title: Early transition in foraging mode promoted greater reproductive effort in widely foraging lizards

abstract: Life-history theory predicts that the optimal reproductive effort of an organism is affected by factors such as energy acquisition and predation risk. Although this is a major focus of study in evolutionary ecology, the empirical evidence consists of conflicting data on a few organisms. For instance, theoretical models within the foraging mode paradigm suggest that widely foraging females have evolved low reproductive effort, because a heavy reproductive load decreases their ability to escape from predators. By contrast, a long-standing prediction of evolutionary theory indicates that organisms subject to high mortality due to predation, as suggested for widely foraging species, should increase their reproductive investment. Here, we revise the available literature on the relationship between foraging mode and reproductive effort of lizards. In doing so, we present evidence that widely foraging species have evolved greater reproductive effort than sit-and wait species. This is the largest comparative analysis of foraging mode and reproductive effort to date: 485 species grouped in 32 families. Based on our findings, we propose a theoretical model derived from the optimal foraging theory that potentially explains the observed patterns in lizards, paving the way for ecologist to test mechanistic hypotheses at the intraspecific level.

category: evolutionary biology


title: PRC2-mediated repression is essential to maintain identity and function of differentiated dopaminergic and serotonergic neurons

abstract: How neurons in the CNS can maintain cellular identity over an entire lifespan remains largely unknown. Here we show that long-term maintenance of identity in differentiated dopaminergic and serotonergic neurons is critically reliant on the Polycomb repressive complex 2 (PRC2). Deletion of the obligate PRC2-component, Eed, in these neurons, resulted in global loss of H3K27me3, followed by a gradual activation of genes harbouring both H3K27me3 and H3K9me3 modifications. Notably, H3K9me3 was also lost at these PRC2-targets prior to gene activation. Neuronal survival was not compromised, instead there was a reduction in subtype specific gene expression as well as a progressive impairment of dopaminergic or serotonergic neuronal function leading to behavioural deficits characteristic of Parkinsons disease (PD) or mood disorders, respectively. Single cell analysis revealed an unexpected subtype specific vulnerability to loss of PRC2-repression in dopamine neurons of the substantia nigra, the neurons primarily affected in PD. Taken together, our study reveals that a PRC2-dependent non-permissive chromatin state is essential to maintain subtype identity and function of dopaminergic and serotonergic neurons.

category: neuroscience


title: The amyloid precursor protein modulates the position and length of the axon initial segment offering a new perspective on Alzheimer's disease genetics

abstract:
The small A{beta} peptide has been hypothesized to be the main driver of Alzheimers disease (AD). A{beta} is a proteolytic cleavage product of a larger protein, the amyloid precursor protein (APP), whose normal functions remain largely unexplored. We report here activities of the full-length APP protein that relate directly to the etiology of AD. Increasing neuronal activity leads to a rapid increase in App gene expression. In both cultures of mouse cortical neurons and human iPSC-derived neurons, elevated APP protein changes the structure of the axon initial segment (AIS), the site of action potential initiation. In neurons with elevated APP, the AIS shortens in length and shifts in position away from the cell body. Both changes would be expected to reduce neuronal excitability. The AIS effects are due to the cell-autonomous actions of APP; exogenous A{beta} - either fibrillar or oligomeric - has no effect. The findings relate directly to AD in several ways. In culture, APP carrying the Swedish familial AD mutation (APPSwe) induces stronger AIS changes than wild type APP. Ankyrin G and {beta}IV-spectrin, scaffolding proteins of the AIS, both physically associate with APP, and APPSwe binds more avidly than wild type APP. Finally, neurons in the frontal cortex of humans with sporadic AD reveal histologically elevated levels of APP protein that invade the domain of the AIS, whose length is significantly shorter than that found in healthy control neurons. The findings offer an alternative explanation for the effects of at least some familial AD mutations.

SignificanceIn familial Alzheimers disease (AD) the linkage between the genetics of APP, the neuropathology of the amyloid plaques and the symptoms of dementia are one of the strongest pieces of evidence supporting the amyloid cascade hypothesis - a conceptualization that marks the A{beta} peptide as the root cause of AD. Yet, formally, the genetics only point to APP, not its A{beta} breakdown product. We report here that the full-length APP protein affects the properties of the axon initial segment and through these changes serves as a dynamic regulator of neuronal activity. We propose that this newly discovered APP function offers a different, A{beta}-independent, view of the genetic evidence.


title: Cell type diversity in a developing octopus brain

abstract:
Octopuses are mollusks that have evolved intricate neural systems comparable with vertebrates in terms of cell number, complexity and size. The cell types within the octopus brain that control their amazingly rich behavioral repertoire are still unknown. Here we profile cell diversity of the paralarval Octopus vulgaris brain to build a comprehensive cell type atlas that comprises mostly neural cells, as well as multiple glial subtypes, endothelial cells and fibroblasts. Moreover, we spatially map cell types within the octopus brain, including vertical and optic lobe cell types. Investigation of cell type conservation reveals a shared gene signature between glial cells of mice, fly and octopus. Genes related to learning and memory are enriched in vertical lobe cells, which show molecular similarities with Kenyon cells in Drosophila. Taken together, our data sheds light on cell type diversity and evolution of the complex octopus brain.

Highlights & Key findingsO_LICharacterization of different cell types present in the early paralarval brain
C_LIO_LICross-species comparisons reveal a conserved glial gene expression signature
C_LIO_LIVertical lobe amacrine cells in octopus have molecular similarities to fly Kenyon cells
C_LIO_LIHomeobox genes are defining transcription factors for cell type identity
C_LIO_LIRecently expanded gene families may underlie cellular diversification
C_LI

category: developmental biology


title: Tracking Genomic Characteristics across Oceanic Provinces: Contrasting Early and Mature Plastic Biofilm Communities

abstract:
While plastic has become omnipresent in the marine environment, knowledge of how plastic biofilm communities develop from functional metabolic and phylogenetic perspectives is nascent, although these data are central to understanding microbial ecology surrounding plastic substrates in the ocean. By incubating virgin microplastics during oceanic transects and comparing with naturally occurring plastic litter at the same locations, we constructed functional gene catalogs to contrast the metabolic differences between early and mature biofilm communities. Early colonization incubations were consistently dominated by Alteromonadaceae and harbored significantly higher proportions of genes associated with adhesion, biofilm formation, chemotaxis, defense, iron acquisition and utilization, and motility. Comparative genomic analyses with Alteromonas, Marinobacter, and Marisediminitalea metagenome assembled genomes (MAGs) spotlighted the importance of the mannose-sensitive hemagglutinin operon, adhesive genes genetically transposed from intestinal pathogens, for early colonization of hydrophobic plastic surfaces. Synteny alignments of the former operon also demonstrated apparent positive selection for mshA alleles across all MAGs. Early colonizers varied little in terms of large-scale genomic characteristics, despite the presence of latitudinal, salinity, and temperature gradients. Mature plastic biofilms, composed of predominantly Rhodobacteraceae followed by Flavobacteriaceae, that are critically important for carbon turnover in oceanic ecosystems, displayed significantly higher proportions of genes involved in oxidative phosphorylation, phosphonate metabolism, photosynthesis, secondary metabolism, and Type IV secretion. Our metagenomic analyses provide insight into early biofilm formation on virgin surfaces in the marine environment, as well as how early colonizers self-assemble, compared to mature, taxonomically, and metabolically diverse biofilms.

Significance StatementLittle is known about plastic biofilm assemblage dynamics and successional changes over time. Our results demonstrate that highly reproducible and predictable types of bacteria, with similar genomic characteristics, can initially colonize plastic in the marine environment across varying environmental gradients. The key gene sets involved in foundational bacterial colonization may have broad impacts for biofilm formation on plastic surfaces used in agriculture, biomedicine, environmental science, and food science. Genomic characteristics of early colonizers may metabolically underpin the origin of the ordered succession observed in marine microbial communities and be useful for predicting microbial community membership and biogeochemical processes.

category: ecology


title: On taming the effect of transcript level intra-condition count variation during differential expression analysis: a story of dogs, foxes and wolves

abstract: The evolution of RNA-seq technologies has yielded datasets of high scientific value that are often generated as condition associated biological replicates within differential expression studies. As the number of replicates increase, so to does confidence in identifying differentially expressed transcripts. With rapidly expanding RNA-seq data archives there is opportunity to augment replicate numbers when conditions of interest overlap at an inter-study level. Despite correction procedures for estimating transcript abundance, a remaining source of error is transcript level intra-condition count variation; as partially indicated by the disjointed results between differential expression analysis tools. Such variation is amplified at an inter-study level. Here, we present TVscript, a tool that removes reference-based transcripts associated with intra-condition variation above specified thresholds. With this tool we explore the effects of removing transcripts associated with varying degrees of intra-condition variation on differential expression analysis. This is done in relation to inter- and intra-study datasets representing brain samples of dogs, wolves and foxes (wolves vs. dogs and aggressive vs. tame foxes). We demonstrate that 20% of the transcripts identified as being differentially expressed are associated with high levels of intra-condition variation. This is an over-representation relative to the reference set. As transcripts harbouring such variation are removed from the reference prior to differential expression analysis a discordance from 26 to 40% in the lists of differentially expressed transcripts is observed when compared to those obtained using the non-filtered reference. For our data, the removal of transcripts possessing intra-condition variation values within (and above) the 97th and 95th percentiles, for wolves vs. dogs and aggressive vs. tame foxes, maximized the detection of differentially expressed transcripts as a result of alterations within gene-wise dispersion estimates. Through this analysis the support for seven genes with potential for being involved with selection for tameness is provided. TVscript is available at: https://sourceforge.net/projects/tvscript/.

category: bioinformatics


title: Alternative Assembly of Qβ Virus Like Particles

abstract: Q{beta} virus like particles (VLPs) are versatile platforms for grafting functional groups for vaccine development. The structure of Q{beta} VLPs at atomic detail are critical for design of more effective vaccines. While the structures of native Q{beta} VLPs have been determined previously, the structure of VLPs assembled from a recombinantly expressed Q{beta} coat protein, which are extensively used as platforms have not been studied. We sought to determine the crystal structures of VLPs assembled from recombinantly expressed Q{beta} coat protein of wild type and two mutants: A38K and A38K/A40C/D102C. The structures of Q{beta} VLPs assembled from recombinantly expressed Q{beta} coat proteins showed that VLPs can be assembled both in T=1 and T=3 symmetry.

category: biophysics


title: In vitro analysis of catalase and superoxide dismutase mimetic properties of blue tattoo ink

abstract: Tattoo inks are comprised of different combinations of bioactive chemicals with combined biological effects that are insufficiently explored. Tattoos have been associated with oxidative stress; however, a recent N-of-1 study suggested that blue tattoos may be associated with suppressed local skin oxidative stress. The present study aimed to explore the attributes of the blue tattoo ink (BTI) that may explain its possible effects on redox homeostasis, namely the catalase (CAT) and superoxide dismutase (SOD)-mimetic properties that have been reported for copper(II) phthalocyanine (CuPC) - the main BTI constituent. Intenze Persian blue (PB) BTI has been used in the experiment. CAT and SOD-mimetic properties of PB and its pigment-enriched fractions were analyzed using the carbonato-cobaltate (III) formation-derived H2O2 dissociation and 1,2,3-trihydroxybenzene autoxidation rate assays utilizing simple buffers and biochemical matrix of normal skin tissue as chemical reaction environments. CuPC-based tattoo ink PB and both its blue and white pigment-enriched fractions demonstrate CAT and SOD-mimetic properties in vitro with effect sizes demonstrating a substantial dependence on the biochemical environment. PB constituents act as inhibitors of CAT but potentiate its activity in the biochemical matrix of the skin. CuPC-based BTI can mimic antioxidant enzymes, however chemical constituents other than CuPC (e.g. the photoreactive TiO2) seem to be at least partially responsible for the BTI redox-modulating properties.

category: pharmacology and toxicology


title: Trichodesmium Erythraeum produces a higher photocurrent than other cyanobacterial species in bio-photo electrochemical cells.

abstract: In recent years, the increase in world energy consumption, and the worries from potential future disasters that may derive from climate change have inspired the motivation to develop renewable energy technologies. One of the promising methods is the utilization of whole bacterial cells to produce photocurrent in a bio-photo electrochemical cell (BPEC). The photocurrent derives from the photosynthesis pathway, while the redox couple NADP+/NADPH perform cyclic electron mediation between photosystem I inside the cells, and the anode. Over the years, various cyanobacterial species were utilized in diverse BPECs setups, while the photocurrent was enhanced by the addition of natural electron mediators such as NAD+, NADP+, Cytochrome C, Vitamin B1, and the artificial mediator potassium ferricyanide. The cyanobacterium Trichodesmium Erythraeum (Te) is a marine species that consist of high content of Phycocyanin and Phycoerythrin pigments that play a major role in photosynthesis enhancement. In this work, we produce for the first-time photocurrent from Te. We apply 2D-fluorescence measurements to detect its NADPH secretion and show that its photocurrent production is enhanced as a function of increasing electrolyte salinity. Finally, we produce photocurrent from additional cyanobacterial species: Synechocystis sp. PCC6803, Synechococcus elongatus PCC 7942, Acaryochloris marina MBIC 11017, and Spirulina, using their cultivation medium as electrolytes in the BPEC. We show that TE produces a photocurrent intensity that is significantly greater than all other species with and without the addition of exogenous electron mediators. The utilization of TE may pave the way toward the establishment of marine clean energy technologies.

category: microbiology


title: Comprehensive visualization of cell-cell interactions in single-cell and spatial transcriptomics with NICHES

abstract:
SummaryRecent years have seen the release of several toolsets that reveal cell-cell interactions from single-cell data. However, all existing approaches leverage mean celltype gene expression values, and do not preserve the single-cell fidelity of the original data. Here, we present NICHES (Niche Interactions and Communication Heterogeneity in Extracellular Signaling), a tool to explore extracellular signaling at the truly single-cell level. NICHES allows embedding of ligand-receptor signal proxies to visualize heterogeneous signaling archetypes within cell clusters, between cell clusters, and across experimental conditions. When applied to spatial transcriptomic data, NICHES can be used to reflect local cellular microenvironment. NICHES can operate with any list of ligand-receptor signaling mechanisms and is compatible with existing single-cell packages and pseudotime techniques. NICHES is also a user friendly and extensible program, allowing rapid analysis of cell-cell signaling at single-cell resolution.

Availability and implementationNICHES is an open-source software implemented in R under academic free license v3.0 and it is available at github.com/msraredon/NICHES.

Contactmichasam.raredon@yale.edu; yuval.kluger@yale.edu


title: A surface morphometrics toolkit to quantify organellar membrane ultrastructure using cryo-electron tomography

abstract: Cellular cryo-electron tomography (cryo-ET) enables 3-dimensional reconstructions of organelles in their native cellular environment at subnanometer resolution. However, quantifying ultrastructural features of pleomorphic organelles in three dimensions is challenging, as is defining the significance of observed changes induced by specific cellular perturbations. To address this challenge, we established a semi-automated workflow to segment organellar membranes and reconstruct their underlying surface geometry in cryo-ET. To complement this workflow, we developed an open source suite of ultrastructural quantifications, integrated into a single pipeline called the surface morphometrics toolkit. This toolkit allows detailed mapping of spacing, curvature, and orientation onto reconstructed membrane meshes, highlighting subtle organellar features that are challenging to detect in three dimensions and allowing for statistical comparison across many organelles. To demonstrate the advantages of this approach, we combine cryo-ET with cryo-fluorescence microscopy to correlate bulk mitochondrial network morphology (i.e., elongated versus fragmented) with membrane ultrastructure of individual mitochondria in the presence and absence of endoplasmic reticulum (ER) stress. Using our toolkit, we demonstrate ER stress promotes adaptive remodeling of ultrastructural features of mitochondria including spacing between the inner and outer membranes, local curvature of the inner membrane, and spacing between mitochondrial cristae. We show that differences in membrane ultrastructure correlate to mitochondrial network morphologies, suggesting that these two remodeling events are coupled. Our toolkit offers opportunities for quantifying changes in organellar architecture on a single-cell level using cryo-ET, opening new opportunities to define changes in ultrastructural features induced by diverse types of cellular perturbations.

category: cell biology


title: Severe acute respiratory disease in American mink (Neovison vison) experimentally infected with SARS-CoV-2

abstract:
An animal model that fully recapitulates severe COVID-19 presentation in humans has been a top priority since the discovery of SARS-CoV-2 in 2019. Although multiple animal models are available for mild to moderate clinical disease, a non-transgenic model that develops severe acute respiratory disease has not been described. Mink experimentally infected with SARS-CoV-2 developed severe acute respiratory disease, as evident by clinical respiratory disease, radiological, and histological changes. Virus was detected in nasal, oral, rectal, and fur swabs. Deep sequencing of SARS-CoV-2 from oral swabs and lung tissue samples showed repeated enrichment for a mutation in the gene encoding for nonstructural protein 6 in open reading frame 1a/1ab. Together, these data indicate that American mink develop clinical features characteristic of severe COVID19 and as such, are uniquely suited to test viral countermeasures.

One Sentence SummarySARS-CoV-2 infected mink develop severe respiratory disease that recapitulates some components of severe acute respiratory disease, including ARDS.


title: The AAA-ATPase Atad1 and its partners promote degradation of desmin intermediate filaments in muscle

abstract: Maintenance of desmin intermediate filaments (IF) is vital for muscle plasticity and function, and their perturbed integrity due to accelerated loss or aggregation causes atrophy and myopathies. Calpain-1-mediated disassembly of ubiquitinated desmin IF is a prerequisite for desmin loss, myofibril breakdown and atrophy. Because calpain-1 does not harbor a bona fide ubiquitin binding domain, the precise mechanism for desmin IF disassembly remains unknown. Here, we demonstrate that the AAA-ATPase, ATAD1, is required to facilitate disassembly and turnover of ubiquitinated desmin IF. We identified PLAA and UBXN4 as ATAD1s interacting partners, and show they are required for desmin filaments turnover because their downregulation attenuated desmin loss upon denervation. The ATAD1-PLAA-UBXN4 complex binds desmin filaments and promotes a release of phosphorylated and ubiquitinated species into the cytosol, presenting ATAD1 as the only known AAA-ATPase that preferentially acts on phosphorylated substrates. Desmin filaments disassembly was accelerated by the coordinated functions of Atad1 and calpain-1, which interact in muscle. Thus, by extracting ubiquitinated desmin from the insoluble filament, ATAD1 may expose calpain-1 cleavage sites on desmin, consequently enhancing desmin solubilization and degradation in the cytosol.


title: Extracellular matrix deposition precedes muscle-tendon integration during murine forelimb morphogenesis

abstract:
The development of a functional vertebrate musculoskeletal system requires the combination of contractile muscle and extracellular matrix (ECM)-rich tendons that transmit muscle-generated force to bone. Despite the different embryologic origins, muscle and tendon integrate at the myotendinous junction (MTJ) to seamlessly connect cells and ECM across this interface. While the cell-cell signaling factors that direct development have received considerable attention, how and when the ECM linking these tissues is deposited remains unknown. To address this gap, we analyzed the 3D distribution of different ECM and the influence of skeletal muscle in forelimbs from wildype (WT) and muscle-less Pax3Cre/Cre mice. At E11.5, prior to MTJ integration, an aligned ECM was present at the presumptive insertion of the long triceps into the WT ulna. Mechanically robust tendon-like and muscle compartmentalization structures, positive for type I collagen, type V collagen, and fibrillin-2, still formed when muscle was knocked out. However, MTJ-specific ECM was not observed when muscle was absent. Our results show that an ECM-based template forms independent of muscle, but muscle is needed for the proper assembly of ECM at the MTJ.

Summary statementAn aligned ECM template connects tendon and muscle during limb development, independent of muscle progenitor migration into the limb; however, the assembly of MTJ-specific ECM requires the presence of muscle.


title: Shelterin is a Dimeric Complex with Extensive Structural Heterogeneity

abstract: Human shelterin is a six-subunit complex - comprised of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1 - that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been generated of the individual structured domains within shelterin, the architecture and stoichiometry of the full complex are currently unknown. Here we report the purification of shelterin subcomplexes and reconstitution of the entire complex using full-length, recombinantly produced components. By combining negative-stain electron microscopy (EM), crosslinking mass spectrometry (XLMS), mass photometry, and native mass spectrometry (MS), we obtain stoichiometries as well as domain-scale architectures of shelterin subcomplexes and determine that they are extensively conformationally heterogenous. For POT1/TPP1 and POT1/TPP1/TIN2, we observe high variability in the positioning of the POT1 DNA-binding domain, the TPP1 OB fold, and the TIN2 TRFH domain with respect to the C-terminal domains of POT1. Truncation of unstructured linker regions in TIN2, TPP1, and POT1 did not reduce the conformational variability of the heterotrimer. Both shelterin and the TRF1/TIN2/TPP1/POT1 subcomplex primarily adopt fully dimeric complexes, even in the absence of DNA substrates. TRF1/TIN2/TPP1/POT1 and shelterin complex showed extensive conformational variability, regardless of the presence of DNA substrates. We conclude that shelterin adopts a multitude of conformations and argue that its unusual architectural variability is beneficial for its many functions at telomeres.

category: biochemistry


title: Ion transfer mechanisms in Mrp-type antiporters from high resolution cryoEM and molecular dynamics simulations

abstract: Multiple resistance and pH adaptation (Mrp) cation/proton antiporters are essential for growth of a variety of halophilic and alkaliphilic bacteria under stress conditions. Mrp-type antiporters are closely related to the membrane domain of respiratory complex I. We determined the structure of the Mrp antiporter from Bacillus pseudofirmus by electron cryo-microscopy at 2.2 [A] resolution. The structure resolves more than 99% of the sidechains of the seven membrane subunits MrpA to MrpG plus 360 water molecules, including [~]70 in putative ion translocation pathways. Molecular dynamics simulations based on the high-resolution structure revealed details of the antiport mechanism. We find that switching the position of a histidine residue between three hydrated pathways in the MrpA subunit is critical for proton transfer that drives gated transmembrane sodium translocation. Several lines of evidence indicate that the same histidine-switch mechanism operates in respiratory complex I.


title: Cryo-EM structure of an active central apparatus

abstract: Accurately regulated ciliary beating in time and space is critical for diverse cellular activities, which impact the survival and development of nearly all eukaryotic species. An essential beating regulator is the conserved central apparatus (CA) of motile cilia, composed of a pair of microtubules (C1 and C2) associated with hundreds of protein subunits per repeating unit. It is largely unclear how CA plays its regulatory roles in ciliary motility. Here, we present high-resolution structures of Chlamydomonas reinhardtii CA by cryo-electron microscopy (cryo-EM) and its dynamic conformational behavior in multiple scales. The structures show how functionally related projection proteins of CA are clustered onto a spring-shaped scaffold of armadillo-repeat proteins, facilitated by elongated rachis-like proteins. The two halves of CA are brought together by elastic chain-like bridge proteins to achieve coordinated activities. We captured an array of kinesin-like protein (KLP1) in two different stepping states, which are actively correlated with beating wave propagation of cilia. These findings establish a structural framework for understanding the role of CA in cilia.


title: Chondroitin sulfates enhances the barrier function of basement membrane assembled by heparan sulfates

abstract: Glycosaminoglycans (GAGs) are ubiquitously expressed polysaccharides attached to proteoglycans, but their functions in the retina are poorly understood. Here we generated conditional knockouts of biosynthetic enzymes for heparan sulfate (HS) and chondroitin sulfate (CS) in retinal progenitor cells. We showed that ablation of HS polymerase Ext1 did not affect initial progression of retinal angiogenesis, but it disrupted the pruning of blood vessels and establishment of arterioles and venules. In the absence of retinal HS, blood vessels were also vulnerable to high oxygen tension in early postnatal stages, which can be rescued by exogenous VEGF, consistent with the role of retinal HS in the fine-tuning of VEGF signaling. Furthermore, we observed that the retinal inner limiting membrane (ILM) was disrupted by deletion of Ext1 in a timing specific manner, suggesting that retinal HS is required for the assembly but not the maintenance of the ILM. Lastly, we showed that further deletion of C4st1, a CS sulfation enzyme, did not affect the assembly of the ILM, but aggravated the ILM permeability when combined with Ext1 deletion. These results demonstrated an important role of CS and HS in establishing the barrier function of basement membrane.


title: Multi-color dSTORM microscopy in Hormad1-/- spermatocytes reveals alterations in meiotic recombination intermediates and synaptonemal complex structure

abstract:
Recombinases RAD51 and its meiosis-specific paralog DMC1 accumulate on single-stranded DNA (ssDNA) of programmed DNA double strand breaks (DSBs) in meiosis. Here we used three-color dSTORM microscopy to study how the two recombinases are organized on the ssDNA at individual DSBs, using a mouse model with severe defects in meiotic DSB formation and synapsis: Hormad1-/-. In accordance with previous analyses, we most frequently observed recombinase configurations with 1 DMC1 and 1 RAD51 nanofocus (D1R1), or two DMC1 nanofoci combined with a single RAD51 nanofocus (D2R1), and established that upon synapsis, recombinase nanofoci localized closer to the synaptonemal complex (SYCP3), in both wild type and Hormad1-/- spermatocytes. In wild type, the D1R1:D2R1 frequency ratio decreased over time, but in the knockout, this ratio did not change due to increased D2R1 and decreased D1R1 foci frequency in leptotene. Also, the Hormad1-/- nanofoci were smaller. Nearest neighbor analysis of RAD51/DMC1 nanofoci showed two preferred distances at [~]300 and [~]900 nm in wild type, but only at [~]300 nm in Hormad1-/-. Combined with the lower total number of DSBs in the mutant, this suggests that a preferred distance of 900 nm represents the distance between DSB sites. We propose models with the D1R1 configuration representing a scenario with only one DSB end containing recombinases, and the other end bound by other ssDNA binding proteins, or with both ends loaded by the two recombinases, but in below-resolution proximity. Surprisingly, the data also revealed a hitherto unknown function of HORMAD1 in inhibiting the formation of coils in the synaptonemal complex. SPO11 plays a similar but weaker role in coiling and SYCP1 had the opposite effect. In conclusion, our data show that HORMAD1 affects the lifetime of recombinase-accumulation patterns at meiotic DSBs, as well as the structure of the synaptonemal complex.

Author summaryMeiosis is a specialized cell division which generates haploid germ cells. In order to correctly pair homologous chromosomes in the first meiotic prophase, repair of programmed double strand breaks (DSBs) is essential. By unravelling molecular details of the protein assemblies at single DSBs, using super-resolution microscopy, we aim to understand the dynamics of repair intermediates and their functions. To this end, we investigated the localization of the two recombinases RAD51 and DMC1 in wild type and HORMAD1-deficient cells. HORMAD1 is involved in multiple aspects of homologous chromosome association: it regulates formation and repair of DSBs, and it stimulates formation of the synaptonemal complex, the macromolecular protein assembly that connects paired chromosomes. RAD51 and DMC1 enable chromosome pairing by promoting the invasions of the intact chromatids by single-stranded DNA ends that result from DSBs. We found that, in the absence of HORMAD1, RAD51 and DMC1 showed small but significant morphological and positional changes, combined with altered kinetics of specific RAD51/DMC1 configurations. We also observed changes in the structure of the synaptonemal complex in Hormad1-/ spermatocytes. This study contributes to a better understanding of the molecular details of meiotic homologous recombination and the role of HORMAD1 in meiotic prophase.


title: Nascent Polypeptide Associated Complex-alpha and Signal Recognition Particle are required for cardiac development and remodeling

abstract: Congenital Heart Disease (CHD) is driven by a strong genetic predisposition, yet only a small subset of patients ([~]20%) are diagnosed with a precise genetic cause. Therefore, expanding the pool of genes associated with CHD and establishing the functional relationships between genes can assemble a more comprehensive genetic network to better understand cardiac development and pathogenesis. In our studies, we identified protein biogenesis cofactors Nascent polypeptide Associated Complex (NAC) and Signal Recognition Particle (SRP) that bind disparate subsets of emerging nascent polypeptides at the ribosome exit site to direct polypeptide fates, as novel regulators of cell differentiation and cardiac morphogenesis. Knockdown (KD) of the alpha- (Nac) or beta- subunit (bicaudal, bic) of NAC in the developing Drosophila heart led to disruption of cardiac remodeling during pupal stages resulting in an adult fly with no heart. Heart loss was rescued by combined KD of Nac with the Hox gene Abd-B. Consistent with a central role for this interaction in the regulation of cardiogenesis, KD of Nac in Cardiac Progenitors derived from human iPSCs impaired cardiac differentiation while co-KD with mammalian Hox genes HOXC12 and HOXD12 rescued this phenotype. The effect of Nac KD on the fly heart was temporally regulated, in that KD in embryo or in pupae caused only a partial loss of the heart, whereas KD during both stages led to heart loss similar to continuous KD throughout life. This suggests that Nac KD already in the embryo may reprogram cells leading to aberrant cardiac remodeling during pupal stages. Lastly, KD of several SRP subunits individually in the fly heart produced a range of cardiac phenotypes that targeted specific segments and cell types, indicating spatially regulated activities of SRP components in the heart. Together, these data suggest that despite NAC and SRP ubiquitous presence, they displayed spatially and temporally fine-tuned activities for proper cardiac morphogenesis. Nacs interaction with cardiac-specific Hox gene functions builds upon the novel role of this pathway and expands our understanding of the complex genetic networks involved in cardiac development and pathogenesis.


title: Single-molecule nanopore dielectrophoretic trapping of α-Synuclein with lipid membranes

abstract: The lipid--Synuclein (-Syn) interaction plays a crucial role in the pathogenesis of Parkinsons disease. Here, we trap -Syn at a conjunction of an -hemolysin (HL) single nanopore-lipid to investigate the folding and unfolding kinetics of -Syn in a lipidic environment. The hybridized -Syn is generated through a reaction between a 5-thiol-modified nucleotide oligo (dC30) and the -Syn mutant (A140C). Owing to an applied voltage, single-molecule hybridized -Syn can be trapped at the single nanopore. The trapping events are associated with dielectrophoretic force. The folding and unfolding events of -Syn can be observed at the pore-membrane junction through interpretation of blockade current amplitudes and dwell time. This can be related to the protein quaternary structure influenced by the -Syn-membrane interaction, allowing further analysis of -Syn conformational dynamics. We studied how disease associated metal ions (Cu2+, Zn2+) modulate folding and unfolding of -Syn at the interface of the membranes and pore, and how -helical peptidomimetics stabilize the helical conformation of -Syn in the presence of a membrane. These studies aid our understanding of the complexity of the interaction of -Syn, lipid membranes and metal ions, and in using peptidomimetics, a new strategy against -Syn toxicity and aggregation is advanced.


title: Rapid Selectivity to Natural Images Across Layers of Primate V1

abstract: Visual systems are thought to have adapted to the statistical properties of natural scenes. However, the extent to which visual neurons respond selectively to natural images, and the stage at which that selectivity emerges remains unclear. To address these questions, we recorded the visual activity of neurons in macaque V1 using high-density electrode arrays (Neuropixels), and compared neuronal responses to images presented at three levels of naturalness. We found that within 60 ms of stimulus onset, neurons in all cortical layers, including input layers 4C alpha and beta, responded more vigorously to natural images than to statistically matched naturalistic texture and noise images. The result remained when residual variations in the local image statistics were factored out. V1 neurons also showed high population and lifetime sparseness for natural images. Across the population of V1 neurons, sensitivity to natural images exceeded the sensitivity to other image categories. The results reveal a rapid and pervasive preference for natural images is present at the earliest stages of cortical processing.


title: Latent circuit inference from heterogeneous neural responses during cognitive tasks

abstract: Higher cortical areas carry a wide range of sensory, cognitive, and motor signals supporting complex goal-directed behavior. These signals are mixed in heterogeneous responses of single neurons tuned to multiple task variables. Dimensionality reduction methods used to analyze neural responses rely merely on correlations, leaving unknown how heterogeneous neural activity arises from connectivity to drive behavior. Here we present a framework for inferring a low-dimensional connectivity structure--the latent circuit--from high-dimensional neural response data. The latent circuit captures mechanistic interactions between task variables and their mixed representations in single neurons. We apply the latent circuit inference to recurrent neural networks trained to perform a context-dependent decision-making task and find a suppression mechanism in which contextual representations inhibit irrelevant sensory responses. We validate this mechanism by confirming the behavioral effects of patterned connectivity perturbations predicted by the latent circuit structure. Our approach can reveal interpretable and causally testable circuit mechanisms from heterogeneous neural responses during cognitive tasks.


title: Reversal of obesogenic feeding and hypometabolism by a bifurcating GABAergic neural circuit

abstract: Diet-induced obesity is characterized by unsatiated consumption of energy-dense diets and impaired metabolism, whereby anti-obesity effect of the high-level of circulating leptin is unknowingly blunted. Emerging evidence suggests that the leptin receptor (LepR) signaling system, residing within the agouti-related protein (AgRP) neurons of the hypothalamus, critically contributes to obesogenic feeding, nutrient partitioning, and energy metabolism. However, the neural circuit mechanism underlying the leptin-dependent control of obesogenic feeding and energy balance remains largely elusive. Here, we show that two distinct subgroups of LepR-expressing AgRP neurons send non-collateral, GABAergic projections to the dorsomedial hypothalamic nucleus (DMH) and to the medial part of the medial preoptic nucleus (MPO) for the differential control of metabolic homeostasis and obesogenic feeding, respectively. We found that the AgRPLepR-DMH neural circuit plays a significant role in leptin-dependent control of metabolic homeostasis through the 3-containing GABAA receptor signaling on the melanocortin 4 receptor neurons within the DMH (MC4RDMH). In contrast, the AgRPLepR-MPO neural circuit elicits dominant effects on the appetitive response to high-fat diet through the 2-containing GABAA receptors on the MC4RMPO neurons. Consistent with these behavioral results, the post-synaptic GABAA neurons located within the DMH and MPO displayed differentiated firing responses under various feeding and nutrient conditions. Our results demonstrate that these novel GABAergic neural circuits exert differentiated control of metabolic hemostasis and obesogenic feeding via distinct post-synaptic targets of leptin-responsive AgRP neurons. The findings of two genetically and anatomically distinct GABAA receptor signaling pathways within the DMH and MPO would undoubtedly accelerate the development of targeted, individualized, anti-obesity therapy.


title: Structural basis for the neurotropic AAV9 and the engineered AAVPHP.eB recognition with cellular receptors

abstract: Clade F adeno-associated virus (AAV) 9 has been utilized as therapeutic gene delivery vector, and it is capable of crossing blood brain barrier (BBB). Recently, an AAV9 based engineering serotype with enhanced BBB crossing ability, AAVPHP.eB, further expand clade F AAVs usages in the central nervous system (CNS) gene delivery. In this study, we determined the cryo-electron microscopy (cryo-EM) structures of the AAVPHP.eB, and its parental serotype AAV9 alone or in complex with their essential receptor Adeno-associated virus receptor (AAVR). These structures reveal the molecular details of their AAVR recognition, where the polycystic kidney disease (PKD) repeat domain 2 (PKD2) of AAVR interact to the 3-fold protrusions and the raised capsid regions between the 2- and 5-fold axes termed the 2/5-fold wall of both AAV9 and AAVPHP.eB virions. The interacting patterns of AAVR to AAV9 and AAVPHP.eB are similar with what was observed in AAV1/AAV2-AAVR complexes. Moreover, we found that AAVPHP.eB variable region VIII (VR-VIII) may independently facilitate the new receptor recognition responsible for enhanced CNS transduction. Our study provides insights into different receptor recognition for engineered AAVPHP.eB and parental serotype AAV9, and further reveal the potential molecular basis underlying their different tropism.


title: Marine invertebrate diversity and distribution; the evidence of human activities in Rommel Bay

abstract: The current note hypothesised that human/tourism beach activities in Rommel Bay, Marsa Matrouh, Egypt (31{degrees}2139.9"N 27{degrees}1512.3"E) affected the marine invertebrates in this area. The note resulted some new findings of arthropod, molluscan, and seagrasses species diversity and distribution. The hypothesised study concluded that human/tourism beach activities did not eliminate the invertebrate communities, but these animals have shifted and moved themselves to escape away from noise and pollution. Further investigations should be taken into consideration about aquatic insects, mites, crustaceans, seagrasses, and molluscans diversity and distribution, to figure out the actual structures and interactions between these animals within the Rommel Bay ecosystem, besides what are the stresses they have, and what are the consequences.


title: Variant to function mapping at single-cell resolution through network propagation

abstract: With burgeoning human disease genetic associations and single-cell genomic atlases covering a range of tissues, there are unprecedented opportunities to systematically gain insights into the mechanisms of disease-causal variation. However, sparsity and noise, particularly in the context of single-cell epigenomic data, hamper the identification of disease- or trait-relevant cell types, states, and trajectories. To overcome these challenges, we have developed the SCAVENGE method, which maps causal variants to their relevant cellular context at single-cell resolution by employing the strategy of network propagation. We demonstrate how SCAVENGE can help identify key biological mechanisms underlying human genetic variation including enrichment of blood traits at distinct stages of human hematopoiesis, defining monocyte subsets that increase the risk for severe coronavirus disease 2019 (COVID-19), and identifying intermediate lymphocyte developmental states that are critical for predisposition to acute leukemia. Our approach not only provides a framework for enabling variant-to-function insights at single-cell resolution, but also suggests a more general strategy for maximizing the inferences that can be made using single-cell genomic data.

category: genomics


title: Augmenting Transcranial Magnetic Stimulation Coil with Magnetic Material: An Optimization Approach

abstract: Transcranial magnetic stimulation (TMS) is a neuromodulation technique that has been approved by the U.S. Food and Drug Administration for several neuropsychiatric disorders, including major depression and obsessive-compulsive disorder. However, the therapeutic efficacy of TMS treatment has been modest, despite decades of research. While there are many potential reasons as to why, one of the most obvious is the limitations of current technologies. One prominent example is the penetration depth-focality tradeoff of existing TMS coils. The most widely used figure-of-8 coils stimulate brain regions just superficially under the coil, missing deep brain regions known to be critically involved in psychiatric disorders; while ring-type coils can stimulate deep into the brain, but stimulate a large brain volume (lack of focality). A new coil design strategy is proposed: magnetic materials encompassing the human head are optimized to shape the electromagnetic field generated by the primary coil. Specifically, a mathematical model was developed to describe the physical problem; the magnetic materials were discretized into unit blocks; Newtons gradient descent method was applied to iteratively optimize the spatial distribution of the unit blocks to achieve a desired electric field distribution inside a head model. Results reveal that the proposed design achieves a coil penetration depth equal to or better than state-of-the art commercial coils, while improving the depth-focality tradeoff by a factor of 2.2 to 2.7. As a proof-of-concept, a prototype coil and a spherical head model were constructed; the spatial distribution of the induced electric field inside the head model was mapped. Results validated the proposed coil design. TMS coils based on this novel design strategy could potentially lead to better therapeutic outcome.

category: bioengineering


title: plotsr: Visualising structural similarities and rearrangements between multiple genomes

abstract:
SummaryThird-generation genomic technologies have led to a sharp increase in the number of high-quality genome assemblies. This allows the comparison of multiple assembled genomes of individual species and demands for new tools for visualising their structural properties. Here we present plotsr, an efficient tool to visualize structural similarities and rearrangements between multiple genomes. It can be used to compare genomes on chromosome level or to zoom in on any selected region. In addition, plotsr can augment the visualisation with regional identifiers (e.g. genes or genomic markers) or histogram tracks for continuous features (e.g. GC content or polymorphism density).

Availability and implementationplotsr is implemented as a python package and uses the standard matplotlib library for plotting. It is freely available under the MIT license at GitHub (https://github.com/schneebergerlab/plotsr) and bioconda (https://anaconda.org/bioconda/plotsr).

ContactManish Goel (manish.goel@lmu.de), Korbinian Schneeberger (k.schneeberger@lmu.de)


title: A robust and fast two-sample test of equal correlations with an application to differential co-expression

abstract: A robust and fast two-sample test for equal Pearson correlation coefficients (PCCs) is important in solving many biological problems, including, e.g., analysis of differential co-expression. However, few existing methods for this test can achieve robustness against deviation from normal distributions, accuracy under small sample sizes, and computational efficiency simultaneously. Here, we propose such a method for testing DIfferential COrrelation using a Saddlepoint Approximation of the Residual bootstrap (DICOSAR). To achieve robustness, accuracy, and efficiency, DICOSAR combines the ideas underlying the pooled residual bootstrap, the signed root of a likelihood ratio statistic, and a multivariate saddlepoint approximation. Through a comprehensive simulation study and a real data analysis of gene co-expression, we demonstrate that DICOSAR is accurate and robust in controlling the type I error rate for detecting differential correlation and provides a faster alternative to the permutation method. We further show that it can also be used for testing differential correlation matrices. These results suggest that DICOSAR provides an analytical approach to facilitate rapid testing for the equality of PCCs in a large-scale analysis.


title: A metric and its derived protein similarity network to analyze function-oriented ortholog

abstract: Orthology prediction is challenging yet rewarding. Orthologs are the cornerstone of almost all comparative genomics studies. Dozens of ortholog resources have been available and broadly used over the past decades. However, the inconsistency between these resources has drawn growing concerns, especially when more proteomes are available and ortholog databases expand. It is no longer easy to decide which ortholog database to use, and it becomes necessary to compare conclusions based on different resources. We are presenting here a metric to assess ortholog functional consistency. Using this metric, we built a network connecting proteins based on their functional similarity. We then detected network communities as ortholog groups, and each protein in our ortholog group inherited the network degree centrality. By benchmarking Quest for Orthologs (QfO) and some representative ortholog resources, we concluded the degree centrality could serve as the index for the reliability of functional consistency. The numerical nature of degree centrality could also open a door for quantitative study in pan-genome and other comparative genomics studies.


title: A metric and its derived protein network for evaluation of ortholog database inconsistency


title: FiMO: Inferring the Temporal Order of Mutations on Clonal Phylogeny under Finite-sites Models

abstract: Determining the temporal order of somatic mutations that drives cancer progression is essential for understanding the intra-tumor heterogeneity (ITH) and designing personalized therapy. Recently emerged single-cell DNA sequencing (SCS) technologies provide high-resolution datasets suitable for elucidating the temporal order of mutation. However, this task is challenged by technical artifacts associated with single-cell sequencing. While computational methods have been developed to account for these errors, these methods rely on using infinite sites assumption which gets violated in human cancers due to deletion, loss of heterozygosity and parallel mutations. Here, we propose a novel method FiMO, which employs a Bayesian frameowrk for inferring the temporal order of somatic mutations from noisy SCS mutational profiles under tumor evolutionary models that account for mutation recurrence and losses. Using synthetic datasets generated under a wide variety of settings, we show that FiMO outperforms the state-of-the-art methods in inferring the temporal order of mutations. We also applied FiMO on two experimental colon cancer datasets for inferring the temporal order of somatic mutations and quantifying their posterior probability. FiMO is publicly available at https://github.com/aveshag/FiMO.


title: Pan-cancer analysis reveals the prognostic potential of THAP9/THAP9-AS1 Sense-Antisense gene pair in human cancer.

abstract: Human THAP9 is a domesticated transposon homologous to the Drosophila P-element transposase (DmTNP). However, the exact functional role of THAP9 is unknown. THAP9-AS1 (THAP9-anti sense1) is a lncRNA involved in various cancers. Together they are arranged head-to-head on opposite DNA strands forming a sense and anti-sense gene pair. No studies have analyzed THAP9 and THAP9-AS1 from a pan-cancer perspective. First, we analyzed the bidirectional promoter region between THAP9 and THAP9-AS1 for core promoter elements and CPG Islands. Moreover, we analyzed the expression, prognosis, and biological function of the two genes across different types of tumors included in The Cancer Genome Atlas and GTEx datasets. The results showed that the expression of both genes varies in different tumors. Expression of the THAP9 and THAP9-AS1 gene pair was observed to be strongly correlated with prognosis in patients with tumors; higher expression of the two genes was usually linked to poor overall and disease-free survival. Therefore, they may serve as a potential biomarker of clinical tumor prognosis. Further, we performed gene coexpression analysis using WGCNA followed by differential gene correlation analysis (DGCA) across 22 cancers to study which genes share the expression pattern THAP9 and THAP9-AS1 and if their interacting partners change in various conditions. Gene ontology & KEGG pathway analysis of genes coexpressed with the two genes identified gene enrichment related to DNA binding and Herpes simplex virus 1 infection.


title: Probabilistic Learning of Treatment Trees in Cancer

abstract: AO_SCPLOWBSTRACTC_SCPLOWAccurate identification of synergistic treatment combinations and their underlying biological mechnisms is critical across many disease domains, especially cancer. In translational oncology research, preclinical systems such as patient-derived xenografts (PDX) have emerged as a unique study design evaluating multiple treatments administered to samples from the same human tumor implanted into genetically identical mice. In this paper, we propose a novel Bayesian probabilistic tree-based framework for PDX data to investigate the hierarchical relationships between treatments by inferring treatment cluster trees, referred to as treatment trees (Rx-tree). The framework motivates a new metric of mechanistic similarity between two or more treatments accounting for inherent uncertainty in tree estimation; treatments with a high estimated similarity have potentially high mechanistic synergy. Building upon Dirichlet Diffusion Trees, we derive a closed-form marginal likelihood encoding the tree structure, which facilitates computationally efficient posterior inference via a new two-stage algorithm. Simulation studies demonstrate superior performance of the proposed method in recovering the tree structure and treatment similarities. Our analyses of a recently collated PDX dataset produce treatment similarity estimates that show a high degree of concordance with known biological mechanisms across treatments in five different cancers. More importantly, we uncover new and potentially effective combination therapies that confer synergistic regulation of specific downstream biological pathways for future clinical investigations. Our accompanying code, data, and shiny application for visualization of results are available at: https://github.com/bayesrx/RxTree.


title: Delineate clonal dynamics of ovarian cancer initiating cells during pre-malignant progression using a mouse genetic mosaic system

abstract:
High-grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy. Early detection remains the most promising strategy to decrease mortality and requires a deep understanding of the cancer initiation process. Recent breakthroughs identified the fallopian tube (FT) Pax8+ cells as a cell-of-origin for HGSOC. However, the progressive trajectory from an initiated Pax8+ cell toward malignancy remains elusive. Here, we use a mouse genetic mosaic system called MADM (Mosaic Analysis with Double Markers), which generates sporadic mutant cells labeled with GFP, to trace the clonal dynamics of HGSOC development. Surprisingly, we observed dichotomous clonal expansion kinetics of individual cancer-initiating Pax8+ cells; while the vast majority stalled immediately, a few significantly expanded. Clonal analysis of wildtype cells showed concordant dichotomy, suggesting that a small fraction of Pax8+ cells intrinsically carry higher expansion potential. Furthermore, although all wildtype clones stalled shortly after development, some mutant clones continued to progress, indicating that oncogenic mutations prolong clonal expansion beyond the cell-intrinsic potential. Finally, intra-clonal fate mapping revealed that progressing mutant clones have a higher propensity to produce Pax8+ cells, linking imbalanced cell differentiation to disease progression. In summary, our studies reveal that the earliest clonal expansion during HGSOC initiation emerges when oncogenic mutations exaggerate the intrinsic expansion potential of a rare Pax8+ subset and bias its differentiation potential, warranting future investigation as a readout for early detection and cancer prevention.

SIGNIFICANCE STATEMENTEffective early detection is the most promising approach to manage cancer, but it requires a thorough understanding of cancer initiation and early progression. Serous ovarian cancer has been found to originate from Pax8+ cells in the fallopian tube. To assess the clonal dynamics of cancer-initiating cells, we established a mouse genetic mosaic model that enables clonal analysis of mutant Pax8+ cells in the fallopian tube. We uncovered that the earliest clonal event in ovarian cancer development emerges from a rare subset of stem/progenitor-like cells with intrinsic expansion potential, and that oncogenic mutations exaggerate and prolong this potential to promote cancer initiation. Our findings pinpoint a precise target for future early-detection and cancer prevention studies.

HIGHLIGHTSO_LIOvarian cancer initiation revealed by a mouse genetic mosaic model.
C_LIO_LIDichotomous expansion capacity of initiated cells at the clonal level.
C_LIO_LISynergies between cell-intrinsic properties and onco-mutations fuels clonal expansion.
C_LIO_LIImbalanced differentiation within highly expansive clones leads to Pax8 clusters.
C_LI

category: cancer biology


title: Clonal Dynamics of Cancer-Initiating Cells in a Mouse Genetic Mosaic Model of Serous Ovarian Cancer


title: Regional mutational signature activities in cancer genomes

abstract: Cancer genomes harbor a catalog of somatic mutations. The type and genomic context of these mutations depend on their causes, and allow their attribution to particular mutational signatures. Previous work has shown that mutational signature activities change over the course of tumor development, but investigations of genomic region variability in mutational signatures have been limited. Here, we expand upon this work by constructing regional profiles of mutational signature activities over 2,203 whole genomes across 25 tumor types, using data aggregated by the Pan-Cancer Analysis of Whole Genomes (PCAWG) consortium. We present GenomeTrackSig as an extension to the TrackSig R package to construct regional signature profiles using optimal segmentation and the expectation-maximization (EM) algorithm. We find that 426 genomes from 20 tumor types display at least one change in mutational signature activities (changepoint), and 306 genomes contain at least one of 107 recurrent changepoints shared by seven or more genomes of the same tumor type. Five recurrent changepoint locations are shared by multiple tumor types. Within these regions, the particular signature changes are often consistent across samples of the same type and some, but not all, are characterized by signatures associated with subclonal expansion. The changepoints we found cannot strictly be explained by gene density, mutation density, or cell-of-origin chromatin state. We hypothesize that they reflect a confluence of factors including evolutionary timing of mutational processes, regional differences in somatic mutation rate, large-scale changes in chromatin state that may be tissue type-specific, and changes in chromatin accessibility during subclonal expansion. These results provide insight into the regional effects of DNA damage and repair processes, and may help us localize genomic and epigenomic changes that occur during cancer development.


title: PyRodentTracks: flexible computer vision and RFID based system for multiple rodent tracking and behavioral assessment.

abstract:
PyRodentTracks (PRT) is a scalable and customizable computer vision and RFID- based system for multiple rodent tracking and behavior assessment that can be set up within minutes in any user-defined arena at minimal cost. PRT is composed of the online Raspberry Pi-based video and RFID acquisition and the subsequent offline analysis tools. The system is capable of tracking up to 6 mice in experiments ranging from minutes to days. PRT maintained a minimum of 88% detections tracked with an overall accuracy >85% when compared to manual validation of videos containing 1-4 mice in a modified home-cage. As expected, chronic recording in home-cage revealed diurnal activity patterns. Moreover, it was observed that novel non-cagemate mice pairs exhibit more similarity in travel trajectory patterns over a 10-minute period in the openfield than cagemates. Therefore, shared features within travel trajectories between animals may be a measure of sociability that has not been previously reported. Moreover, PRT can interface with open-source packages such as Deeplabcut and Traja for pose estimation and travel trajectory analysis, respectively. In combination with Traja, PRT resolved motor deficits exhibited in stroke animals. Overall, we present an affordable, open-sourced, and customizable/scalable rodent-specific behavior recording and analysis system.

Statement of SignificanceAn affordable, customizable, and easy-to-use open-source rodent tracking system is described. To tackle the increasingly complex questions in neuroscience, researchers need a flexible system to track rodents of different coat colors in various complex experimental paradigms. The majority of current tools, commercial or otherwise, can only be fully automated to track multiple animals of the same type in a single defined environment and are not easily setup within custom arenas or cages. Moreover, many tools are not only expensive but are also difficult to set up and use, often requiring users to have extensive hardware and software knowledge. In contrast, PRT is easy to install and can be adapted to track rodents of any coat color in any user-defined environment with few restrictions. We believe that PRT will be an invaluable tool for researchers that are quantifying behavior in identified animals.


title: A brain atlas of the camouflaging dwarf cuttlefish, Sepia bandensis

abstract: The dwarf cuttlefish, Sepia bandensis, a small cephalopod that exhibits dynamic camouflage, is an emerging model organism in neuroscience. Coleoid cephalopods (cuttlefish, octopus, and squid) evolved large, complex brains capable of learning, problem-solving, and memory. We used high-resolution magnetic resonance imaging (MRI), deep learning, and fluorescent histology to generate a dwarf cuttlefish brain atlas and built an interactive web tool (cuttlebase.org) to host the data. Guided by observations in other cephalopods, we identified 38 brain lobes. The dwarf cuttlefish brain is partially encased in cartilage and includes two large optic lobes (74% the total volume of the brain), chromatophore lobes whose motor neurons directly innervate the skin, and a vertical lobe that has been implicated in learning and memory. Motor neurons emerging from the chromatophore lobe modulate the color, pattern, and texture of the skin to elicit camouflage. This brain atlas provides a valuable tool for exploring the neural basis of cuttlefish behavior.


title: Reduced resilience of functional state transitions in patients with anti-NMDA receptor encephalitis

abstract:
BackgroundPatients with anti-NMDA receptor (NMDAR) encephalitis suffer from a severe neuropsychiatric syndrome, yet most patients show no abnormalities in routine magnetic resonance imaging (MRI). In contrast, advanced neuroimaging studies have consistently identified disrupted functional connectivity in these patients, with recent work suggesting increased volatility of functional state dynamics. Here, we investigate these network dynamics through the spatiotemporal trajectory of meta-state transitions, yielding a time-resolved account of brain state exploration in NMDAR encephalitis.

MethodsResting-state functional MRI data were acquired in 73 patients with NMDAR encephalitis and 73 age- and sex-matched healthy controls. Time-resolved functional connectivity was clustered into brain meta-states, giving rise to a time-resolved transition network graph with states as nodes and transitions between brain meta-states as weighted, directed edges. Network topology, robustness, and transition cost of these transition networks were compared between groups.

ResultsTransition networks of patients showed significantly lower local efficiency (t = -2.43, p = 0.016), lower robustness (t = -2.03, p = 0.044) and higher leap size (t = 2.21, p = 0.029) compared to controls. Furthermore, the ratio of within-to-between module transitions and state similarity was significantly lower in patients. Importantly, these metrics were correlated with disease severity and disease duration.

ConclusionsThese findings reveal systematic alterations of transition networks in patients, suggesting that NMDAR encephalitis is characterized by reduced stability of brain state transitions and that this reduced resilience of transition networks plays a clinically relevant role in the manifestation of the disease.


title: RNA-Targeting CRISPR/Cas13d System Eliminates Disease-Related Phenotypes in Pre-clinical Models of Huntington's Disease.

abstract: Huntingtons disease (HD) is a fatal, dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in exon 1 of the huntingtin (HTT) gene. Although the pathogenesis of HD remains complex, the CAG-expanded (CAGEX) HTT mRNA and protein ultimately causes disease through a toxic gain-of-function mechanism. As the reduction of pathogenic mutant HTT mRNA is beneficial as a treatment, we developed a CAGEX RNA-eliminating CRISPR-Cas13d system (Cas13d/CAGEX) that binds and eliminates toxic CAGEX RNA in HD patient iPSC-derived striatal neurons. We show that intrastriatal delivery of Cas13d/CAGEX via a single adeno-associated viral vector, serotype 9 (AAV9) mediates significant and selective reduction of mutant HTT mRNA and protein levels within the striatum of heterozygous zQ175 mice, an established mouse model of HD. Moreover, the reduction of mutant HTT mRNA renders a sustained reversal of HD phenotypes, including improved motor coordination, attenuated striatal atrophy, and reduction of mutant HTT protein aggregates. Importantly, phenotypic improvements were durable for at least 8 months without gross or behavioral adverse effects, and with minimal off-target interactions of Cas13d/CAGEX in the mouse transcriptome. Taken together, we demonstrate a proof-of-principle of an RNA-targeting CRISPR/Cas13d system as a therapeutic approach for HD, a strategy with broad implications for the treatment of other dominantly inherited neurodegenerative disorders.


title: Imprinted genes Cdkn1c and Igf2 interact to promote terminal differentiation of adult NSCs

abstract: Genomic imprinting is implicated in the control of gene dosage in neurogenic niches. Insulin-like growth factor 2 (Igf2), is an imprinted gene that shows biallelic expression only in the vascular compartment contributing to neural stem cells (NSCs) maintenance in the subventricular zone (SVZ) niche. The effects of this factor in adult NSCs differentiation are not well defined. We show here that IGF2 promotes cell fate commitment of NSCs by inducing the expression of another imprinted gene, the maternally expressed gene cyclin-dependent kinase inhibitor 1c (Cdkn1c), which encodes for p57 protein, eliciting cell cycle exit and terminal differentiation into astrocytes, neurons and oligodendrocytes. Using a conditional mouse model with Cdkn1c-deficient neural progenitors, we confirm that IGF2 and p57 interact in a common pathway to regulate the differentiation program of adult NSCs. This occurs through a mechanism involving the PI3K-Akt pathway that mediates regulation of p57 expression. We also show that the imprinted state of the Cdkn1c gene is not altered after IGF2 treatment confirming maternal expression of the gene in NSCs. Our results identify a molecular mechanism by which a paracrine factor produced and secreted by the neurogenic niche compartment can modulate Cdkn1c dosage to trigger differentiation of adult NSCs.


title: A novel neuroelectrophysiological age index implicates brain health and sleep disorders

abstract: Sleep architecture and microstructures alter with aging and sleep disorder-led accelerated aging. We proposed a sleep electroencephalogram (EEG) based brain age prediction model using convolutional neural networks. We then associated the estimated brain age index (BAI) with brain structural aging features, sleep disorders and various sleep parameters. Our model also showed a higher BAI (predicted brain age minus chronological age) is associated with cortical thinning in various functional areas. We found a higher BAI for sleep disorder groups compared to healthy sleepers, as well as significant differences in the spectral pattern of EEG among different sleep disorders (lower power in slow and{vartheta} waves for sleep apnea vs. higher power in {beta} and{sigma} for insomnia), suggesting sleep disorder-dependent pathomechanisms of aging. Our results demonstrate that the new EEG-BAI can be a biomarker reflecting brain health in normal and various sleep disorder subjects, and may be used to assess treatment efficacy.


title: Single cell profiling reveals functional heterogeneity and serial killing in human peripheral and ex vivo-generated CD34+ progenitor derived Natural Killer cells

abstract: Increasing evidence suggest that Natural killer (NK) cells are composed of distinct functional subsets. This multi-functional role displayed by NK cells have made them an attractive choice for anti-cancer immunotherapy. A functional NK cell repertoire is generated through cellular education, resulting in heterogeneous NK cell population with distinct capabilities to respond to different stimuli. The application of a high-throughput droplet-based microfluidic platform allows monitoring of NK cell-target cell interactions at single-cell level and in real-time. Through fluorescence-based screening of around 80,000 droplets, with different Effector:Target ratios, a fully automated image analysis allows for the assessment of individual killing events in each droplet over time. We observed a variable response of single NK cells towards different target cells and identified a distinct population of NK cells capable of inducing multiple target lysis, coined as serial killers. To meet the increasing clinical demand for NK cells several sources, such as umbilical cord blood (UCB), have successfully been explored. By assessing the cytotoxic dynamics, we showed that single UCB-derived CD34+ hematopoietic progenitor (HPC)-NK cells display superior anti-tumor cytotoxicity. Additionally, with an integrated analysis of cytotoxicity and cytokine secretion we showed that target cell interactions augmented cytotoxic as well as secretory behavior of NK cells. By providing an in-depth assessment over NK cell functions, this study provides crucial information on diversity and functional characteristics of peripheral blood NK cells and ex vivo-generated HPC-NK cells to develop and improve of NK cell-based cancer immunotherapy.

category: immunology


title: Multiplexed strain phenotyping defines consequences of genetic diversity in Mycobacterium tuberculosis for infection and vaccination outcomes

abstract: There is growing evidence that genetic diversity in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, contributes to the outcomes of infection and public health interventions, such as vaccination. Epidemiological studies suggest that among the phylogeographic lineages of Mtb, strains belonging to Lineage 2 (L2) are associated with concerning clinical features including hypervirulence, treatment failure, and vaccine escape. The global expansion and increasing prevalence of L2 has been attributed to the selective advantage conferred by these characteristics, yet confounding host and environmental factors make it difficult to identify the bacterial determinants driving these associations in human studies. Here, we developed a molecular barcoding strategy to facilitate high-throughput, experimental phenotyping of Mtb clinical isolates. This approach allowed us to characterize growth dynamics for a panel of genetically diverse Mtb strains during infection and after vaccination in the mouse model. We found that L2 strains exhibit distinct growth dynamics in vivo and are resistant to the immune protection conferred by Bacillus Calmette-Guerin (BCG) vaccination. The latter finding corroborates epidemiological observations and demonstrates that mycobacterial features contribute to vaccine efficacy. To investigate the genetic and biological basis of L2 strains distinctive phenotypes, we performed variant analysis, transcriptional studies, and genome-wide transposon sequencing. We identified functional genetic changes across multiple stress- and host-response pathways in a representative L2 strain that are associated with variants in regulatory genes. These adaptive changes may underlie the distinct clinical characteristics and epidemiological success of this lineage.


title: Diet outweighs genetics in shaping gut microbiomes in Asian honeybee

abstract:
BackgroundThe gut microbiome is a crucial element that facilitates a hosts adaptation to a changing environment. Host-specificity often coincides with distinctions in gut microbes, suggesting a co-evolution of the holobionts. However, it is unclear how gut microbiota shared by a common host ancestor would co-diversify with hosts and eventually become distinct among sister hosts. In this context, understanding the evolutionary pathway of gut microbiomes of the same host species could provide insight on how holobionts co-adapt along environmental gradients. Specifically, we ask which factor, nature or nurture, i.e., genetics or diets, contributes more to the shaping of gut microbiome, along with host diversification and range expansion.

ResultsWe compared and analyzed the gut microbiomes of 99 Asian honeybees, Apis cerana, from genetically diverged populations covering 13 provinces across China. Bacterial composition varied significantly across populations at phylotype, sequence-discrete population (SDP), and strain levels, but with extensive overlaps, indicating the diversity of microbial community among A. cerana populations is driven by nestedness. Taken together, genetics exhibited tangential impacts, while pollen diets were significantly correlated with both the composition and function of gut microbiome. Core bacteria, Gilliamella and Lactobacillus Firm-5, showed antagonistic turnovers and contributed to the enrichment in carbohydrate transport and metabolism. By feeding and inoculation bioassays, we confirmed that the variations in pollen polysaccharide composition contributed to the trade-off of these core bacteria.

ConclusionsProgressive change, i.e., nestedness, is the foundation of gut microbiome evolution in the Asian honeybee. Such a transition during the co-diversification of gut microbiomes is shaped primarily by environmental factors, diets in general, pollen polysaccharide in particular.


title: Vaccinia virus attenuation by codon deoptimization of the A24R gene for vaccine development

abstract:
Poxviruses have large DNA genomes and they are able to infect multiple vertebrate and invertebrate animals, including humans. Despite the eradication of smallpox, poxvirus infections still remain a significant public health concern. Vaccinia virus (VV) is the prototypic member in the poxviridae family and it has been used extensively for different therapeutic applications, including the generation of vaccines against multiple infectious diseases and/or for oncolytic treatment. Many attempts have been pursued to develop novel attenuated forms of VV with improved safety profiles for their implementation as vaccines and/or vaccines vectors. We and others have previously demonstrated how RNA viruses encoding codon-deoptimized viral genes are attenuated, immunogenic and able to protect, upon a single administration, against challenge with parental viruses. In this study, we employed the same experimental approach based on the use of misrepresented codons for the generation of a recombinant (r)VV encoding a codon-deoptimized A24R gene, which is a key component of the viral RNA polymerase. Similar to our previous studies with RNA viruses, the A24R codon-deoptimized rVV (v-A24cd) was highly attenuated in vivo but able to protect, after a single intranasal dose administration, against an otherwise lethal challenge with parental VV. These results indicate that poxviruses can be effectively attenuated by synonymous codon deoptimization and open the possibility of using this methodology alone or in combination with other experimental approaches for the development of attenuated vaccines for the treatment of poxvirus infection, or to generate improved VV-based vectors. Moreover, this approach could be applied to other DNA viruses.

IMPORTANCEThe family poxviridae includes multiple viruses of medical and veterinary relevance, being vaccinia virus (VV) the prototypic member in the family. VV was used during the smallpox vaccination campaign to eradicate variola virus (VARV), which is considered a credible bioterrorism threat. Because of novel innovations in genetic engineering and vaccine technology, VV has gained popularity as a viral vector for the development of vaccines against several infectious diseases. Several approaches have been used to generate attenuated VV for its implementation as vaccine and/or vaccine vector. Here, we generated a rVV containing a codon-deoptimized A24R gene (v-A24cd), which encodes a key component of the viral RNA polymerase. v-A24cd was stable in culture cells and highly attenuated in vivo but able to protect against a subsequent lethal challenge with parental VV. Our findings support the use of this approach for the development of safe, stable, and protective live-attenuated VV and/or vaccine vectors.


title: The first horse gut microbiome gene catalog reveals that rare microbiome ensures better cardiovascular fitness in endurance horses

abstract: Emerging evidence indicates that the gut microbiome contributes to endurance exercise performance, but the extent of their functional and metabolic potential remains unknown. Using elite endurance horses as a model system for exercise responsiveness, we built the first equine gut microbial gene catalog comprising more than 25 million non-redundant genes representing 4,696 genera spanning 95 phyla. The unprecedented resolution unrevealed functional pathways relevant for both the structure of the microbiome and the host health and recovered 369 novel metagenome-assembled bacterial genomes, providing useful reference for future studies. Integration of microbial and host omic datasets suggested that microbiomes harboring rare species were functionally dissimilar from those enriched in Lachnospiraceae taxa. Moreover, they offered expanded metabolic pathways to fine-tune the cardiovascular capacity through mitochondria-mediated mechanisms. The results identify an associative link between horse endurance capability and its microbiome gene function, laying the basis for nutritional interventions that could benefit endurance athletes.


title: Type IV pili is a critical virulence factor in clinical isolates of Paenibacillus thiaminolyticus

abstract:
Hydrocephalus, the leading indication for childhood neurosurgery worldwide, is particularly prevalent in low-and-middle-income countries (LMICs). Hydrocephalus preceded by an infection, or postinfectious hydrocephalus (PIH), accounts for up to 60% of hydrocephalus in LMICs. Since many children with hydrocephalus suffer poor long-term outcomes despite surgical intervention, prevention of hydrocephalus remains paramount. Our previous studies implicated a novel bacterial pathogen, Paenibacillus thiaminolyticus, as a contributor to PIH in Uganda. Here we report the isolation of three P. thiaminolyticus strains, Mbale, Mbale2, and Mbale3, from patients with PIH and the demonstration that the three clinical isolates exhibit virulence in mice while P. thiaminolyticus type strain, B-4156, does not. We constructed complete genome assemblies of the clinical isolates as well as the reference strain and performed comparative genomics and proteomics analyses to identify potential virulence factors. One candidate virulence factor is a cluster of genes carried on a mobile genetic element that encodes a type IV pilus and is present in all three PIH patient strains but absent in the type strain. Proteomic and transcriptomic data confirmed the expression of this cluster of genes in the Mbale strain, while CRISPR-mediated deletion of the gene cluster substantially reduced the virulence of this strain. Our comparative proteogenomic analysis also identified various antibiotic resistance loci in the virulent strains. These results provide insight into the mechanism of virulence of Paenibacillus thiaminolyticus and suggest avenues for the diagnosis and treatment of this novel bacterial pathogen.

Author SummaryPostinfectious hydrocephalus (PIH), a devastating sequela of neonatal infection, is associated with increased childhood mortality and morbidity. Paenibacillus thiaminolyticus was recently identified as the dominant organism highly associated with PIH in an African cohort. Our whole-genome sequencing, RNA sequencing and proteomics of three clinical isolates and a type strain in combination with CRISPR editing has revealed the type IV pili (T4P), encoded in a mobile genetic element, as a critical virulence factor for P. thiaminolyticus infection. Given the widespread presence of T4P in pathogens, the presence of T4P operon could serve as an important diagnostic and therapeutic target in P. thiaminolyticus and related bacteria.


title: Impaired ERK MAPK activation in mature osteoblasts enhances bone formation via the mTOR pathway

abstract: Emerging evidence supports that osteogenic differentiation of skeletal stem cells (SSCs) is a key determinant of overall bone formation and bone mass. Despite extensive studies showing mitogen-activated protein kinase (MAPK) function in osteoblast differentiation, none of these studies properly show in vivo evidence of impacting post-lineage commitment and subsequent maturation. Here, we describe how the extracellular signal-regulated kinase (ERK) pathway in osteoblasts controls bone formation by suppressing the mechanistic target of rapamycin (mTOR) pathway. We also show that, while ERK inhibition blocks the differentiation of osteogenic precursors when initiated at an early stage, ERK inhibition surprisingly promotes the later stages of osteoblast differentiation. Accordingly, inhibition of the ERK pathway using a small compound inhibitor or conditional deletion of the MAP2Ks Mek1 and Mek2, in mature osteoblasts and osteocytes (Mek1/2Dmp1), markedly increased bone formation due to augmented osteoblast differentiation. Mice with inducible deletion of the ERK pathway in mature osteoblasts (Mek1/2Ocn-Ert) also displayed similar phenotypes, demonstrating that this phenotype reflects continuous postnatal inhibition of late-stage osteoblast maturation. Mechanistically, ERK inhibition increases mitochondrial function and SGK1 phosphorylation via mTOR2 activation, which leads to osteoblast differentiation and production of angiogenic and osteogenic factors to promote bone formation. This phenotype was partly reversed by inhibiting mTOR. Our study uncovers a surprising dichotomy of ERK pathway functions in osteoblasts, whereby ERK activation promotes the early differentiation of osteoblast precursors, but inhibits the subsequent differentiation of committed osteoblasts via mTOR-mediated regulation of mitochondrial function and SGK1.

category: molecular biology


title: Analysis of non-coding RNAs in  Methylorubrum extorquens  reveals a novel small RNA specific to Methylobacteriaceae

abstract: Methylorubrum extorquens metabolizes methanol, a cheap raw material that can be derived from waste. It is a facultative methylotroph, making it a model organism to study the metabolism of one carbon compounds. Despite a considerable interest to exploit this bacteria as a biotechnological tool in a methanol-based bioeconomy, little is known about its non-coding sRNA. Small RNAs play well-documented essential roles in Escherichia coli for post-transcriptional regulation; and have important functions in many bacteria, including other Alphaproteobacteria like Agrobacterium tumefaciens. M. extorquens is expected to contain many sRNAs, especially since it also encodes for the protein Hfq, a chaperone protein important in the interaction between sRNAs and their target, but also critical for the stabilization of sRNAs themselves. Few sRNAs are annotated in the genome of this Alphaproteobacteria and they were never validated. In this study, formerly annotated sRNAs ffh, CC2171, BjrC1505 were confirmed by Northern blot, validating the expression of sRNAs in M. extorquens. Moreover, analysis of RNA-sequencing data established a considerable list of potential sRNAs. Interesting candidates selected after bioinformatic analysis were tested by Northern blot, revealing a novel sRNA specific to Methylobacteriaceae, sRNA Met2624. Its expression patterns and genomic context were analyzed. This research is the first experimental validation of sRNAs in M. extorquens and paves the way for other sRNA discoveries.


title: Mechano-signaling feedback underlies precise inner hair cell patterning in the organ of Corti

abstract: The mammalian hearing organ, the organ of Corti, is one of the most organized tissues in the mammalian body plan. It contains precisely positioned array of alternating sensory hair cells (HC) and non-sensory supporting cells that emerge during embryonic development from an initially disordered pro-sensory domain. While much is known on the genetics and biochemistry underlying this process, it is still unclear how such precise alternating patterns emerge during embryonic development. Here, we combine live imaging of mouse inner ear explants with hybrid mechano-regulatory models to elucidate the mechanisms underlying the formation of a single row of inner HC (IHC). We show that a narrow strip of initially disordered salt-and-pepper pattern, generated by Notch-mediated lateral inhibition, is dynamically refined by coordinated intercalations, delaminations, and differential adhesion. We identify a new morphological transition, termed  hopping intercalation, that allows nascent IHC to  hop under the apical plane into their final position. We further show that IHC patterning is associated with boundary localization of the cell adhesion molecules, Nectin-3 and Nectin-1. Our experimental results and modeling support a mechanism for precise patterning based on a feedback between Notch-mediated differentiation and mechanically driven cellular reorganization that is likely relevant for many developmental processes.


title: Assessing flower-visiting arthropod diversity in apple orchards through environmental DNA flower metabarcoding and visual census

abstract: Arthropods are essential to maintaining healthy and productive agricultural systems. Apples are cultivated worldwide and rely on pollination. Honey bees are used for pollination but wild bees and other arthropods also contribute to pollination. Flower visitors can also be natural enemies or herbivores. In some cases, such as Syrphids, a group can have more than one role, adults being pollinators and the larvae being natural enemies of pests. In the present study, we assessed the biodiversity of arthropod flower visitors in four Danish apple orchards and compared the use of molecular and non-molecular techniques to study arthropod communities in agricultural ecosystems. Arthropod DNA collected from apple flowers was analysed by metabarcoding and pollinators were recorded through visual assessment in the orchards. These techniques resulted in two complementary lists of arthropods detected. Non-bee arthropods constituted a big part of the community of apple flower visitors by both methods. Metabarcoding detected 14 taxa and had 72% species resolution while visual census identified 7 different taxa with 14% species resolution. This study showed the importance of using different sampling methodologies to obtain a more accurate picture of fauna present. It also revealed the high presence of non-bee arthropods visiting flowers in apple orchards. The outcome of our study provides information regarding the effects of management practices on arthropod biodiversity, which can contribute to informing on suitable management practices to increase crop yield and maintain healthy agricultural systems.


title: Relation between the number of peaks and the number of reciprocal sign epistatic interactions

abstract: Empirical essays of fitness landscapes suggest that they may be rugged, that is having multiple fitness peaks. Such fitness landscapes, those that have multiple peaks, necessarily have special local structures, called reciprocal sign epistasis ([14]). Here, we investigate the quantitative relationship between the number of fitness peaks and the number of reciprocal sign epistatic interactions. Previously it has been shown ([14]) that pairwise reciprocal sign epistasis is a necessary but not sufficient condition for the existence of multiple peaks. Applying discrete Morse theory, which to our knowledge has never been used in this context, we extend this result by giving the minimal number of reciprocal sign epistatic interactions required to create a given number of peaks.


title: Genomic instability caused by Arp2/3 complex inactivation results in micronucleus biogenesis and cellular senescence

abstract:
The Arp2/3 complex is a ubiquitous actin nucleator with well-characterized activities in cell organization and movement, but its roles in chromatin-associated and cell cycle-related processes are relatively understudied. We investigated how the Arp2/3 complex affects genomic integrity, mitosis, and cell proliferation using mouse fibroblasts containing an inducible knockout (iKO) of the ArpC2 subunit. We show that permanent Arp2/3 ablation results in DNA damage, the formation of cytosolic micronuclei, and cellular senescence. Upon Arp2/3 depletion, cells undergo an abrupt proliferation arrest that is accompanied by activation of the tumor suppressor p53, upregulation of its downstream cell cycle inhibitor Cdkn1a/p21, and recognition of micronuclei by the cytosolic DNA sensor cGAS. Micronuclei arise in ArpC2 iKO cells due to chromosome segregation defects during mitosis and premature mitotic exits. Such phenotypes are explained by the presence of damaged chromatin fragments that fail to attach to the mitotic spindle, abnormalities in actin assembly during metaphase, and asymmetric microtubule architecture during anaphase. These studies establish functional requirements for the mammalian Arp2/3 complex in genome stability and mitotic spindle organization. They further expand our understanding of the intracellular mechanisms that lead to senescence and suggest that cytoskeletal dysfunction is an underlying factor in biological aging.

Author SummaryThe actin cytoskeleton consists of protein polymers that assemble and disassemble to control the organization, shape, and movement of cells. However, relatively little is understood about how the actin cytoskeleton affects genome maintenance, cell multiplication, and biological aging. In this study, we show that knocking out the Arp2/3 complex, a core component of the actin assembly machinery, causes DNA damage, genomic instability, defects in chromosome partitioning during mitosis, and a permanent cell proliferation arrest called senescence. Since senescent cells are major contributors to both age-associated diseases and tumor suppression, our findings open new avenues of investigation into how natural or experimental alterations of cytoskeletal proteins impact the process of aging and the regulation of cancer.

category: genetics


title: Genome-wide analysis identifies Homothorax and Extradenticle as regulators of insulin in Drosophila Insulin-Producing cells

abstract: Drosophila Insulin-Producing Cells (IPCs) are the main production site of the Drosophila Insulin-like peptides or Dilps which have key roles in regulating growth, development, reproduction, lifespan and metabolism. To better understand the signalling pathways and transcriptional networks that are active in the IPCs we queried publicly available transcriptome data of over 180 highly inbred fly lines for dilp expression and used this as the input for a Genome-wide association study (GWAS). This resulted in the identification of variants in 125 genes that were associated with variation in dilp expression. The function of 57 of these genes in the IPCs was tested using an RNAi-based approach. We found that IPC-specific depletion of most genes resulted in differences in expression of one or more of the dilps. We then elaborated further on homothorax, one of the candidate genes with the strongest effect on dilp expression. We found that Homothorax and its binding partner Extradenticle are involved in regulating dilp2, -3 and -5 expression and that genetic depletion of both transcription factors leads to phenotypes associated with reduced insulin signalling. Furthermore, we provide evidence that other transcription factors involved in eye development are also functional in the IPCs. In conclusion, we showed that this GWAS approach using gene expression levels as input identified genetic regulators implicated in IPC function and dilp expression.


title: Inversion of a topological domain leads to restricted changes in its gene expression and affects inter-domain communication

abstract: The interplay between the topological organization of the genome and the regulation of gene expression remains unclear. Depletion of molecular factors underlying genome topology, such as CTCF and cohesin, leads to modest alterations in gene expression, while genomic rearrangements involving boundaries of topologically associating domains (TADs) disrupt normal gene expression and can lead to pathological phenotypes. Here we inverted an almost entire TAD (245kb out of 300kb) within the X-inactivation centre (Xic), leaving its boundaries intact. This led to a significant rearrangement of topological contacts within the TAD, mostly in accordance to the orientation of underlying CTCF binding sites but suggesting heterogeneity in the "contact" potential of different CTCF sites. The inversion also led to increased contact insulation with the neighbouring TAD. Expression of most genes within the inverted TAD remained unaffected in mouse embryonic stem cells and during differentiation. Interestingly, expression in the neighbouring TAD of the noncoding transcript Xist, which controls X-chromosome inactivation, was ectopically upregulated. The same inversion in mouse embryos led to a bias in Xist expression, but X-inactivation choice ratios did not significantly deviate from wild type. Smaller deletions and inversions of specific clusters of CTCF sites within the TAD led to similar results: rearrangement of contacts, limited changes in local gene expression but significant changes in Xist expression. Our study suggests that the wiring of regulatory interactions within a TAD can influence the expression of genes in neighbouring TADs, highlighting the existence of mechanisms for inter-TAD communication.


title: The monosialoganglioside GM1a protects trophoblasts, erythrocytes and endothelial cells against complement attack

abstract:
The complement system is a part of the innate immune system in the fluid phase and efficiently eliminates pathogens. However, its activation requires tight regulation on the host cell surface in order not to compromise cellular viability. Previously, we showed that loss of placental cell surface sialylation in mice in vivo leads to a maternal complement attack at the fetal-maternal interface, ultimately resulting in loss of pregnancy. To gain insight into the regulatory function of sialylation in complement activation, we here generated trophoblast stem cells devoid of sialylation, which also revealed complement sensitivity and cell death in vitro. Glycolipid-analysis by xCGE-LIF allowed us to identify the monosialoganglioside GM1a as a key element of cell surface complement regulation. Exogenously administered GM1a integrated into the plasma membrane of trophoblasts, dramatically increased binding of complement factor H and was sufficient to protect the cells from complement attack and cell death. Furthermore, GM1a treatment rescued sensitized human erythrocytes and endothelial cells from complement attack in a concentration dependent manner. This study demonstrates for the first time the complement regulatory potential of exogenously administered gangliosides and paves the way for sialoglycotherapeutics as a novel substance class for membrane-targeted complement regulators.

Key pointsO_LIThe naturally occurring sialic acid containing glycosphingolipid GM1a is a potent regulator of complement activation on mammalian trophoblast, erythrocyte and endothelial cell surfaces.
C_LIO_LIIncorporated GM1a recruits complement factor H and thus could represent a novel membrane-targeted complement therapeutic.
C_LI