysis in a Jupyter Notebook like environment, dubbed Command Line Interface (CLI) by programming in Python and/or R directly without limiting themselves to functional modules available via graphical user interface (GUI). Finally, it pioneers methods to visualize multi-modal data, such as spatial transcriptomics embedding aligned with histological image on one slice or multiple slices in a grid format, and the latest 10x Genomic Multiome dataset where both DNA accessibility and gene expression in the same cells are measured, under the same framework in an integrative way to fully leverage the functionalities mentioned above. Taken together, the open-source tool makes large scale single cell data visualization and analysis more accessible to biologists in a user-friendly manner and fosters computational reproducibility by simplifying data and code reuse through the CLI. Going forward, it has the potential to become a crowdsourcing ecosystem for the scientific community to contribute even more modules to the Swiss Army knife of single cell data exploration tools.


title: Real-time single-cell characterizationof the eukaryotic transcription cyclereveals correlations between RNAinitiation, elongation, and cleavage

abstract: The eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript. Although each of these steps can be regulated as well as coupled with each other, their in vivo dissection has remained challenging because available experimental readouts lack sufficient spatiotemporal resolution to separate the contributions from each of these steps. Here, we describe a novel application of Bayesian inference techniques to simultaneously infer the effective parameters of the transcription cycle in real time and at the single-cell level using a two-color MS2/PP7 reporter gene and the developing fruit fly embryo as a case study. Our method enables detailed investigations into cell-to-cell variability in transcription-cycle parameters as well as single-cell correlations between these parameters. These measurements, combined with theoretical modeling, suggest a substantial variability in the elongation rate of individual RNA polymerase molecules. We further illustrate the power of this technique by uncovering a novel mechanistic connection between RNA polymerase density and nascent RNA cleavage efficiency. Thus, our approach makes it possible to shed light on the regulatory mechanisms in play during each step of the transcription cycle in individual, living cells at high spatiotemporal resolution.

category: biophysics


title: Imaging transmembrane dynamics of biomolecules at live cell plasma membranes using quenchers in extracellular environment

abstract: It is a big challenge to measure position changes of biomolecules in the direction normal to the plasma membranes of living cells. We developed a one donor-multiple quenchers F[o]rster resonance energy transfer method by using non-fluorescent quenchers in the extracellular environment. It senses subnanometer position changes of a fluorophore-labeled biomolecule in the plasma membrane. The method was validated by monitoring flip-flops of individual lipid molecules incorporated in plasma membranes. We studies membrane perforation by a host defense peptide from the extracellular side and found that the pore-forming peptide is dynamic, switching among different insertion depths. The method is especially useful in studying interactions of membrane proteins with the inner surfaces of plasma membranes. Our method will find wide applications in systematic analysis of fundamental cellular processes at plasma membranes.


title: Capitalizing on paradoxical activation of the MAPK pathway for treatment of Imatinib-resistant mast cell leukemia and chronic myelogenous leukemia

abstract: Prevention of fatal side effects during cancer therapy of cancer patients with high-dosed pharmacological inhibitors is to date a major challenge. Moreover, the development of drug resistance poses severe problems for the treatment of patients with leukemia or solid tumors. Particularly drug-mediated dimerization of RAF kinases can be the cause of acquired resistance, also called "paradoxical activation". Here we re-analyzing the effects of different tyrosine kinase inhibitors (TKIs) on the proliferation, metabolic activity, and survival of the Imatinib-resistant, KITV560G,D816V-expressing human mast cell (MC) leukemia (MCL) cell line HMC-1.2. We observed that low concentrations of the TKIs Nilotinib and Ponatinib resulted in enhanced proliferation, suggesting paradoxical activation of the MAPK pathway. Indeed, these TKIs caused BRAF-CRAF dimerization, resulting in ERK1/2 activation. The combination of Ponatinib with the MEK inhibitor Trametinib, at nanomolar concentrations, effectively suppressed HMC-1.2 proliferation, metabolic activity, and induced apoptotic cell death. Effectiveness of this drug combination was recapitulated in the human KIT D816V MC line ROSA KITD816V and in KIT D816V hematopoietic progenitors obtained from in patient-derived induced pluripotent stem cells (iPS cells). In conclusion, mutated KIT-driven Imatinib resistance can be efficiently bypassed by a low concentration combination of the TKI Ponatinib and the MEK inhibitor Trametinib, potentially reducing the negative side effects associated to MCL therapy.

category: cancer biology


title: Head-Direction Coding in the Hippocampal Formation of Birds

abstract: Birds strongly rely on spatial memory and navigation. Therefore, it is of utmost interest to reveal how space is represented in the avian brain. Here we used tetrodes to record neurons from the hippocampal formation of Japanese quails - a ground-dwelling species - while the quails roamed a 1x1-meter arena (>2,100 neurons from 23 birds). Whereas spatially-modulated cells (place-cells, border-cells, etc.) were generally not encountered, the firing-rate of 12% of the neurons was unimodally and significantly modulated by the head-azimuth - i.e. these were head-direction cells (HD cells). Typically, HD cells were maximally active at one preferred-direction and minimally at the opposite null-direction, with preferred-directions spanning all 360{degrees}. The HD tuning was relatively broad (mean width [~]130{degrees}), independent of the animals position and speed, and was stable during the recording-session. Similarly to findings in rodents, the HD tuning usually rotated with the rotation of a salient visual cue in the arena. These findings support the existence of an allocentric head-direction representation in the quail hippocampal formation, and provide the first demonstration of head-direction cells in birds.


title: DARPP-32 Distinguishes a Subset of Adult-Born Neurons in Zebra Finch HVC

abstract: Adult male zebra finches (Taeniopygia guttata) continually incorporate adult-born neurons into HVC, a telencephalic brain region necessary for the production of learned song. These neurons express immediate early genes during song production, suggesting a role for neurogenesis in song production throughout the lifespan. Half of these adult-born HVC neurons (HVC NNs) can be backfilled from the robust nucleus of the arcopallium (RA) and are a part of the vocal motor pathway underlying learned song production, but the other half do not backfill from RA, and they remain to be characterized. Here we used cell birth-dating, retrograde tract tracing, and immunofluorescence to demonstrate that half of all HVC NNs express thephosphoprotein DARPP-32, a protein associated with dopamine (DA) receptor expression. We also demonstrate that DARPP-32+ HVC NNs are contacted by tyrosine hydroxylase immunoreactive fibers suggesting that they receive catecholaminergic input, have transiently larger nuclei than DARPP-32- HVC NNs, and do not backfill from RA. Taken together, these findings help characterize a group of HVC NNs that have no apparent projections to RA and so far have eluded any positive identification other than HVC NN status.


title: A Population Receptive Field Model of the Magnetoencephalography Response

abstract: 1Computational models which predict the neurophysiological response from experimental stimuli have played an important role in human neuroimaging. One type of computational model, the population receptive field (pRF), has been used to describe cortical responses at the millimeter scale using functional magnetic resonance imaging (fMRI) and electrocorticography (ECoG). However, pRF models are not widely used for non-invasive electromagnetic field measurements (EEG/MEG), because individual sensors pool responses originating from several centimeter of cortex, containing neural populations with widely varying spatial tuning. Here, we introduce a forward-modeling approach in which pRFs estimated from fMRI data are used to predict MEG sensor responses. Subjects viewed contrast-reversing bar stimuli sweeping across the visual field in separate fMRI and MEG sessions. Individual subjects pRFs were modeled on the cortical surface at the millimeter scale using the fMRI data. We then predicted cortical time series and projected these predictions to MEG sensors using a biophysical MEG forward model, accounting for the pooling across cortex. We compared the predicted MEG responses to observed visually evoked steady-state responses measured in the MEG session. We found that pRF parameters estimated by fMRI could explain a substantial fraction of the variance in steady-state MEG sensor responses (up to 60% in individual sensors). Control analyses in which we artificially perturbed either pRF size or pRF position reduced MEG prediction accuracy, indicating that MEG data are sensitive to pRF properties derived from fMRI. Our model provides a quantitative approach to link fMRI and MEG measurements, thereby enabling advances in our understanding of spatiotemporal dynamics in human visual field maps.


title: Serotonergic Effects on Interoception

abstract: Interoception is the signalling, perception, and interpretation of internal physiological states. Much of the psychopharmacology of interoception is still undiscovered. However, psychiatric disorders associated with changes of interoception, including depressive, anxiety, and eating disorders are often treated with selective serotonin reuptake inhibitors (SSRIs). The causal effect of acute changes of serotonin transmission on interoceptive cognition was tested by a within-participant, crossover, placebo-controlled study. Forty-seven healthy human volunteers (31 female, 16 male) were tested both on and off a 20mg oral dose of the commonly prescribed SSRI citalopram. For each randomly ordered session, participants made judgments on the synchrony of their heartbeat to auditory tones and expressed confidence in each of these judgments. Citalopram enhanced insight into the likelihood that ones interoceptive judgment had been correct, driven primarily by enhanced confidence for correct responses. This effect was independent of measured cardiac and subjective effects of the drug. This novel result is evidence that acute serotonin changes can alter metacognitive insight into the reliability of inferences based on interoceptive information, which is a foundation for considering effects of serotonin on cognition and emotion in terms of effective top-down regulation of interoceptive influence on mental states.


title: Synaptic Zn2+ potentiates the effects of cocaine on striatal dopamine neurotransmission and behavior

abstract: Cocaine binds to the dopamine (DA) transporter (DAT) to regulate cocaine reward and seeking behavior. Zinc (Zn2+) also binds to the DAT, but the in vivo relevance of this interaction is unknown. We found that Zn2+ concentrations in postmortem brain (caudate) tissue from humans who died of cocaine overdose were significantly lower than in control subjects. Moreover, the level of striatal Zn2+ content in these subjects negatively correlated with plasma levels of benzoylecgonine, a cocaine metabolite indicative of recent use. In mice, repeated cocaine exposure increased synaptic Zn2+ concentrations in the caudate putamen (CPu) and nucleus accumbens (NAc). Cocaine-induced increases in Zn2+ were dependent on the Zn2+ transporter 3 (ZnT3), a neuronal Zn2+ transporter localized to synaptic vesicle membranes, as ZnT3 knockout (KO) mice were insensitive to cocaine-induced increases in striatal Zn2+. ZnT3 KO mice showed significantly lower electrically-evoked DA release and greater DA clearance when exposed to cocaine compared to controls. ZnT3 KO mice also displayed significant reductions in cocaine locomotor sensitization, conditioned place preference (CPP), self-administration, and reinstatement compared to control mice and were insensitive to cocaine-induced increases in striatal DAT binding. Finally, dietary Zn2+ deficiency in mice resulted in decreased striatal Zn2+ content, cocaine locomotor sensitization, CPP, and striatal DAT binding. These results indicate that cocaine increases synaptic Zn2+ release and turnover/metabolism in the striatum, and that synaptically-released Zn2+ potentiates the effects of cocaine on striatal DA neurotransmission and behavior and is required for cocaine-primed reinstatement. In sum, these findings reveal new insights into cocaines pharmacological mechanism of action and suggest that Zn2+ may serve as an environmentally-derived regulator of DA neurotransmission, cocaine pharmacodynamics, and vulnerability to cocaine use disorders.


title: Polygenic Scores for Plasticity: A New Tool for Studying Gene-Environment Interplay

abstract: Outcomes of interest to demographers--fertility; health; education--are the product of both an individuals genetic makeup and his or her social environment. Yet Gene x Environment research (GxE) currently deploys a limited toolkit on the genetic side to study gene-environment interplay: polygenic scores (PGS, or what we call mPGS) that reflect the influence of genetics on levels of an outcome. The purpose of the present paper is to develop a genetic summary measure better suited for GxE research. We develop what we call variance polygenic scores (vPGS), or polygenic scores that reflect genetic contributions to plasticity in outcomes. The first part of the analysis uses the UK Biobank (N ~ 326,000 in the training set) and the Health and Retirement Study (HRS) to compare four approaches for constructing polygenic scores for plasticity. The results show that widely-used methods for discovering which genetic variants affect outcome variability fail to serve as distinctive new tools for GxE. Then, using the polygenic scores that do capture distinctive genetic contributions to plasticity, we analyze heterogeneous effects of a UK education reform on health and educational attainment. The results show the properties of a new tool useful for population scientists studying the interplay of nature and nurture and for population-based studies that are releasing polygenic scores to applied researchers.

category: genetics


title: Anatomy, transcription dynamics and evolution of wheat ribosomal RNA loci deciphered by a multi-omics approach

abstract:
Background and AimsThree out of four RNA components of ribosomes are encoded by 45S rDNA loci, whose transcripts are processed into 18S, 5.8S and 26S ribosomal RNAs. The loci are organized as long head-to-tail tandem arrays of nearly identical units spanning over several megabases of sequence. Due to this peculiar structure, the number of rRNA genes, their sequence composition and expression status remain unclear, especially in complex polyploid genomes harbouring multiple loci. Here we conducted a complex study to decipher structure and activity of both major and minor rRNA loci in hexaploid bread wheat (Triticum aestivum).

MethodsWe employed an original, multi-omics approach, combining chromosome flow sorting and optical mapping with transcriptome and methylome sequencing.

Key ResultsThe former two techniques enabled unbiased quantification of rDNA units in particular loci of the wheat genome. Total number of rRNA genes organized in tandem arrays was 4388, with 64.1, 31.4, 3.9 and 0.7% located in short arms of chromosomes 6B, 1B, 5D and 1A, respectively. At the expression level, only 1B and 6B loci contributed to transcription at roughly 2:1 ratio. The 1B:6B ratio varied among five analysed tissues (embryo, coleoptile, root tip, primary leaf, mature leaf), being the highest (2.64:1) in mature leaf and lowest (1.72:1) in coleoptile. Cytosine methylation was considerably higher in CHG contexts in the silenced 5D locus compared to the active 1B and 6B loci.

ConclusionsA fine genomic organization and tissue-specific expression of rRNA loci were deciphered, for the first time, in a complex polyploid species. We documented various mechanisms of rRNA dosage control, including gene elimination and stable inactivation related to nucleolar subdominance of A and D-genome loci, and a subtle, developmentally regulated silencing of one of the major loci. The results are discussed in the context of wheat evolution and transcription regulation.

category: genomics


title: Multi-regional module-based signal transmission in the mouse visual cortex

abstract: The visual cortex is organized hierarchically, but the presence of extensive recurrent and parallel pathways make it challenging to decipher how signals flow between neuronal populations. Here, we tracked the flow of spiking activity recorded from six interconnected levels of the mouse visual hierarchy. By analyzing leading and lagging spike-timing relationships among pairs of simultaneously recorded neurons, we created a cellular-scale directed network graph. Using a module-detection algorithm to cluster neurons based on shared connectivity patterns, we uncovered two multi-regional communication modules distributed across the hierarchy. The direction of signal flow between and within these modules, differences in layer and area distributions, and distinct temporal dynamics suggest that one module is positioned to transmit feedforward sensory signals, whereas the other integrates inputs for recurrent processing. These results suggest that multi-regional functional modules may be a fundamental feature of organization beyond cortical areas that supports signal propagation across hierarchical recurrent networks.


title: Multi-regional module-based signal transmission in mouse visual cortex


title: cFos ensembles in the dentate gyrus rapidly segregate over time and do not form a stable map of space

abstract: Transgenic cFos reporter mice are used to identify and manipulate neurons that store contextual information during fear learning. It is not clear, however, how spatial information acquired over several training days is integrated in the hippocampus. Using a water maze task, we observed that cFos expression patterns in the dentate gyrus are temporally unstable and shift daily. Surprisingly, cFos patterns did not get more stable with increasing spatial memory precision. Despite the fact that cFos was no longer expressed, optogenetic inhibition of neurons that expressed cFos on the first training day affected performance days later. Triggered by training, {Delta}FosB accumulates and provides a negative feedback mechanism that makes the cFos ensemble in the dentate gyrus dependent on the history of activity. Shifting cFos expression to a different set of granule cells every day may aid the formation of episodic memories.


title: RetroCHMP3 Blocks Budding of Enveloped Viruses Without Blocking Cytokinesis

abstract: Many enveloped viruses require the endosomal sorting complexes required for transport (ESCRT) pathway to exit infected cells. This highly conserved pathway mediates essential cellular membrane fission events, which restricts the acquisition of adaptive mutations to counteract viral co-option. Here, we describe duplicated and truncated copies of the ESCRT-III factor CHMP3 that block ESCRT-dependent virus budding and that arose independently in New World monkeys and mice. When expressed in human cells, these retroCHMP3 proteins potently inhibit release of retroviruses, paramyxoviruses, and filoviruses. Remarkably, retroCHMP3 proteins have evolved to reduce interactions with other ESCRT-III factors and to have little effect on cellular ESCRT processes, revealing routes for decoupling cellular ESCRT functions from viral exploitation. The repurposing of duplicated ESCRT-III proteins thus provides a mechanism to generate broad-spectrum viral budding inhibitors without blocking highly conserved essential cellular ESCRT functions.


title: A hybrid stochastic-deterministic approach to explore multiple infection and evolution in HIV

abstract: To study viral evolutionary processes within patients, mathematical models have been instrumental. Yet, the need for stochastic simulations of minority mutant dynamics can pose computational challenges, especially in heterogeneous systems where very large and very small subpopulations coexist. Here, we describe a hybrid stochastic-deterministic algorithm to simulate mutant evolution in large viral populations, such as acute HIV-1 infection, and further include the multiple infection of cells. We demonstrate that the hybrid method can approximate the fully stochastic dynamics with sufficient accuracy at a fraction of the computational time, and quantify evolutionary end points that cannot be expressed by deterministic models, such as the mutant distribution or the probability of mutant existence at a given infected cell population size. We apply this method to study the role of multiple infection and intracellular interactions among different virus strains (such as complementation and interference) for mutant evolution. Multiple infection is predicted to increase the number of mutants at a given infected cell population size, due to a larger number of infection events. We further find that viral complementation can significantly enhance the spread of disadvantageous mutants, but only in select circumstances: it requires the occurrence of direct cell-to-cell transmission through virological synapses, as well as a substantial fitness disadvantage of the mutant, most likely corresponding to defective virus particles. This, however, likely has strong biological consequences because defective viruses can carry genetic diversity that can be incorporated into functional virus genomes via recombination. Through this mechanism, synaptic transmission in HIV might promote virus evolvability.


title: Subfunctionalisation of paralogous genes and evolution of differential codon usage preferences: the showcase of polypyrimidine tract binding proteins

abstract:
AO_SCPLOWBSTRACTC_SCPLOWGene paralogs are copies of an ancestral gene that appear after gene or full genome duplication. When the two sister gene copies are maintained in the genome, redundancy may release certain evolutionary pressures, allowing one of them to access novel gene functions. Here we focused on the evolutionary history of the three polypyrimidine tract binding protein (PTBP) paralogs and their concurrent evolution of differential codon usage preferences in vertebrate species.

PTBP1-3 show high identity at the amino acid level (up to 80%), but display strongly different nucleotide composition, divergent CUPrefs and distinct tissue-specific expression levels. Phylogenetic inference suggests that the duplication events leading to the three extant PTBP1-3 lineages predate the basal diversification within vertebrates. We identify a distinct substitution pattern towards GC3-enriching mutations in PTBP1, concurrent with a trend for the use of common codons and for a tissue-wide expression. Genomic context analysis shows that GC3-rich nucleotide composition for PTBP1s is driven by local mutational processes. In contrast, PTBP2s are enriched in AT-ending, rare codons, and display tissue-restricted expression. Nucleotide composition and CUPrefs of PTBP2 are only partly driven by local mutational forces, and could have been shaped by selective forces. Interestingly, trends for use of UUG-Leu codon match those of AT-ending codons.

Our interpretation is that a combination of directional mutation-selection has differentially shaped CUPrefs of PTBPs in Vertebrates: GC-enrichment of PTBP1 is linked to the strong and broad tissue expression, while AT-enrichment of PTBP2 and PTBP3 are linked to rare CUPrefs and specialized spatio-temporal expression. This scenario is compatible with a gene subfunctionalisation process by differential expression regulation associated to the evolution of specific CUPrefs.

1 Significance StatementIn vertebrates, PTBP paralogs display strong differences in gene composition, gene expression regulation, and their expression in cell culture depends on their codon usage preferences. We show that placental mammals PTBP1 have become GC-rich because of local mutational pressures, resulting in an enrichment of frequently used codons and in a strong, tissue-wide expression. On the contrary, PTBP2 in vertebrates are AT-rich, with a lower contribution of local mutational processes to their specific nucleotide composition, show high frequency of rare codons and in placental mammals display a restricted expression pattern contrasting to that of PTBP1. The systematic study of composition and expression patterns of gene paralogs can help understand the complex mutation-selection interplay that shape codon usage bias in multicellular organisms.


title: Feedback from retinal ganglion cells to the inner retina

abstract: Retinal ganglion cells (RGCs) are thought to be strictly postsynaptic within the retina. They carry visual signals from the eye to the brain, but do not make chemical synapses onto other retinal neurons. Nevertheless, they form gap junctions with other RGCs and amacrine cells, providing possibilities for RGC signals to feed back into the inner retina. Here we identified such feedback circuitry in the salamander and mouse retinas. First, using biologically inspired circuit models, we found mutual inhibition among RGCs of the same type. We then experimentally determined that this effect is mediated by gap junctions with amacrine cells. Finally, we found that this negative feedback lowers RGC visual response gain without affecting feature selectivity. The principal neurons of the retina therefore participate in a recurrent circuit much as those in other brain areas, not being a mere collector of retinal signals, but are actively involved in visual computations.


title: A genetic screen identifies new steps in oocyte maturation that enhance proteostasis in the immortal germ lineage

abstract: Somatic cells age and die, but the germ-cell lineage is immortal. In C. elegans, germline immortality involves proteostasis renewal at the beginning of each new generation, when oocyte-maturation signals from sperm trigger the clearance of carbonylated proteins and protein aggregates. Here, we explore the cell biology of this proteostasis renewal in the context of a whole-genome RNAi screen. Oocyte maturation signals are known to trigger protein-aggregate removal via lysosome acidification. Our findings suggest that lysosomes are acidified as a consequence of changes in ER activity that permit assembly of the lysosomal V-ATPase, which in turn allows lysosomes to clear the aggregates via microautophagy. We define two functions for mitochondria, both of which appear to be independent of ATP generation. Many genes from the screen also regulate lysosome acidification and age-dependent protein aggregation in the soma, suggesting a fundamental mechanistic link between proteostasis renewal in the germline and somatic longevity.


title: DNA methylation signatures of duplicate gene evolution in angiosperms

abstract: Gene duplication is an ongoing source of genetic novelty and evolutionary innovation. DNA methylation has been proposed as a factor in duplicate gene evolution. However, previous studies have largely been limited to individual species, apply differing methods, and have predominantly focused on CG methylation. The generalizability of these relationships at phylogenetic and population scales remains unknown. Here, we apply a consistent comparative epigenomics approach across 43 diverse angiosperm species and a population of 928 Arabidopsis thaliana accessions examining both CG and non-CG methylation contexts n whole-genome duplicate (WGD) and single-gene duplicate (SGD) genes. We observe several overarching trends, demonstrating that genic DNA methylation is differentially associated with the type of gene duplication, age of gene duplication, sequence evolution, and gene expression. WGDs are typically unmethylated or marked by mCG-only gene-body methylation, while SGDs typically are enriched for unmethylated genes or methylation in both mCG and non-CG contexts (transposon-like methylation or teM). TeM in particular was associated with relatively more recent SGDs and higher sequence divergence. However, we find variation across the phylogeny, as well as phylogenetic effects, which could be key to a deeper understanding of these relationships. Within the A. thaliana population, differences in duplicate age and sequence evolution were observed based on the frequency of genic methylation. Collectively, these results indicate that genic methylation states differently mark duplicate gene evolution.


title: Transcriptional kinetic synergy: a complex landscape revealed by integrating modelling and synthetic biology

abstract: 1Gene regulation involves synergistic interactions between transcription factors (TFs). Classical thermodynamic models offer a biophysical understanding of synergy based on binding cooperativity and regulated recruitment of RNA polymerase. However, transcription requires polymerase to transition through multiple states. Accordingly, recent work has suggested that "kinetic synergy" can arise through TFs differentially regulating distinct steps of the transcription cycle. Disentangling both sources of synergy has been challenging. Here, we combine theory and experiment to analyze TFs binding to a single shared site, thereby removing simultaneous specific DNA binding. Using the graph-based linear framework, we integrate TF binding with regulation of the transcription cycle, and reveal a complex kinetic synergy landscape dependent on TF concentration, DNA binding and transcriptional activity. We exploit synthetic zinc-finger TF fusions to experimentally interrogate these predictions. Our results confirm that transcription cycle regulation must be integrated with recruitment for a quantitative understanding of transcriptional control.

category: systems biology


title: AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation

abstract: The heat stress response activates the transcription factor heat shock factor 1 (HSF1), which subsequently upregulates heat shock proteins to maintain the integrity of the proteome. HSF1 activation requires nuclear localization, trimerization, DNA binding, phosphorylation, and gene transactivation. Phosphorylation at S326 is an important regulator of HSF1 transcriptional activity. Phosphorylation at S326 is mediated by AKT1, mTOR, p38, and MEK1. Here, we observe that AKT1 activates HSF1 independent of mTOR. AKT2 also phosphorylated S326 of HSF1 but showed weak ability to activate HSF1. Similarly, mTOR, p38, and MEK1 all phosphorylated S326 but AKT1 was the more potent activator. Mass spectrometry showed that AKT1 also phosphorylated HSF1 at T142, S230, and T527 in addition to S326 whereas the other kinases did not. Subsequent investigation revealed that phosphorylation at T142 is necessary for HSF1 trimerization and that S230, S326, and T527 are required for HSF1 gene transactivation and recruitment of TFIIB and CDK9. This study suggests that HSF1 hyperphosphorylation is targeted and these specific residues have direct function in regulating HSF1 transcriptional activity.

category: biochemistry


title: Sex change in aquarium systems establishes the New Zealand spotty wrasse (Notolabrus celidotus) as a temperate model species for the investigation of sequential hermaphroditism

abstract:
The stunning sexual transformation commonly triggered by age, size or social context in some fishes is one of the best examples of phenotypic plasticity thus far described. To date our understanding of this process is dominated by studies on a handful of subtropical and tropical teleosts, often in wild settings because sex change has been challenging to achieve in captivity. Here we have established the protogynous New Zealand spotty wrasse, Notolabrus celidotus, as a temperate model for the experimental investigation of sex change. Captive fish were induced to change sex using either aromatase inhibition or manipulation of social groups. Complete transition from female to male occurred over 60 days and time-series sampling was used to quantify changes in hormone production, gene expression and gonadal cellular anatomy using radioimmunoassay, nanoString nCounter mRNA and histological analyses, respectively. Early-stage decreases in plasma 17{beta}-estradiol (E2) concentrations or gonadal aromatase (cyp19a1a) expression were not detected in spotty wrasse, despite these being commonly associated with the onset of sex change in subtropical and tropical protogynous (female-to-male) hermaphrodites. In contrast, expression of the masculinising factor amh (anti-Mullerian hormone) increased during early sex change, implying a potential role as a proximate trigger for masculinisation. Expression of male-related genes responsible for androgen production cyp11c1 and hsd11b2 increased from mid sex change. Gonadal expression of the glucocorticoid and mineralocorticoid receptors nr3c1 and nr3c2, putative mediators of the stress hormone cortisol, increased in late stages of sex change. Collectively, these data provide a foundation for the spotty wrasse as a temperate teleost model to study sex change and cell fate in vertebrates.

Summary statementThe spotty wrasse, Notolabrus celidotus, is a new temperate model for the study of vertebrate sex change, this work characterises endocrine and genetic markers based on laboratory induced sex change.

category: physiology


title: Genome-wide identification of tomato xylem sap fitness factors for three plant-pathogenic Ralstonia species

abstract:
Plant pathogenic Ralstonia spp. colonize plant xylem and cause wilt diseases on a broad range of host plants. To identify genes that promote growth of diverse Ralstonia strains in xylem sap from tomato plants, we performed genome-scale genetic screens (random barcoded transposon mutant sequencing screens; RB-TnSeq) in three strains spanning the genetic, geographical, and physiological range of plant pathogenic Ralstonia: R. solanacearum IBSBF1503, R. pseudosolanacearum GMI1000, and R. syzygii PSI07. Contrasting mutant fitness phenotypes in culture media versus in xylem sap suggest that Ralstonia strains are adapted to ex vivo xylem sap and that culture media impose foreign selective pressures. Although wild-type Ralstonia grew in sap and in rich medium with similar doubling times and to a similar carrying capacity, more genes were essential for growth in sap than in rich medium. Each strain required many genes associated with envelope remodeling and repair processes for full fitness in xylem sap. These genes were associated with peptidoglycan peptide formation (murI), secretion of periplasmic proteins (tatC), periplasmic protein folding (dsbA), synthesis of osmoregulated periplasmic glucans (mdoGH), and LPS biosynthesis. Mutants in four genes had strong, sap-specific fitness defects in all strain backgrounds: murI, thiC, purU, and a lipoprotein (RSc2007). Many amino acid biosynthesis genes were required for fitness in both minimal medium and xylem sap. Multiple mutants with insertions in virulence regulators had gains-of-fitness in culture media and neutral fitness in sap. Our genome-scale genetic screen identified Ralstonia fitness factors that promote growth in xylem sap, an ecologically relevant condition.

ImportanceTraditional transposon mutagenesis genetic screens pioneered molecular plant pathology and identified core virulence traits like the type III secretion system. TnSeq approaches that leverage next-generation sequencing to rapidly quantify transposon mutant phenotypes are ushering in a new wave of biological discovery. Here we have adapted a genome-scale approach, random barcoded transposon mutant sequencing (RB-TnSeq), to discover fitness factors that promote growth of three related bacterial strains in a common niche, tomato xylem sap. Fitness of wild-type and mutants show that Ralstonia spp. are adapted to grow well in xylem sap from their natural host plant, tomato. Our screen identified multiple sap-specific fitness factors with roles in maintaining the bacterial envelope. These factors include putative adaptations to resist plant defenses that may include antimicrobial proteins and specialized metabolites that damage bacterial membranes.


title: Database and deep learning toolbox for noise-optimized, generalized spike inference from calcium imaging data

abstract: Calcium imaging is a key method to record patterns of neuronal activity across populations of identified neurons. Inference of temporal patterns of action potentials ( spikes) from calcium signals is, however, challenging and often limited by the scarcity of ground truth data containing simultaneous measurements of action potentials and calcium signals. To overcome this problem, we compiled a large and diverse ground truth database from publicly available and newly performed recordings. This database covers various types of calcium indicators, cell types, and signal-to-noise ratios and comprises a total of >35 hours from 298 neurons. We then developed a novel algorithm for spike inference (CASCADE) that is based on supervised deep networks, takes advantage of the ground truth database, infers absolute spike rates, and outperforms existing model-based algorithms. To optimize performance for unseen imaging data, CASCADE retrains itself by resampling ground truth data to match the respective sampling rate and noise level. As a consequence, no parameters need to be adjusted by the user. To facilitate routine application of CASCADE we developed systematic performance assessments for unseen data, we openly release all resources, and we provide a user-friendly cloud-based implementation.


title: Identification of activity-induced Egr3-dependent genes reveals genes associated with DNA damage response and schizophrenia

abstract: Bioinformatics and network studies have identified the immediate early gene transcription factor early growth response 3 (EGR3) as a master regulator of genes differentially expressed in the brains of patients with neuropsychiatric illnesses ranging from schizophrenia and bipolar disorder to Alzheimers disease. However, few studies have identified and validated Egr3-dependent genes in the mammalian brain. We have previously shown that Egr3 is required for stress-responsive behavior, memory, and hippocampal long-term depression in mice. To identify Egr3-dependent genes that may regulate these processes, we conducted an expression microarray on hippocampi from wildtype (WT) and Egr3-/- mice following electroconvulsive seizure (ECS), a stimulus that induces maximal expression of immediate early genes including Egr3. We identified 71 genes that were differentially expressed between WT and Egr3-/- mice one hour following ECS. Bioinformatic analyses showed that many of these are altered in schizophrenia. Ingenuity pathway analysis revealed the GADD45 (growth arrest and DNA-damage-inducible) family (Gadd45b, Gadd45g) as the leading category of differentially expressed genes. Together with differentially expressed genes in the AP-1 transcription factor family genes (Fos, Fosb), and the centromere organization protein Cenpa, these results revealed that Egr3 is required for activity-dependent expression of genes involved in the DNA damage response. Our findings implicate EGR3 as gene critical for the expression of genes that are disrupted in schizophrenia and reveal a novel requirement for EGR3 in the expression of genes involved in activity-induced DNA damage response.


title: The spectrum of covariance matrices of randomly connected recurrent neuronal networks

abstract: A key question in theoretical neuroscience is the relation between the connectivity structure and the collective dynamics of a network of neurons. Here we study the connectivity-dynamics relation as reflected in the distribution of eigenvalues of the covariance matrix of the dynamic fluctuations of the neuronal activities, which is closely related to the networks Principal Component Analysis (PCA) and the associated effective dimensionality. We consider the spontaneous fluctuations around a steady state in a randomly connected recurrent network of stochastic neurons. An exact analytical expression for the covariance eigenvalue distribution in the large-network limit can be obtained using results from random matrices. The distribution has a finitely supported smooth bulk spectrum and exhibits an approximate power-law tail for coupling matrices near the critical edge. We generalize the results to include connectivity motifs and discuss extensions to Excitatory-Inhibitory networks. The theoretical results are compared with those from finite-size networks and and the effects of temporal and spatial sampling are studied. Preliminary application to whole-brain imaging data is presented. Using simple connectivity models, our work provides theoretical predictions for the covariance spectrum, a fundamental property of recurrent neuronal dynamics, that can be compared with experimental data.


title: Strategies of grain number determination differentiate barley row-types

abstract:
Gaining knowledge on intrinsic interactions of various yield components is crucial to improve the yield potential in small grain cereals. It is well known in barley that increasing the grain number (GN) preponderantly improves their yield potential; however, the yield components determining GN and their association in barley row-types are less explored. In this study, we assessed different yield components like potential spikelet number (PSN), spikelet survival (SSL), spikelet number (SN), grain set (GS), and grain survival (GSL), as well as their interactions with GN by using a selected panel of two- and six-rowed barley types. Also, to analyze the stability of these interactions, we performed the study in two growth conditions, greenhouse and field. From this study, we found that in two-rowed, GN determination is strongly influenced by PSN rather than SSL and/or GS in both growth conditions. Conversely, in six-rowed, GN is associated with SSL instead of PSN and/or GS. Thus, our study exemplified that increasing GN might be possible by augmenting PSN in two-rowed genotypes, while for six-rowed genotypes, the ability of SSL needs to be improved. We speculate that this disparity of GN determination in barley row-types might be due to the fertility of lateral spikelets. Collectively, this study revealed that the GN of two-rowed largely depends on the developmental trait, PSN, while in six-rowed, it mainly follows the ability of SSL.

HighlightIn cereals, understanding the interactions of different yield components that influence the grain number is essential to increase their yield by modulating the components. We show in this study that the grain number of two-rowed barley is predominantly determined by the potential spikelet number while in six-rowed by spikelet survival.


title: Molecular and evolutionary mechanisms of yeast thermotolerance

abstract: Many familiar traits in the natural world--from lions manes to the longevity of bristlecone pine trees--arose in the distant past, and have long since fixed in their respective species. A key challenge in evolutionary genetics is to figure out how and why species-defining traits have come to be. We used the thermotolerance growth advantage of the yeast Saccharomyces cerevisiae over its sister species Saccharomyces paradoxus as a model for addressing these questions. Analyzing loci at which the S. cerevisiae allele promotes thermotolerance, we detected robust evidence for positive selection, including amino acid divergence between the species and conservation within S. cerevisiae populations. Since such signatures were particularly strong at the chromosome segregation gene ESP1, we used this locus as a case study for focused mechanistic follow-up. Experiments revealed that, in culture at high temperature, the S. paradoxus ESP1 allele conferred a qualitative defect in biomass accumulation and cell division relative to the S. cerevisiae allele. Only genetic divergence in the ESP1 coding region mattered phenotypically, with no functional impact detectable from the promoter. Together, these data support a model in which an ancient ancestor of S. cerevisiae, under selection to boost viability at high temperature, acquired amino acid variants at ESP1 and many other loci, which have been constrained since then. Complex adaptations of this type hold promise as a paradigm for interspecies genetics, especially in deeply diverged traits that may have taken millions of years to evolve.


title: VeTra: a tool for trajectory inference based on RNA velocity

abstract: Trajectory inference (TI) for single cell RNA sequencing (scRNAseq) data is a powerful approach to interpret dynamic cellular processes such as cell cycle and development. Still, however, accurate inference of trajectory is challenging. Recent development of RNA velocity provides an approach to visualize cell state transition without relying on prior knowledge. To perform TI and group cells based on RNA velocity we developed VeTra. By applying cosine similarity and merging weakly connected components, VeTra identifies cell groups from the direction of cell transition. Besides, VeTra suggests key regulators from the inferred trajectory. VeTra is a useful tool for TI and subsequent analysis.


title: Reduced anterior cingulate cortex volume induced by chronic stress correlates with increased behavioral emotionality and decreased synaptic puncta density

abstract:
Clinical and preclinical studies report that chronic stress induces behavioral deficits as well as volumetric and synaptic alterations in corticolimbic brain regions including the anterior cingulate cortex (ACC), amygdala (AMY), nucleus accumbens (NAc) and hippocampus (HPC). Here, we aimed to investigate the volumetric changes associated with chronic restraint stress (CRS) and link these changes to the CRS-induced behavioral and synaptic deficits. We first confirmed that CRS increases behavioral emotionality, defined as collective scoring of anxiety- and anhedonia-like behaviors. We then demonstrated that CRS induced a reduction of total brain volume which negatively correlated with behavioral emotionality. Region-specific analysis identified that only the ACC showed significant decrease in volume following CRS (p<0.05). Reduced ACC correlated with increased behavioral emotionality (r=-0.56; p=0.0003). Although not significantly altered by CRS, AMY and NAc (but not the HPC) volumes were negatively correlated with behavioral emotionality. Finally, using structural covariance network analysis to assess shared volumetric variances between the corticolimbic brain regions and associated structures, we found a progressive decreased ACC degree and increased AMY degree following CRS. At the cellular level, reduced ACC volume correlated with decreased PSD95 (but not VGLUT1) puncta density (r=0.35, p<0.05), which also correlated with increased behavioral emotionality (r=-0.44, p<0.01), suggesting that altered synaptic strength is an underlying substrate of CRS volumetric and behavioral effects. Our results demonstrate that CRS effects on ACC volume and synaptic density are linked to behavioral emotionality and highlight key ACC structural and morphological alterations relevant to stress-related illnesses including mood and anxiety disorders.

HighlightsO_LIChronic restraint stress (CRS) decreases anterior cingulate cortex (ACC) volume
C_LIO_LIACC and amygdala (AMY) volumes negatively correlate with behavioral emotionality
C_LIO_LICRS decreased the strength and degree of the ACC structural covariance network
C_LIO_LICRS increased the strength and degree of the AMY structural covariance network
C_LIO_LIPSD95 puncta density correlates with behavioral emotionality and ACC volume.
C_LI


title: mtDNA replication in dysfunctional mitochondria promotes deleterious heteroplasmy via the UPRmt

abstract: The accumulation of deleterious mitochondrial genomes ({Delta}mtDNAs) underlies inherited mitochondrial diseases and contributes to the aging-associated decline in mitochondrial function. In heteroplasmic cells, oxidative phosphorylation (OXPHOS) function declines as the population of {Delta}mtDNAs increase relative to wildtype mtDNAs. In response to mitochondrial perturbations, the bZIP protein ATFS-1 induces a transcription program to promote the recovery of mitochondrial function. Paradoxically, ATFS-1 is also required to maintain {Delta}mtDNAs in heteroplasmic worms. However, the mechanism(s) by which ATFS-1 promotes {Delta}mtDNA accumulation relative to wildtype mtDNAs is unclear. Here, we show that mitochondrial-localized ATFS-1 binds almost exclusively to {Delta}mtDNAs in heteroplasmic worms. Moreover, we demonstrate that mitochondrial ATFS-1 promotes the preferential binding of the mtDNA replicative polymerase (POLG) to {Delta}mtDNAs. Interestingly, inhibition of the mtDNA-bound protease LONP-1 increased ATFS-1 and POLG binding to wildtype mtDNAs. Furthermore, LONP-1 inhibition in C. elegans and human cybrid cells improved the heteroplasmy ratio and restored OXPHOS function. Our findings suggest that ATFS-1 promotes mtDNA replication by recruiting POLG to mtDNA in a manner that is antagonized by LONP-1. We speculate that this mechanism promotes the repair and expansion of the mitochondrial network by synchronizing mtDNA replication with UPRmt activation driven by nuclear ATFS-1 activity. However, this repair mechanism cannot resolve OXPHOS defects in mitochondria harboring {Delta}mtDNAs, resulting in an accumulation of ATFS-1 in dysfunctional mitochondria and constitutive replication of {Delta}mtDNAs.


title: Stochastic survival of the densest and mitochondrial DNA clonal expansion in ageing

abstract: The expansion of deleted mitochondrial DNA molecules has been associated with ageing 1,2, particularly in skeletal muscle fibres 3-5; its mechanism has remained unclear for three decades. Previous accounts have assigned a replicative advantage to the deletions 6-8, but there is evidence that cells can, instead, selectively remove defective mitochondrial DNA 9. Here we present a spatial model that, without a replicative advantage, but instead through a combination of enhanced density for mutants and noise, produces a wave of expanding mutations with speeds consistent with experimental data 10. A standard model based on replicative advantage yields waves that are too fast. We provide a formula that predicts that wave-speed drops with copy number, consonant with experimental data. Crucially, our model yields travelling waves of mutants even if mutants are preferentially eliminated. Additionally, we predict that experimentally observed mutant loads can be produced by de novo mutation rates that are drastically lower than previously thought for neutral models 11. Given this exemplar of how noise, density and spatial structure affect muscle age-ing, we introduce the mechanism of stochastic survival of the densest, an alternative to replicative advantage, that may underpin other evolutionary phenomena.


title: The actin nucleator Spir-1 is a virus restriction factor that promotes IRF3 activation

abstract:
Cellular proteins often have multiple and diverse functions. This is illustrated with protein Spir-1 that is an actin nucleator, but, as shown here, also functions to enhance IRF3 activation downstream of RNA sensing by RIG-I/MDA-5. In human and mouse cells lacking Spir-1, IRF3 activation is impaired, whereas Spir-1 overexpression enhanced IRF3 activation. Furthermore in Spir-1-/- cells, the infectious virus titres and sizes of plaques formed by two viruses that are sensed by RIG-I, vaccinia virus (VACV) and Zika virus, are increased. These observations demonstrate the biological importance of Spir-1 in the response to virus infection. Like cellular proteins, viral proteins also have multiple and diverse functions. Here, we also show that VACV virulence factor K7 binds directly to Spir-1 and that a diphenylalanine motif of Spir-1 is needed for this interaction and for Spir-1-mediated enhancement of IRF3 activation. Thus, Spir-1 is a new virus restriction factor and is targeted directly by an immunomodulatory viral protein that enhances virus virulence and diminishes IRF3 activation.

Author SummaryInfection of cells by viruses is sensed by host molecules called pattern recognition receptors (PRRs) that activate signalling pathways leading to an anti-viral response. In turn, viruses express proteins that negate these host responses to mediate escape from the anti-viral response. Here, we report that protein K7 from a large DNA virus called vaccinia virus (VACV), binds to a host cell protein called Spir-1. Spir-1 is known to regulate the assembly of actin filaments inside cells, but here we show that Spir-1 also functions to activate the host response to virus infection and to limit the replication and spread of both RNA and DNA viruses. Thus, this study has uncovered new functions of cellular protein Spir-1 as an activator of innate immunity and as a restriction factor for diverse viruses. Further, it shows that Spir-1 is targeted by a virus protein during infection.


title: A genome-wide CRISPR-Cas9 screen identifies CENPJ as a host regulator of altered microtubule organization during Plasmodium liver infection.

abstract: Prior to initiating symptomatic malaria, a single Plasmodium sporozoite infects a hepatocyte and develops into thousands of merozoites, in part by scavenging host resources. We show that host microtubules dynamically reorganize around the developing liver stage (LS) parasite. Using a genome-wide CRISPR-Cas9 screen, we identified host regulators of cytoskeleton organization, vesicle trafficking, ER/Golgi stress and lipid biogenesis that regulate Plasmodium LS development. These novel regulators of infection, including Centromere Protein J (CENPJ), led us to interrogate how microtubule organizing centers (MTOCs) are regulated during infection. Foci of {gamma}-tubulin localized to the parasite periphery; depletion of CENPJ exacerbated this re-localization and increased infection. Further, we show that the Golgi acts as a non-centrosomal MTOC by organizing {gamma}-tubulin and stimulating microtubule nucleation at the parasite periphery. Collectively, we show that the Plasmodium LS recruits the host Golgi to form MT mediated conduits along which host organelles are recruited to the PVM, to support liver stage development. Our findings suggest many host-targeted pharmacological inhibitors may inhibit LS infection.


title: Maintaining robust size across environmental conditions through plastic growth dynamics

abstract:
Organ growth is tightly regulated across environmental conditions to generate appropriate final size. While the size of some organs is free to vary, others need to maintain constant size to function properly. This poses a unique problem: how is robust final size achieved when environmental conditions can alter some major growth processes? While we know that brain growth is "spared" from the effects of the environment from humans to fruit flies, we do not understand how this process alters growth dynamics across brain compartments. Here, we explore how this robustness in brain size is achieved by examining differences in growth patterns between the larval body, the brain, and a brain compartment - the mushroom bodies - in Drosophila melanogaster across both thermal and nutritional conditions. We identify key differences in patterns of growth between the whole brain and mushroom bodies that are likely to underlie robustness of final organ shape. Further, we show that these differences produce distinct brain shapes across environments.

Significance of StudyA long-standing question in Biology has been how fully functional multicellular organisms with highly specialized organs are generated, given that organs initiate growth at different times across development. Although the genetic mechanisms that underlie growth has been studied extensively, we are yet to understand how growth pattern of organs produces distinct final shapes across changing environmental conditions. We use the Drosophila brain, to reveal that key differences in growth dynamics are likely to underlie robustness of final organ shape and are tuned by nutrition and temperature. Further deepening our knowledge of how final organ shape is maintained across environmental conditions.


title: RecT recombinase expression enables efficient gene editing in Enterococcus

abstract:
Enterococcus faecium is a ubiquitous Gram-positive bacterium that has been recovered from the environment, food, and microbiota of mammals. Commensal strains of E. faecium can confer beneficial effects on host physiology and immunity, but antibiotic usage has afforded antibiotic-resistant and pathogenic isolates from livestock and humans. However, the dissection of E. faecium functions and mechanisms has been restricted by inefficient gene editing methods. To address these limitations, here we report the expression of E. faecium RecT recombinase significantly improves the efficiency of recombineering technologies in both commensal and antibiotic-resistant strains of E. faecium and other Enterococcus species such as E. durans and E. hirae. Notably, the expression of RecT in combination with clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 and guide RNAs (gRNAs) enabled highly efficient scar-less single-stranded DNA recombineering to generate specific gene editing mutants in E. faecium. Moreover, we demonstrate that E. faecium RecT expression facilitated chromosomal insertions of double-stranded DNA templates encoding antibiotic selectable markers to generate gene deletion mutants. As further proof-of-principle, we use CRISPR-Cas9 mediated recombineering to knock out both sortase A genes in E. faecium for downstream functional characterization. The general RecT-mediated recombineering methods described here should significantly enhance genetic studies of E. faecium and other closely related species for functional and mechanistic studies.

ImportanceEnterococcus faecium is widely recognized as an emerging public health threat with the rise of drug resistance and nosocomial infections. Nevertheless, commensal Enterococcus strains possess beneficial health functions in mammals to upregulate host immunity and prevent microbial infections. This functional dichotomy of Enterococcus species and strains highlights the need for in-depth studies to discover and characterize the genetic components underlining its diverse activities. However, current genetic engineering methods in E. faecium still require passive homologous recombination from plasmid DNA. This involves the successful cloning of multiple homologous fragments into a plasmid, introducing the plasmid into E. faecium, and screening for double-crossover events that can collectively take up to multiple weeks to perform. To alleviate these challenges, we show that RecT recombinase enables rapid and efficient integration of mutagenic DNA templates to generate substitutions, deletions, and insertions in genomic DNA of E. faecium. These improved recombineering methods should facilitate functional and mechanistic studies of Enterococcus.


title: Rabaptin5 targets autophagy to damaged early endosomes and Salmonella containing vacuoles by interaction with FIP200 and ATG16L1

abstract: Selective autophagy of damaged organelles is an important process for cellular homeostasis. The mechanisms how autophagy selects specific targets is often poorly understood. Rabaptin5 was previously known as a major regulator of early endosome identity and maturation. Here we identified two novel Rabaptin5 interactors: FIP200, a subunit of the ULK1 autophagy initiator complex, and ATG16L1, a central component of the E3-like enzyme in LC3 lipidation. Indeed, autophagy of early endosomes damaged by chloroquine or monensin treatment was found to require Rabaptin5 and particularly a specific short sequence motif binding to the WD domain of ATG16L1. Rabaptin5 and this interaction with ATG16L1 is further required for much of autophagic elimination of Salmonella enterica in phagosomes with early endosomal characteristics early after infection. Our results demonstrate a novel function of Rabaptin5 in quality control of early endosomes in the selective recruitment of autophagy to damaged early endosomes and phagosomes.


title: Mosquito sound communication: are male swarms loud enough to attract females?

abstract: Given the unsurpassed sound-sensitivity of mosquitoes among arthropods and the sound-source power required for long-range hearing, we investigated the distance over which female mosquitoes detect species-specific cues in the sound of station-keeping mating swarms. A common misunderstanding, that mosquitoes cannot hear at long-range because their hearing organs are  particle-velocity receptors, has clouded the fact that particle-velocity is an intrinsic component of sound whatever the distance to the sound source. We exposed free-flying Anopheles coluzzii females to pre-recorded sounds of male An. coluzzii and An. gambiae s.s. swarms over a range of natural sound-levels. Sound-levels tested were related to equivalent distances between the female and the swarm for a given number of males, enabling us to infer distances over which females might hear large male-swarms. We show that females do not respond to swarm sound up to 48 dB SPL and that louder SPLs are not ecologically relevant for a swarm. Considering that swarms are the only mosquito sound-source that would be loud enough to be heard at long-range, we conclude that inter-mosquito acoustic communication is restricted to close-range pair interactions. We also showed that the sensitivity to sound in free-flying males is much enhanced compared to that of tethered ones.


title: Tree-Aggregated Predictive Modeling of Microbiome Data

abstract: Modern high-throughput sequencing technologies provide low-cost microbiome survey data across all habitats of life at unprecedented scale. At the most granular level, the primary data consist of sparse counts of amplicon sequence variants or operational taxonomic units that are associated with taxonomic and phylogenetic group information. In this contribution, we leverage the hierarchical structure of amplicon data and propose a data-driven and scalable tree-guided aggregation framework to associate microbial subcompositions with response variables of interest. The excess number of zero or low count measurements at the read level forces traditional microbiome data analysis workflows to remove rare sequencing variants or group them by a fixed taxonomic rank, such as genus or phylum, or by phylogenetic similarity. By contrast, our framework, which we call trac (tree-aggregation of compositional data), learns data-adaptive taxon aggregation levels for predictive modeling, greatly reducing the need for user-defined aggregation in preprocessing while simultaneously integrating seamlessly into the compositional data analysis framework. We illustrate the versatility of our framework in the context of large-scale regression problems in human gut, soil, and marine microbial ecosystems. We posit that the inferred aggregation levels provide highly interpretable taxon groupings that can help microbiome researchers gain insights into the structure and functioning of the underlying ecosystem of interest.


title: Tapping into a phospholipid-LRH-1 axis yields a powerful anti-inflammatory agent with in vivo activity against colitis

abstract: Phospholipids are ligands for nuclear hormone receptors (NRs) and regulate transcriptional programs relevant to normal physiology and disease. Here, we demonstrate that mimicking phospholipid-NR interactions greatly improves agonists of liver receptor homolog-1 (LRH-1), a promising therapeutic target for diabetes and colitis. Conventional LRH-1 modulators partially occupy the binding pocket, leaving vacant a region important for phospholipid binding and allostery. Therefore, we constructed a set of hybrid molecules with elements of natural phospholipids appended to a synthetic LRH-1 agonist. The phospholipid-mimicking group improves binding affinity, increases LRH-1 transcriptional activity, promotes coregulator recruitment, and interacts with the targeted LRH-1 residues in crystal structures. The best new agonist markedly improves colonic histopathology and disease-related weight loss in a humanized LRH-1 murine T-cell transfer model of colitis. This is the first evidence of in vivo efficacy for an LRH-1 modulator in colitis, a leap forward in agonist development.