title: Replication-dependent organisation constrains positioning of long DNA repeats in bacterial genomes

abstract: Bacterial genome organisation is primarily driven by chromosome replication from a single origin of replication. However chromosomal rearrangements, which can disrupt such organisation, are inevitable in nature. Long DNA repeats are major players mediating rearrangements, large and small, via homologous recombination. Since changes to genome organisation affect bacterial fitness - and more so in fast-growing than slow-growing bacteria - and are under selection, it is reasonable to expect that genomic positioning of long DNA repeats is also under selection. To test this, we identified identical DNA repeats of at least 100 base pairs across ~6,000 bacterial genomes and compared their distribution in fast and slow growing bacteria. We find that long identical DNA repeats are distributed in a non-random manner across bacterial genomes. Their distribution differs in their overall number, orientation and proximity to the origin of replication, in fast and slow growing bacteria. We show that their positioning - which might arise from a combination of the processes that produce repeats and selection on rearrangements that recombination between repeat elements might cause - permits minimum disruption to the replication-dependent genome organisation of bacteria, thus acting as a major constraint.

category: genomics


title: Genomes of Two Flying Squid Species Provide Novel Sights into Adaptations of Cephalopods to Pelagic Life

abstract: Pelagic cephalopods have evolved a series of fascinating traits, such as excellent visual acuity, high-speed agility, and photophores for adaptation to open pelagic oceans. However, the genetic mechanisms underpinning these traits are not well understood. Thus, in this study, we obtained high-quality genomes of two purpleback flying squid species (Sthenoteuthis oualaniensis and Sthenoteuthis sp.), with sizes of 5450 and 5651 Mb. Comparative genomic analyses revealed a common expansion of the S-crystallin subfamily SL20-1 associated with visual acuity in the purpleback flying squid lineage and showed that evolution of high-speed agility for the species was accompanied by significant positive selection pressure on genes related to energy metabolism. These molecular signals might have contributed to the evolution of their adaptative predatory and anti-predatory traits. In addition, transcriptomic analysis provided clear indications of the evolution for the photophores of purpleback flying squids, inter alia that recruitment of new genes and energy metabolism genes may have played key functional roles in the process.


title: Improved sensitivity and resolution of ATAC-seq differential DNA accessibility analysis

abstract: Eukaryotic genomes are packaged into chromatin, and the extent of its compaction must be modulated to allow several biological processes such as gene transcription. The regulatory elements of expressed genes are typically in relatively accessible chromatin, and several studies have revealed a reliable correlation between the abundance of mRNA transcripts and the degree of DNA accessibility at the regulatory elements of their coding genes. In consequence, the genome-wide profiling of DNA accessibility by methods such as ATAC-seq can help in the study of gene regulatory networks by serving as a proxy for gene expression and by helping identify important gene cis-regulatory elements and the trans-acting factors that bind them. The predominant approach used to identify differentially accessible genomic loci from ATAC-seq data obtained in two conditions of interest is comparable to that employed in RNA-seq gene expression profiling studies: accessible regions are identified through peak calling and treated like "genes", then sequenced DNA fragments (originating from two neighboring transposase insertion events) that overlap them are counted and subjected to abundance modeling, which then allows to identify those that have a significant difference between the two conditions. We reasoned that this approach could be improved in terms of sensitivity and resolution by introducing two changes: bypassing peak calling, using instead a genome-wide sliding window quantification approach, and counting transposase insertion sites, instead of fragments originating from two neighboring insertion sites. We present the development of this approach, which we term "widaR", for Window- and Insertion-based Differential Accessibility in R, using a murine skeletal myoblast differentiation dataset. Reproducible R code is provided.


title: Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain

abstract: Nucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an in-depth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (FnCas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a. We probed the structural states of FnCas12a variants altered in helix 1 and/or the BH using single-molecule FRET measurements and assayed the pre-crRNA processing, cis- and trans-DNA cleavage activity. We show that helix 1 and not the BH is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the BH upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site.

category: biochemistry


title: The orphan ligand, Activin C, signals through activin receptor-like kinase 7

abstract: Activin ligands are formed from two disulfide-linked inhibin {beta} subunit chains. They exist as homodimeric proteins, as in the case of activin A (ActA; Inh{beta}A/Inh{beta}A) or activin C (ActC; Inh{beta}C/Inh{beta}C), or as heterodimers, as with activin AC (ActAC; Inh{beta}A:Inh{beta}C). While the biological functions of ActA and activin B (ActB) have been well-characterized, little is known about the biological function of ActC or ActAC. One thought is that the Inh{beta}C chain functions to interfere with ActA production by forming less active ActAC heterodimers. Here, we assessed and characterized the signaling capacity of ligands containing the Inh{beta}C chain. ActC and ActAC activated SMAD2/3-dependent signaling via the type I receptor, activin receptor-like kinase 7 (ALK7). Relative to ActA and ActB, ActC exhibited lower affinity for the cognate activin type II receptors and was resistant to neutralization by the extracellular antagonist, follistatin. In mature adipocytes, which exhibit high ALK7 expression, ActC elicited a SMAD2/3 response similar to ActB, which can also signal via ALK7. Collectively, these results establish that ActC and ActAC are active ligands that exhibit a distinct signaling receptor and antagonist profile compared to other activins.


title: Interpreting the contributions of transcript features to protein abundance prediction models

abstract: Protein and mRNA levels correlate only moderately across samples. The availability of proteogenomics data sets with protein and transcript measurements from matching samples is providing new opportunities to assess the degree to which protein levels in a system can be predicted from mRNA information. Here we revisited the protein level prediction problem and analyzed large proteogenomics data from 8 cancer types within the Clinical Proteomic Tumor Analysis Consortium (CPTAC) data set. We trained models to predict the abundance of 13,637 proteins using matching transcriptome data from up to 958 tumor or normal adjacent tissue samples each, and compared predictive performances across algorithms and input features. As data depth increased, the incorporation of additional transcripts as input features increasingly improved the ability to predict sample protein abundance. We found widespread occurrences where the abundance of a protein is considerably less well explained by its own cognate transcript level than that of one or more trans locus transcripts, suggesting post-transcriptional regulations broadly influence protein and mRNA correlation. Transcripts that contribute to non-cognate protein abundance primarily involve those encoding known interaction partners and protein complex members of the protein of interest. Network analysis reveals a proteome-wide interdependency of protein abundance on the transcript levels of interacting proteins. Proteogenomic co-expression analysis may have utility for finding gene interactions and predicting expression changes in biological systems.

category: bioinformatics


title: Induction of cortical Par complex polarity by designed proteins causes cytoskeletal symmetry breaking in unpolarized mammalian cells

abstract: Polarized cells rely on a polarized cytoskeleton for polarized trafficking, oriented migration and spindle orientation during asymmetric cell division. While cytoskeleton remodeling machineries have been extensively characterized at the molecular level, how polarity signaling at the cortex controls remodeling of the cytoskeleton in the cytosol remains elusive. In particular, how the Par complex, the conserved mastermind of polarity during asymmetric cell division, gets assembled and functions is not understood at the molecular level. Here, we dissected the logic of the Par complex pathway by capitalizing on designed proteins able to induce spontaneous symmetry breaking of the cortex in populations of naive, unpolarized cells. We found that the primary kinetic barrier to Par complex assembly is the relief of Par6 autoinhibition, and that inducing Par complex cortical polarity was sufficient to induce two key hallmarks of asymmetric cell division in unpolarized cells: spindle orientation and central spindle asymmetry. These two outputs of the Par complex are separately controlled: spindle orientation is determined by Par3 and does not require the kinase activity of aPKC, while central spindle asymmetry solely depends on an asymmetric activity of aPKC at the cortex. Our work shows how polarity information flows between the cortex and the cytosol despite its diffusive nature, and paves the way towards induction of asymmetric cell division in cultured cells.

category: cell biology


title: Artificial Hsp100-mediated systems for re-localizing protein aggregates

abstract: Spatial Protein Quality Control (sPQC) sequesters misfolded proteins into specific, organelle-associated inclusions within the cell to harness their toxicity. To approach the role of sPQC in cellular fitness, neurodegenerative diseases and aging, we report on the construction of Hsp100-based systems in yeast cells, which can artificially target protein aggregates to non-canonical locations. We demonstrated that aggregates of mutant Huntingtin (mHtt), the disease-causing agent of Huntingtons disease can be artificially targeted to daughter cells as well as to eisosomes and endosomes with this approach. Removing aggregates from mother cells did not significantly affect their lifespan and targeting mHtt to multiple smaller aggregates rather than one large inclusion did not alter its toxicity. We demonstrated that this approach is able to manipulate mHtt inclusion formation also in human cells and has the potential to be a useful complementation to present therapeutic approaches aimed at alleviating age-related neurodegenerative diseases.


title: Strain stiffening of the Ndc80 complex

abstract:
In the mitotic spindle, microtubules attach to chromosomes via kinetochores. The microtubule-binding Ndc80 complex is an integral part of kinetochores, and is essential for kinetochores to attach to microtubules and to transmit forces from dynamic microtubule ends to the chromosomes. The Ndc80 complex has a rod-like appearance with globular domains at its ends that are separated by a long coiled coil. Its mechanical properties are considered important for the dynamic interaction between kinetochores and microtubules. Here, we present a novel method that allows us to time-trace the effective stiffness of Ndc80 complexes following shortening microtubule ends against applied force in optical trap experiments. Applying this method to wild type Ndc80 and three variants (CH-domains mutated or Hec1-tail unphosphorylated, phosphorylated, or truncated), we reveal that each variant exhibits strain stiffening, i.e., the effective stiffness increases under tension that is built up by a depolymerizing microtubule. The strain stiffening relation is roughly linear and independent of the state of the microtubule. We introduce a polymer model for the Ndc80 complex, which shows that the strain stiffening can be traced back to the specific architecture of the Ndc80 complex with a characteristic flexible kink and the bending elasticity of flaring protofilaments, which exert force to move Ndc80 complexes. Our model accounts for changes in the amount of load-bearing attachments at various force levels and reproduces the roughly linear strain stiffening behavior, highlighting the importance of force-dependent binding affinity.

SIGNIFICANCEBy time-tracing the stiffness of microtubule end-tracking Ndc80 complexes in optical trap experiments, we detect strain stiffening, and, thereby, provide new insights into the elastic properties of the Ndc80 complex. The strain stiffening is robust against mutations in the Ndc80 complex. We relate strain stiffening to the structure of the Ndc80 complex by means of a simple polymer model, and to the flexibility of force-generating flaring protofilaments at the tip of the microtubule. Since Ndc80 complexes play a major role for transmitting force from microtubule ends to the kinetochore, their elastic properties are of great interest for a deeper understanding of chromosome dynamics in the mitotic spindle.

category: biophysics


title: Merging of ventral fibers at adhesions drives the remodeling of cellular contractile systems in fibroblasts.

abstract: Ventral stress fibers (VSFs) are contractile actin fibers present in the ventral plane of the cell and existing in a dynamic attachment with cell-matrix focal adhesions. VSFs are critical in cellular mechanobiological functions such as traction force production, cell polarization, and migration. VSF within their intracellular network vary from short, thinner fibers that are randomly oriented to long, thick fibers that span along the whole long axis of a cell. De novo VSF formation was shown to occur by condensation from the cortical actin mesh or by crosslinking of other stress fiber subtypes (dorsal stress fibers and transverse arcs) at the cell front. However, formation of long VSFs that extend across the whole cell axis is not well understood. Here, we report a novel phenomenon of VSF merging in migratory fibroblast cells, which is guided by mechanical force balance and contributes to VSF alignment along the long cell axis. The mechanism of VSF merging involves two steps: connection of two ventral fibers by an emerging myosin II bridge at an intervening adhesion and intervening adhesion dissolution to form a cohesive, contractile VSF. Our data indicate that these two steps are interdependent, since under conditions where adhesion disassembly is slowed, formation of the myosin bridge is slowed as well. Cellular data and computational modeling show that the angle of contact between merging fibers decides successful merging, with angles closer to 180 yielding merging events and shallower angles leading to merge failure. Our data and modeling further show that merging increases the share of uniformly aligned long VSFs, which would contribute to directional traction force production. Thus, we thoroughly characterize merging as process for dynamic reorganization of VSFs in steady state, investigating the steps and variants of the process as well as its functional significance in migratory cells.


title: Targeting A-Kinase Anchoring Protein 12 Phosphorylation in Hepatic Stellate Cells Regulates Liver Injury and Fibrosis in Mouse Models

abstract: Trans-differentiation of hepatic stellate cells (HSCs) to activated state potentiates liver fibrosis through release of extracellular matrix (ECM) components, distorting the liver architecture. Since limited antifibrotics are available, pharmacological intervention targeting activated HSCs may be considered for therapy. A-kinase anchoring protein 12 (AKAP12) is a scaffolding protein that directs protein kinases A/C (PKA/PKC) and cyclins to specific locations spatiotemporally controlling their biological effects. It has been shown that AKAP12s scaffolding functions are altered by phosphorylation. In previously published work, observed an association between AKAP12 phosphorylation and HSC activation. In this work we demonstrate that AKAP12s scaffolding activity towards the endoplasmic reticulum (ER)-resident collagen chaperone, heat-shock protein 47 (HSP47) is strongly inhibited by AKAP12s site-specific phosphorylation in activated HSCs. CRISPR-directed gene editing of AKAP12s phospho- sites restores its scaffolding towards HSP47, inhibiting HSP47s collagen maturation functions and HSC activation. AKAP12 phospho-editing dramatically inhibits fibrosis, ER stress response, HSC inflammatory signaling and liver injury in mice. Our overall findings suggest a pro-fibrogenic role of AKAP12 phosphorylation that may be targeted for therapeutic intervention in liver fibrosis.


title: Zfp281 inhibits the pluripotent-to-totipotent state transition in mouse embryonic stem cells

abstract: The cell-fate transition between pluripotent and totipotent states determines embryonic development and the first cell-lineage segregation. However, limited by the scarcity of totipotent embryos, regulators on this transition remain largely elusive. A novel model to study the transition has been recently established, named the 2-cell-like (2C-like) model. The 2C-like cells are rare totipotent-like cells in the mouse embryonic stem cell (mESC) culture. Pluripotent mESCs can spontaneously transit into and out of the 2C-like state. We previously dissected the transcriptional roadmap of the transition. In this study, we revealed that Zfp281 is a novel regulator for the pluripotent-to-totipotent transition in mESCs. Zfp281 is a transcriptional factor involved in the cell-fate transition. Our study shows that Zfp281 represses transcripts upregulated during the 2C-like transition via Tet1 and consequentially inhibits mESCs from transiting into the 2C-like state. Interestingly, we found that the inhibitory effect of Zfp281 on the 2C-like transition leads to an impaired 2C-like-transition ability in primed-state mESCs. Altogether, our study reveals a novel mediator for the pluripotent-to-totipotent state transition in mESCs and provides insights into the dynamic transcriptional control of the transition.


title: FGF10 triggers de novo alveologenesis in a BPD model: impact on the resident mesenchymal niche cells

abstract: Bronchopulmonary dysplasia (BPD) is a neonatal lung disease developing in premature babies characterized by arrested alveologenesis and associated with decreased Fibroblast growth factor 10 (FGF10) expression. One-week hyperoxia (HYX) exposure of newborn mice leads to a permanent arrest in alveologenesis. To test the role of Fgf10 signaling to promote de novo alveologenesis following hyperoxia, we used transgenic mice allowing inducible expression of Fgf10 and recombinant FGF10 (rFGF10) protein delivered intraperitoneally. We carried out morphometry analysis, and IF on day 45. Alveolospheres assays were performed co-culturing AT2s from normoxia (NOX) with FACS-isolated Sca1Pos resident mesenchymal cells (rMC) from animals exposed to NOX, HYX+PBS, or HYX+FGF10. scRNAseq between rMC-Sca1Pos isolated from NOX and HYX+PBS were also carried out. Transgenic overexpression of Fgf10 and rFGF10 administration rescued the alveologenesis defects following HYX. Alveolosphere assays indicate that the activity of rMC-Sca1Pos is negatively impacted by HYX and partially rescued by rFGF10 treatment. Analysis by IF demonstrates a significant impact of rFGF10 on the activity of resident mesenchymal cells. scRNAseq results identified clusters expressing Fgf10, Fgf7, Pdgfra, and Axin2, which could represent the rMC niche cells for the AT2 stem cells. In conclusion, we demonstrate that rFGF10 administration is able to induce de-novo alveologenesis in a BPD mouse model and identified subpopulations of rMC-Sca1Pos niche cells potentially representing its cellular target.


title: CyberSco.Py: open-source software for event-based, conditional microscopy

abstract: Timelapse fluorescence microscopy imaging is routinely used in quantitative cell biology. However, microscopes could become much more powerful investigation systems if they were endowed with simple unsupervised decision-making algorithms to transform them into fully responsive and automated measurement devices. Here, we report CyberSco.Py, Python software for advanced automated timelapse experiments. We provide proof-of-principle of a user-friendly framework that increases the tunability and flexibility when setting up and running fluorescence timelapse microscopy experiments. Importantly, CyberSco.Py combines real-time image analysis with automation capability, which allows users to create conditional, event-based experiments in which the imaging acquisition parameters and the status of various devices can be changed automatically based on the image analysis. We exemplify the relevance of CyberSco.Py to cell biology using several use case experiments with budding yeast. We anticipate that CyberSco.Py could be used to address the growing need for smart microscopy systems to implement more informative quantitative cell biology experiments.


title: Cellular redox metabolism is modulated by the distinct localization of cyclic nucleotide phosphodiesterase 5A isoforms

abstract:
3-5 cyclic nucleotide phosphodiesterases (PDEs) are a family of evolutionary conserved cAMP and/or cGMP hydrolysing enzymes, components of transduction pathways regulating crucial aspects of cell life. Among them, cGMP-specific PDE5, being a regulator of vascular smooth muscle contraction, is the molecular target of several drugs used to treat erectile dysfunction and pulmonary hypertension.

Production of full-length murine PDE5A isoforms in the milk-yeast Kluyveromyces lactis showed that the quaternary assembly of MmPDE5A1 is a mixture of dimers and tetramers, while MmPDE5A2 and MmPDE5A3 only assembled as dimers. We showed that the N-terminal peptide is responsible for the tetramer assembly of MmPDE5A1, while that of MmPDE5A2 for its mitochondrial localization.

Overexpression of the three isoforms alters at different levels the cAMP/cGMP equilibrium as well as the NAD(P)+/NAD(P)H balance and induces a metabolic switch from oxidative to fermentative. In particular, the mitochondrial localization of MmPDE5A2 unveiled the existence of a cAMP-cGMP signaling cascade in this organelle, for which we propose a metabolic model that could explain the role of PDE5 in some cardiomyopathies and some of the side effects of its inhibitors.


title: Aftiphilin regulation of myosin light chain kinase activity promotes actin dynamics and intestinal epithelial barrier function

abstract: The expression levels of aftiphilin (AFTPH) are significantly lower in inflamed colonic tissues from patients with ulcerative colitis (UC) and mice with experimental colitis. During colonic inflammation, the selective permeability of the colonic epithelium is compromised largely due to dysregulation of proteins associated with either the tight junction (TJ) complex and actin-myosin contraction rings. Here, we hypothesized that inflammation-associated reduction in AFTPH levels might cause an increase in the selective permeability of the colonic epithelium. In this study, we measured the transepithelial electric resistance (TEER), sodium (Na+) ion flux and dextran permeability in polarized colonic epithelial cells after manipulation of AFTPH. Silencing of AFTPH reduced TEER, increased Na+ ion flow and dextran permeability. Examination of mRNA and protein levels of multiple TJ proteins and Na+ ion transporters suggested that AFTPH deficiency did not significantly change expression of most of these transmembrane proteins. While the gross structure of the TJs in AFTPH gene-silenced cells appeared normal, elevated levels of junctional Occludin were observed. Most notably we observed that AFTPH co-localized with myosin light chain kinase (MLCK) and attenuated cellular MLCK activity as observed by phospho-myosin light chain 2 (pMLC2) western blots. Importantly, inhibition of MLCK activity reversed the reduction of TEER in AFTPH-deficient monolayers. Lastly, examination on transmission electron microcopy on microvilli and immunofluorescent microscopy on actin filament arrangement showed that AFTPH deficiency also affected filament arrangement in colonic epithelial cells. Taken together, these results suggest that AFTPH regulates intestinal epithelial permeability and actin polymerization in colonic epithelium through interfering MLCK/MLC interactions.


title: A Unique Cellular Organization of Human Distal Airways and Its Disarray in Chronic Obstructive Pulmonary Disease

abstract: In the human lung, terminal bronchioles (TBs), the most distal conducting airways, open to respiratory bronchioles (RBs) that lead to the alveolar region where gas exchange takes place. This transition occurs in pulmonary lobules, lung tissue units supplied by pre-TBs, which give rise to TBs. Accumulating evidence suggests that remodeling and loss of pre-TBs and TBs underlies progressive airflow limitation in chronic obstructive pulmonary disease (COPD), the third leading cause of death worldwide. Understanding the nature of these changes at the single-cell level has so far been limited by poor accessibility of pre-TBs and TBs. Here, we introduce a novel method of region-precise airway dissection, which enables capture of the entire anatomical continuum of peripheral airways, from pre-TBs to RBs, and the associated alveolar region within the lobule. This approach allowed us to identify terminal airway-enriched secretory cells (TASCs), a unique epithelial cell population of distal airways expressing secretoglobin 3A2 (SCGB3A2) and/or surfactant protein B (SFTPB). TASCs were enriched in TBs, particularly, in areas of TB-RB transition and exhibited an intermediate, broncho-alveolar molecular pattern. TASC frequency was markedly decreased in pre-TBs and TBs of COPD patients compared to those in non-diseased lungs, accompanied by changes in cellular composition of vascular and immune microenvironments. In vitro regeneration assays identified basal cells (BCs) of pre-TBs and TBs as a cellular origin of TASCs in the human lung. Generation of TASCs by these region-specific progenitors was suppressed by IFN-{gamma} signaling that was augmented in distal airways of COPD patients. Thus, altered maintenance of region-specific cellular organization of pre-TBs and TBs represents a key component of distal airway pathology in COPD.


title: KIF24 controls the clustering of supernumerary centrosomes in pancreatic ductal adenocarcinoma cells

abstract: Clustering of supernumerary centrosomes, potentially leading to cell survival and chromosomal instability, is frequently observed in cancers. However, the molecular mechanisms by which centrosome clustering is controlled in cancer cells remain largely unknown. A centrosomal kinesin, KIF24, was previously shown to restrain the assembly of primary cilia in mammalian cells. Here, we revealed that KIF24 depletion suppresses multipolar spindle formation by clustering centrosomes in pancreatic ductal adenocarcinoma (PDAC) cells harboring supernumerary centrosomes. KIF24 depletion also induced hyperproliferation and improved the mitotic progression in PDAC cells. On the other hand, disruption of primary cilia failed to affect the proliferation and spindle formation in KIF24-depleted cells. These results represent a novel role of KIF24 in suppressing centrosome clustering independent of primary ciliation in centrosome-amplified PDAC cells.


title: Cell size and actin architecture determine force generation in optogenetically activated adherent cells

abstract: Adherent cells use actomyosin contractility to generate mechanical force and to sense the physical properties of their environment, with dramatic consequences for migration, division, differentiation and fate. However, the organization of the actomyosin system within cells is highly variable, with its assembly and function being controlled by small GTPases from the Rho-family. How activation of these regulators translates into cell-scale force generation and the corresponding sensing capabilities in the context of different physical environments is not understood. Here we probe this relationship combining recent advances in non-neuronal optogenetics with micropatterning and traction force microscopy on soft elastic substrates. We find that after whole-cell RhoA-activation by the CRY2/CIBN optogenetic system with a short pulse of 100 milliseconds, single cells contract before returning to their original tension setpoint with near perfect precision on a time scale of several minutes. To decouple the biochemical and mechanical elements of this response, we introduce a mathematical model that is parametrized by fits to the dynamics of the substrate deformation energy. We find that the RhoA-response builds up quickly on a time scale of 20 seconds, but decays slowly on a time scale of 50 seconds. The larger the cells and the more polarized their actin cytoskeleton, the more substrate deformation energy is generated. RhoA-activation starts to saturate if optogenetic pulse length exceeds 50 milliseconds, revealing the intrinsic limits of biochemical activation. Together our results suggest that adherent cells establish tensional homeostasis by the RhoA-system, but that the setpoint and the dynamics around it are strongly determined by cell size and the architecture of the actin cytoskeleton, which both are controlled by the extracellular environment.


title: CDKL5 deficiency slows synaptic vesicle endocytosis

abstract: Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe early-onset epileptic encephalopathy resulting mainly from de novo mutations in the X-linked CDKL5 gene. To determine whether loss of presynaptic CDKL5 function contributes to CDD, we examined synaptic vesicle (SV) recycling in primary hippocampal neurons generated from a Cdkl5 knockout rat model. Using a genetically-encoded reporter, we revealed that CDKL5 is selectively required for efficient SV endocytosis. We showed that CDKL5 kinase activity is both necessary and sufficient for optimal SV endocytosis, since kinase-inactive mutations failed to correct endocytosis in CDKL5 knockout neurons, whereas the isolated CDKL5 kinase domain fully restored SV endocytosis kinetics. Finally, we demonstrated that CDKL5-mediated phosphorylation of amphiphysin 1, a putative presynaptic target, is not required for CDKL5-dependent control of SV endocytosis. Overall, our findings reveal a key presynaptic role for CDKL5 kinase activity and enhance our insight into how its dysfunction may culminate in CDD.

category: neuroscience


title: Dual role of an essential HtrA2/Omi protease in the human malaria parasite: maintenance of mitochondrial homeostasis and induction of apoptosis like cell death under cellular stress

abstract: HtrA family of serine protease is known to play role in mitochondrial homeostasis as well as in the programmed cell death. Mitochondrial homeostasis and metabolism are crucial for survival and propagation of malaria parasite within the host. Here we have functionally characterized a Plasmodium falciparum HtrA2 (PfHtrA2) protein, which harbours trypsin like protease activity that can be inhibited by its specific inhibitor, ucf-101. Transgenic parasite line was generated, using HA-glmS C-terminal tagging approach, for localization as well as for inducible knockdown of PfHtrA2. The PfHtrA2 was localized in the parasite mitochondrion during asexual life cycle. Genetic ablation of PfHtrA2 caused significant parasite growth inhibition, decreased replication of mtDNA, increased mitochondrial ROS production, caused mitochondrial fission/fragmentation, and hindered parasite development. However, ucf-101 treatment had no effect on the parasite growth, suggesting non-protease/chaperone role of PfHtrA2 in the parasite. Under cellular stress conditions, inhibition of PfHtrA2 by ucf-101 reduced activation of the caspase like protease as well as parasite cell death, suggesting involvement of protease activity of PfHtrA2 in apoptosis like cell death in the parasite. Under these cellular stress conditions, the PfHtrA2 gets processed but remains localized in the mitochondrion, suggesting that it acts within the mitochondrion by cleaving intra-mitochondrial substrate(s). This was further supported by trans-expression of PfHtrA2 protease domain in the parasite cytosol, which was unable to induce any cell death in the parasite. Overall, we show specific role of PfHtrA2 in maintaining mitochondrial homeostasis as well as in regulating the stress induced cell death.


title: Molecular Crowding Facilitates Bundling of IMPDH Polymers and Cytoophidium Formation

abstract: The cytoophidium is a unique type of membraneless compartment comprising of filamentous protein polymers. Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step of de novo GTP biosynthesis and plays critical roles in active cell metabolism. However, the molecular regulation of cytoophidium formation is poorly understood. Here we show that human IMPDH2 polymers bundle up to form cytoophidium-like aggregates in vitro when macromolecular crowders are present. The self-association of IMPDH polymers is suggested to rely on electrostatic interactions. In cells, the increase of molecular crowding with hyperosmotic medium induces cytoophidia, while the decrease of that by the inhibition of RNA synthesis perturbs cytoophidium assembly. In addition to IMPDH, CTPS and PRPS cytoophidium could be also induced by hyperosmolality, suggesting a universal phenomenon of cytoophidium-forming proteins. Finally, our results indicate that the cytoophidium can prolong the half-life of IMPDH, which is proposed to be one of conserved functions of this subcellular compartment.


title: The X chromosome is a potential polarising signal for asymmetric cell divisions in meiotic cells of a nematode

abstract: The unequal partition of molecules and organelles during cell division results in daughter cells with different fates. Asymmetric cell divisions have been best characterised in systems in which extrinsic signals polarise the mother cell during cell division. However, the mechanisms of asymmetric cell division mediated by intrinsic signals, and the nature of these signals, are mostly unknown. Here we report an asymmetric cell division in the nematode Auanema rhodensis that may be cued by the X chromosome. In the wildtype XO male, the spermatocyte divides asymmetrically to generate X-bearing spermatids that inherit components necessary for sperm viability, and nullo-spermatids that inherits components to be discarded. We found that in XX mutant pseudomales, sperm components co-segregate with the X chromosome, supporting the hypothesis that the X chromosome is employed as a polarising signal for partitioning essential cytoplasmic components for sperm function.

category: developmental biology


title: Lipid-correlated alterations in the transcriptome are enriched in several specific pathways in the postmortem prefrontal cortex of Japanese patients with schizophrenia

abstract:
BackgroundSchizophrenia is a chronic relapsing psychiatric disorder that is characterized by many symptoms and has a high heritability. A previous study showed that specific lipid molecules belong to phosphatidylinositol (PI) and phosphatidylserine (PS) was reduced in the postmortem prefrontal cortex of patients with schizophrenia1. However, signaling pathways contributing to the lipid changes remain unknown. Here we performed two types of transcriptome analyses in patients with schizophrenia: an un-biased transcriptome analysis solely based on RNA-seq data and a correlation analysis between levels of gene expression and lipids.

Methodology/Principal FindingsRNA-Seq analysis was performed in the postmortem prefrontal cortex from 10 subjects with schizophrenia and 5 controls. Correlation analysis between the transcriptome and lipidome from 9 subjects which are the same samples in the previous lipidomics study1 (Table 1). Extraction of differentially expressed genes (DEGs) and further sequence and functional group analysis revealed changes of gene expression levels in phosphoinositide 3-kinase (PI3K)-Akt signaling and the complement system. In addition, a correlation analysis clarified alterations in several signaling/metabolic pathways including lipid-correlated genes, most of which are not found as DEGs in transcriptome analysis alone.

O_TBL View this table:
org.highwire.dtl.DTLVardef@80d74eorg.highwire.dtl.DTLVardef@b780b7org.highwire.dtl.DTLVardef@1637f28org.highwire.dtl.DTLVardef@1315439org.highwire.dtl.DTLVardef@190a230_HPS_FORMAT_FIGEXP  M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONCharacteristics of patients from whom postmortem brain samples were obtained.

Abbreviations: PMI, postmortem interval, the time that has elapsed since a person has died; DOI, duration of illness, The samples used in correlation analysis are shown by black circles.

C_TABLECAPTION C_TBL

ConclusionsThis study provided results of the first integrated analysis of the schizophrenia-associated transcriptome and lipidome within the PFC and revealed that lipid-correlated alterations in the transcriptome are enriched in specific pathways including PI3K-Akt signaling.

category: pathology


title: Concurrent separation of phase-locked and non-phase-locked activity

abstract: Brain dynamics recorded via electroencephalography (EEG) is conceptualized as a sum of two components, "phase-locked" and "non-phase-locked" to the stimulus. Both activities are understood to be stemming from different neuronal mechanisms and hence accurately characterizing them is of immense importance in neuroscientific studies. Here, we discuss the drawbacks of currently used methods to separate the phase-locked and non-phase-locked activity and propose a new method that decomposes the two components simultaneously. First, we demonstrate that single-trial separation of phase-locked and non-phase-locked power is an ill-posed problem. Second, using simulations where ground truth validation is possible, we elucidate the drawbacks of the widely used averaging method and efficacy of the proposed concurrent phaser method (CPM). Using two experimental datasets, audio oddball EEG data and auditory steady-state responses (ASSR) we show how the empirical signal-to-noise estimates warrant the usage of CPM to separate phase-locked and non-phase-locked activity.


title: Geometrical factors determining dendritic domain intersection between neurons: a modeling study

abstract:
Overlap between dendritic trees of neighboring neurons is a defining feature of central nervous systems. Dendritic domains sharing a given locus are arguably more likely to share afferent inputs, and proximity is a requirement for dendro-dendritic communication. Dendritic overlap also differentiates neuronal circuits characterized by redundancy and integration from those characterized by a more specific and selective connectivity. We examined the geometrical factors that affect intersection between pairs of neurons by, specifically, modelling the influence of cell-to-cell distance, and dendritic domain size and shape. We developed MATLAB scripts to determine intersected volume between pairs of three-dimensionally reconstructed neurons using their convex hull (CH) polyhedrons as proxies for dendritic domain. The influence of neuron-to-neuron distance on intersection was not straightforward and reflected the diverse and anisotropic geometry of neurons, and the ostensible displacement of cell bodies from the CH centroid. The influence of size on pair intersection was tested by giving each neuronal CH pair the same (average) volume without modifying their shape. Conversely, the influence of neuronal shape on pair intersection was proved by giving each neuronal pair an average shape without modifying volumes. While both normalization increased intersection, shape averaging was remarkably more effective than size averaging in increasing pair intersection. We conclude that intersection does not only depend on neuron-to-neuron proximity, but critically on neuron-to-neuron differences in size and shape. The results predict that circuits characterized by selective connectivity exhibit greater heterogeneity in size, but especially shape, of their constituent neuronal elements.

Author summaryNeurons communicate with each other using specialized domains called axons and dendrites. Throughout the brain, dendrites from adjacent neurons overlap. This dendritic overlap helps neurons to share common input information coming from axons and also permits communication between dendrites themselves. One way to study dendritic overlap is using the geometrical representation of the dendrites  field of view, using simplified three-dimensional geometrical shapes (polyhedrons) called convex hulls. In this computational study, we used these convex hulls to analyze the geometrical factors underlying intersection between neurons. For that, we developed methods to obtain the intersected volume of pairs of convex hulls from neurons, to then independently modify the location, size and shape of convex hulls. We observed high variability in intersected volume, even when the neurons were at a similar distance between each other. Relevantly, we also found that variability in volume, but especially variability in convex hull shape is the main factor that determines the degree of intersection between pairs of neurons. These data suggest that differences in dendritic size, but especially differences in shape between neurons have a strong effect on overlap between neurons, and therefore on the potential for neurons to share input information and communicate with each other.


title: Sharp wave ripples in macaque V1 and V4 are modulated by top-down visual attention

abstract: Sharp-wave ripples (SWRs) are highly synchronous neuronal activity events. They have been predominantly observed in the hippocampus during offline states such as pause in exploration, slow-wave sleep and quiescent wakefulness. SWRs have been linked to memory consolidation, spatial navigation, and spatial decision-making. Recently, SWRs have been reported during visual search, a form of remote spatial exploration, in macaque hippocampus. However, the association between SWRs and multiple forms of awake conscious and goal-directed behavior is unknown. We report that ripple activity occurs in macaque visual areas V1 and V4 during focused spatial attention. The frequency of ripples is modulated by characteristics of the stimuli, by spatial attention directed toward a receptive field, and by the size of the attentional focus. Critically, the monkeys reaction times in detecting behaviorally relevant stimulus changes was affected on trials with SWRs. These results show that ripple activity is not limited to hippocampal activity during offline states, rather they occur in the neocortex during active attentive states and vigilance behaviors.


title: Integrated Diffusion Image Operator (iDIO): A tool for automated configuration and processing of diffusion MRI data

abstract: The preprocessing of diffusion magnetic resonance imaging (dMRI) data involves numerous steps, including the corrections for head motion, susceptibility distortion, low signal-to-noise ratio, and signal drifting. Researchers or clinical practitioners often need to configure different preprocessing steps depending on disparate image acquisition schemes, which increases the technical threshold for dMRI analysis for non-expert users. This could cause disparities in data processing approaches and thus hinder the comparability between studies. To make the dMRI data processing steps transparent and adapt to various dMRI acquisition schemes for researchers, we propose a semi-automated pipeline tool for dMRI named integrated Diffusion Image Operator or iDIO. This pipeline integrates features from a wide range of advanced dMRI software tools and targets at providing a one-click solution for dMRI data analysis, via automatic configuration for a set of optimal processing steps based on the image header of the input data. Additionally, the pipeline provides options for post-processing, such as estimation of diffusion tensor metrics and whole-brain tractography-based connectomes reconstruction using common brain atlases. The iDIO pipeline also outputs an easy-to-interpret quality control report to facilitate users to assess the data quality. To keep the transparency of data processing, the execution log and all the intermediate images produced in the iDIOs workflow are accessible. The goal of iDIO is to reduce the barriers for clinical or non-specialist users to adopt the state-of-art dMRI processing steps.


title: Regulation of presynaptic Ca2+ channel abundance at active zones through a balance of delivery and turnover

abstract: AbstractVoltage-gated Ca2+ channels (VGCCs) mediate Ca2+ influx to trigger neurotransmitter release at specialized presynaptic sites termed active zones (AZs). The abundance of VGCCs at AZs regulates neurotransmitter release probability (Pr), a key presynaptic determinant of synaptic strength. Although biosynthesis, delivery and recycling cooperate to establish AZ VGCC abundance, experimentally isolating these distinct regulatory processes has been difficult. Here we describe how the AZ levels of Cacophony (Cac), the sole VGCC mediating synaptic transmission in Drosophila, are determined. We also analyzed the relationship between Cac, the conserved VGCC regulatory subunit 2{delta}, and the core AZ scaffold protein Bruchpilot (BRP) in establishing a functional AZ. We find Cac and BRP are independently regulated at growing AZs, as Cac is dispensable for AZ formation and structural maturation, and BRP abundance is not limiting for Cac accumulation. Additionally, AZs stop accumulating Cac after an initial growth phase, whereas BRP levels continue to increase given extended developmental time. AZ Cac is also buffered against moderate increases or decreases in biosynthesis, whereas BRP lacks this buffering. To probe mechanisms that determine AZ Cac abundance, intravital FRAP and Cac photoconversion were used to separately measure delivery and turnover at individual AZs over a multi-day period. Cac delivery occurs broadly across the AZ population, correlates with AZ size, and is rate-limited by 2{delta}. Although Cac does not undergo significant lateral transfer between neighboring AZs over the course of development, Cac removal from AZs does occur and is promoted by new Cac delivery, generating a cap on Cac accumulation at mature AZs. Together these findings reveal how Cac biosynthesis, synaptic delivery, and recycling set the abundance of VGCCs at individual AZs throughout synapse development and maintenance.


title: Beetles provide directed dispersal of viable spores of a keystone wood decay fungus

abstract: Wood decay fungi are considered to be dispersed by wind, but dispersal by animals may also be important, and more so in managed forests where dead wood is scarce. We investigated whether beetles could disperse spores of the keystone species Fomitopsis pinicola. Beetles were collected on sporocarps and newly felled spruce logs, a favourable habitat for spore deposition. Viable spores (and successful germination) of F. pinicola were detected by dikaryotization of monokaryotic bait mycelium from beetle samples. Viable spores were on the exoskeleton and in the faeces of all beetles collected from sporulating sporocarps. On fresh spruce logs, nine beetle species transported viable spores, of which several bore into the bark. Our results demonstrate that beetles can provide directed dispersal of wood decay fungi. Potentially, it could contribute to a higher persistence of some species in fragmented forests where spore deposition by wind on dead wood is less likely.

category: ecology


title: The chemical basis of metabolic interdependence in microbial communities

abstract: Microbial communities play a crucial role in determining the dynamics of soil and marine ecosystems. They strongly influence the physiological functioning of plants and animals, for instance, nutrient uptake, stress tolerance, immune responses in the gut, lung, skin, etc. The diverse species in such communities interact both competitively as well as cooperatively. Cross-feeding, the exchange of metabolites between a pair of microbial species for mutual benefit is a common interaction that probably explains why 99% of natural bacterial species are unculturable on their own in the laboratory. Here, we provide a theoretical, network-level understanding of the conditions under which cross-feeding between a pair of microbial species can be beneficial to both. Using the known microbial repertoire of metabolic reactions, as represented in the KEGG database, we construct a large ensemble of metabolic networks designed to synthesize a set of biomass precursors from specified nutrients. We construct both autonomous networks, that can perform this task on their own, as well as pairs of cross-feeding networks that can only perform this task together but not alone. Surprisingly, we find that there exist cross-feeding pairs that produce higher biomass or energy yields than even the best autonomous networks. We show that such "outperforming" cross-feeding pairs exist only because of certain nonlinearities in the way metabolic flux is distributed in these networks. By analyzing patterns in our ensemble of networks, we propose a set of necessary and (almost) sufficient conditions that the metabolic networks have to satisfy for cross-feeding to be beneficial. These conditions are based partly on the structure of the networks and partly on the chemical and thermodynamic properties of the underlying chemical reactions, phenomenologically quantified in terms of the effect of donating or accepting metabolites on the yield of our constructed networks. Our analysis not only provides a mechanistic understanding of why cross-feeding is prevalent in microbial communities, but also provides a theoretical basis for understanding the benefit of compartmentalization of chemical reactions in a variety of contexts, for instance with mitochondrial vs. cytoplasmic metabolism in eukaryotic cells, or multi-enzyme cascade reactions in industrial contexts.


title: Eco-evolutionary dynamics of clonal multicellular life cycles

abstract: The evolution of multicellular life cycles is a central process in the course of the emergence of multicellularity. The simplest multicellular life cycle is comprised from the growth of the propagule into a colony and its fragmentation to give rise to new propagules. The majority of theoretical models assume selection among life cycles to be driven by internal properties of multicellular groups resulting in growth competition. At the same time, the influence of interactions between groups on the evolution of life cycles is rarely even considered. Here, we present a model of colonial life cycles evolution taking into account group interactions. Our work shows that the outcome of evolution could be coexistence between multiple life cycles or that the outcome may depend on the initial state of the population - scenarios impossible without group interactions. At the same time, we found that some results of these simpler models remain relevant: Evolutionary stable strategies in our model are restricted to binary fragmentation - the same class of life cycles which contains all evolutionarily optimal life cycles in the model without interactions. Our results demonstrate that while models neglecting interactions can capture short-term dynamics, they fall short in predicting the population-scale picture of the evolution.

category: evolutionary biology


title: Little skate genome exposes the gene regulatory mechanisms underlying the evolution of vertebrate locomotion

abstract: The little skate Leucoraja erinacea, a cartilaginous fish, displays pelvic fin driven walking-like behaviors using genetic programs and neuronal subtypes similar to those of land vertebrates. However, mechanistic studies on little skate motor circuit development have been limited, due to a lack of high-quality reference genome. Here, we generated an assembly of the little skate genome, containing precise gene annotation and structures, which allowed post-genome analysis of spinal motor neurons (MNs) essential for locomotion. Through interspecies comparison of mouse, skate and chicken MN transcriptomes, shared and divergent MN expression profiles were identified. Conserved MN genes were enriched for early-stage nervous system development. Comparison of accessible chromatin regions between mouse and skate MNs revealed conservation of the potential regulators with divergent transcription factor (TF) networks through which expression of MN genes is differentially regulated. TF networks in little skate MNs are much simpler than those in mouse MNs, suggesting a more fine-grained control of gene expression operates in mouse MNs. These findings suggest conserved and divergent mechanisms controlling MN development system of vertebrates during evolution and the contribution of intricate gene regulatory networks in the emergence of sophisticated motor system in tetrapods.


title: Genetic affinities and adaptation of the South West coast populations of India

abstract: Evolutionary event has not only transformed the genetic structure of human populations but also associated with social and cultural transformation. South Asian populations were formed as a result of such evolutionary events of migration and admixture of genetically and culturally distinct groups. Most of the genetic studies pointed to large-scale admixture event between Ancestral North Indian (ANI) and Ancestral South Indian (ASI) groups, also additional layers of recent admixture. In the present study we have analyzed 213 individuals inhabited in South West coast India with traditional warriors and feudal lord status and historically associated with recent migrations events and possible admixture with Indo-Scythians, Saka, Huns and Kushans, whose genetic links are still missing. Analysis of autosomal SNP markers suggests that these groups possibly derived their ancestry from some groups of North West India having additional Middle Eastern genetic component and also their separation history suggests very early separation from North West Indian and Gangetic plain Indo-Europeans during late bronze or Iron age, most probably following central India and Godavari basin to South West coast. Higher distribution of west Eurasian mitochondrial haplogroups also points to admixture through maternal lineage. Selection screen using genome wide genealogy approach revealed genetic signatures related to their long-term coastal food habits. Thus, our study suggests that the South West coastal groups with traditional warriors and feudal lords status are of a distinct lineage compared to Dravidian and Gangetic plain Indo-Europeans and are remnants of very early migrations from North West India following Godavari basin to Karnataka and Kerala.

category: genetics


title: Assessing behavioral sex differences to chemical cues of predation risk while provisioning nestlings in a hole-nesting bird

abstract: Birds can assess nest predation risk and adjust their parental activity accordingly. Risk taking behavior should be related to investment in reproduction as well as to confidence in parenthood that often differ between sexes. In those cases, sexual differences in risk taking behavior may be expected. For example, in blue tits, females invest more time and energy than males in nest-building, egg laying and incubation. Furthermore, confidence in parenthood is supposed to be higher for females, as extrapair paternity is common in this species. Therefore, the reproductive value of nestlings may be higher for females than for males and the former may assume greater risks to ensure nestling growth and maximize their reproductive success. We examined potential sexual differences in the risk assumed by parents in relation to perceived risk of predation inside the nest cavity, where predation risk perception may be higher. We increased perceived predation risk by adding predator chemical cues inside blue tit (Cyanistes caeruleus) nest-boxes, and we tested whether female and male parents differed in the risk assumed when taking care of nestlings. Females and males did not differ in the risk assumed in response to perceived predation risk. However, females reduced time devoted to nest sanitation activities when predator chemical cues were detected inside the nest-box, likely as an anti-predatory strategy to minimize their own risk of predation. Therefore, these results add to the evidence that birds can detect chemical cues of predators inside the nest cavity and suggest that the behavioral response to an increase in risk of predation perceived through olfactory cues is not sex-dependent in blue tit.

category: animal behavior and cognition


title: The physical properties of the stick insect pad secretion are independent of body size

abstract: Many insects use adhesive organs to climb. The ability to cling to surfaces is advantageous but is increasingly challenged as animals grow, due to the associated reduction in surface-to-volume ratio. Previous work has demonstrated that some climbing animals overcome this scaling problem by systematically altering the maximum force per area their adhesive pads can sustain; their adhesive organs become more efficient as they grow, an observation which is also of substantial relevance for the design of bio-inspired adhesives. What is the origin of this change in efficiency? In insects, adhesive contact is mediated by a thin film of a liquid, thought to increase adhesive performance via capillary and viscous forces. Here, we use interference reflection microscopy and dewetting experiments to measure the contact angle and dewetting speed of the pad secretion of Indian stick insects varying in mass by over two orders of magnitude. Neither contact angle nor dewetting speed change significantly with body mass, suggesting that the key physical properties of the pad secretion - its surface tension and viscosity - are size-invariant. Thus, the observed change in pad efficiency is unlikely to arise from systematic changes of the physical properties of the pad secretion; the functional role of the secretion remains unclear.

category: bioengineering


title: Neq2X7: a multi-purpose and open-source fusion DNA polymerase for advanced DNA engineering and diagnostics PCR

abstract:
Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, other properties than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus. The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA and that is unaffected by replacing dTTP with dUTP in PCR. This makes it an attractive DNA polymerase for use e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Furthermore, Neq2X7 is easy to produce, and the expression plasmid is freely available.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: Development of a single layer microfluidic device for dynamic stimulation, culture and imaging of mammalian cells

abstract: The possibility to tightly control the cellular microenvironment within microfluidic devices represents an important step forward precision analysis of cellular phenotypes in vitro. In particular, microfluidic platforms that allow both long-term mammalian cell culture and dynamic modulation of the culture environment can support quantitative studies of cells responses to drugs and fate choices. Here, we report the design and testing of a novel microfluidic device of easy production (single Polydimethylsiloxane layer), which integrates a micromixer with vacuum assisted cell loading for long-term cell culture and dynamic mixing of culture media with 4 different inputs. Finite element modelling was used to predict the flow rates and device dimensions to achieve diffusion-based fluid mixing. The device showed efficient diffusion-based mixing and dynamic exchange of media in the cell trapping chambers. This work represents the first attempt to integrate single layer microfluidic mixing devices with vacuum assisted cell loading systems for long-term mammalian cell culture and dynamic stimulation.


title: Unleashing alternative polyadenylation analyses with REPAC

abstract: Alternative polyadenylation (APA) is an important post-transcriptional mechanism that has major implications in biological processes and diseases. Although specialized sequencing methods for polyadenylation exist, their presence in public repositories is extremely limited when compared to traditional RNA-sequencing. To overcome this, we developed REPAC, a framework for the analysis of APA from RNA-sequencing data. REPAC implements a new method for detection of APA and is designed to take advantage of recount3 which enables a streamlined way to analyze over 750,000 publicly available samples. Using REPAC, we investigated the landscape of APA caused by activation of B cells. Our analysis revealed that during this process, hundreds of genes are regulated by APA, most notably genes involved in the secretion pathway which is central for the transition to antibody-secreting B-cells. Moreover, we also showed that many genes associated with interferon response are also shortened, suggesting that APA might also play a significant role in the immune response. We also show that REPAC is faster than alternative methods by at least 7-fold and that it scales well to analysis involving hundreds of samples. Overall, the REPAC method offers an accurate, easy, and convenient solution for the exploration of APA across many phenotypes.


title: HCLC-FC: a novel statistical method for phenome-wide association studies

abstract:
The emergence of genetic data coupled to longitudinal electronic medical records (EMRs) offers the possibility of phenome-wide association studies (PheWAS). In PheWAS, the whole phenome can be divided into numerous phenotypic categories according to the genetic architecture across phenotypes. Currently, statistical analyses for PheWAS are mainly univariate analyses, which test the association between one genetic variant and one phenotype at a time. In this article, we derived a novel and powerful multivariate method for PheWAS. The proposed method involves three steps. In the first step, we apply the bottom-up hierarchical clustering method to partition a large number of phenotypes into disjoint clusters within each phenotypic category. In the second step, the clustering linear combination method is used to combine test statistics within each category based on the phenotypic clusters and obtain p-values from each phenotypic category. In the third step, we propose a new false discovery rate (FDR) control approach. We perform extensive simulation studies to compare the performance of our method with that of other existing methods. The results show that our proposed method controls FDR very well and outperforms other methods we compared with. We also apply the proposed approach to a set of EMR-based phenotypes across more than 300,000 samples from UK Biobank. We find that the proposed approach not only can well-control FDR at a nominal level but also successfully identify 1,244 significant SNPs that are reported to be associated with some phenotypes in the GWAS catalog. Our open-access tools and instructions on how to implement HCLC-FC are available at https://github.com/XiaoyuLiang/HCLCFC.

Author summaryAs a complementary approach to genome-wide association studies, phenome-wide association studies (PheWAS) have been an efficient tool for testing associations between genetic variations and a wide range of phenotypes utilizing all available phenotypic information. For instance, the first PheWAS has demonstrated that rs3135388 on HLA-DRB1 associated with atrial fibrillation and multiple sclerosis. A challenging step in performing large-scale multiple testing of PheWAS is to control the false discovery rate (FDR). In this work, we propose a novel and powerful multivariate method, HCLC-FC, to test the association between a genetic variant with a large number of phenotypes simultaneously controlling FDR. Within each phenotypic category, a newly proposed method clusters phenotypes into different groups and the combined test statistic within each category based on the phenotypic clusters has an asymptotic distribution which avoids the computational burden of simulation. Furthermore, the newly developed FDR controlling process is based on p-values and does not depend on test statistics. Therefore, it is more general and can be applied to other multiple testing procedures to control FDR.


title: New β-propellers are continuously amplified from single blades in all major lineages of the β-propeller superfamily

abstract: {beta}-Propellers are toroidal folds, in which consecutive supersecondary structure units of four anti-parallel {beta}-strands - called blades - are arranged radially around a central axis. Uniquely among toroidal folds, blades span the full range of sequence symmetry, from near identity to complete divergence, indicating an ongoing process of amplification and differentiation. We have proposed that the major lineages of {beta}-propellers arose through this mechanism and that therefore their last common ancestor was a single blade, not a fully formed {beta}-propeller. Here we show that this process of amplification and differentiation is also widespread within individual lineages, yielding {beta}-propellers with blades of more than 60% median pairwise sequence identity in most major {beta}-propeller families. In some cases, the blades are nearly identical, indicating a very recent amplification event, but even in cases where such recently amplified {beta}-propellers have more than 80% overall sequence identity to each other, comparison of their DNA sequence shows that the amplification occurred independently.


title: Comparison and evaluation of data-driven protein stability prediction models

abstract: Predicting protein stability is important to protein engineering yet poses unsolved challenges. Computational costs associated with physics-based models, and the limited amount of data available to support data-driven models, have left stability prediction behind the prediction of structure. New data and advancements in modeling approaches now afford greater opportunities to solve this challenge. We evaluate a set of data-driven prediction models using a large, newly published dataset of various synthetic proteins and their experimental stability data. We test the models in two separate tasks, exercising extrapolation to new protein classes and prediction of the effects on stability of small mutations. Small convolutional neural networks trained from scratch on stability data and large protein embedding models passed through simple downstream models trained on stability data are both able to predict stability comparably well. The largest of the embedding models yields the best performance in all tasks and metrics. We also explored the marginal performance gains seen with two ensemble models.


title: Identification of m6A and m5C RNA modifications at single-molecule resolution from Nanopore sequencing

abstract: The expanding field of epitranscriptomics might rival the epigenome in the diversity of the biological processes impacted. However, the identification of modifications in individual RNA molecules remains challenging. We present CHEUI, a new method that detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) at single-nucleotide and single-molecule resolution from Nanopore signals. CHEUI predicts methylation in Nanopore reads and transcriptomic sites in a single condition, and differential m6A and m5C methylation between any two conditions. Using extensive benchmarking with Nanopore data derived from synthetic and natural RNA, CHEUI showed higher accuracy than other existing methods in detecting m6A and m5C sites and quantifying the site stoichiometry levels, while maintaining a lower proportion of false positives. CHEUI provides a new capability to detect RNA modifications with high accuracy and resolution that can be cost-effectively expanded to other modifications to unveil the full span of the epitranscriptome in normal and disease conditions.


title: iSparse kmeans: a two-step clustering approach for big dynamic functional network connectivity data

abstract:
BackgroundDynamic functional network connectivity (dFNC) estimated from resting-state functional magnetic imaging (rs-fMRI) studies the temporally varying of functional integration between brain networks. In a typical dFNC pipeline, a clustering stage to summarize the connectivity patterns that are transiently but reliably realized over the course of a scanning session. However, identifying the right number of clusters through a conventional clustering criterion computed by running the algorithm repeatedly, over a large range of cluster numbers is time-consuming and requires substantial computational power even for typical dFNC datasets, and the computational demands become prohibitive as datasets become larger and scans longer. Here we developed a new dFNC pipeline, called iterative sparse kmeans or iSparse kmeans, to analyze large dFNC data without having access to huge computational power.

MethodIn iSparse kmeans, we implement two-step clustering. In the first step, we randomly use a sub-sample dFNC data and identify several sets of states at different model orders. In the second step, we aggregate all dFNC states estimated from all iterations in the first step and use this to identify the optimum number of clusters using the elbow criteria. Additionally, we use this new reduced dataset and estimate a final set of states by performing a second kmeans clustering on the aggregated dFNC states from the first k-means clustering. To validate the reproducibility of iSparse kmeans, we analyzed four dFNC datasets from the human connectome project (HCP).

ResultsWe found that both conventional kmeans and iSparse kmeans generate similar brain dFNC states while iSparse kmeans is 27 times faster than the traditional method in finding the optimum number of clusters. We show that the results are replicated across four different datasets from HCP.

ConclusionWe developed a new analytic pipeline which facilitates analysis of large dFNC datasets without having access to a huge computational power source. We validated the reproducibility of the result across multiple datasets.


title: Hyperglycemic O-GlcNAc transferase activity drives cancer stem cell induction in TNBC

abstract:
Enhanced glucose metabolism is a feature of almost all cancers, but downstream functional effects of aberrant glucose flux are difficult to mechanistically determine. The objective of this study is to characterize a mechanism by which elevated glucose level drives a tumorigenic pathway in triple negative breast cancer (TNBC). We used chemical biology methods to track how a metabolite of glucose, N-acetylglucosamine (GlcNAc), is linked to the transcriptional regulatory protein tet-methylcytosine dioxygenase 1 (TET1) as an O-linked GlcNAc post translational modification (O-GlcNAc). In this work, we revealed that intracellular protein glycosylation by O-GlcNAc is driven by high glucose levels in TNBC models, including on TET1. A single enzyme, O-GlcNAc transferase (OGT), is responsible for catalyzing protein modification of O-GlcNAc. We showed that OGT activity is higher in TNBC cell lines compared to non-tumor breast cell lines and is associated with hyperglycemia. Furthermore, enhanced OGT activity activated a pathway for cancer stem-like cell (CSC) reprogramming in TNBC cells. In our model, O-GlcNAcylated TET1 upregulated expression of splicing factor TAR-DNA binding protein (TARDBP), which drives CSC induction as well as higher OGT levels. We show that this OGT-TET1-TARDBP axis "feeds-forward" in hyperglycemic conditions both in cell lines and diet-induced obese mice, which displayed higher blood glucose levels and tumor O-GlcNAc levels than lean littermates. This data converges on a novel pathway whereby hyperglycemia drives aberrant OGT activity, activating a pathway for CSC induction in TNBC. Our findings partially explain a key aspect of how obesity is associated with TNBC risk and negative outcomes.

Statement of ImplicationThis work presents a novel mechanism to explain how obesity is a risk factor for triple-negative breast cancer via elevated sugar-transfer activity by O-GlcNAc transferase in hyperglycemia, leading to the induction of a cancer stem-like cells and revealing a targetable pathway in obesity-associated tumors.

category: cancer biology


title: Human bone marrow organoids for disease modelling, discovery and validation of therapeutic targets in hematological malignancies

abstract:
A lack of models that recapitulate the complexity of human bone marrow has hampered mechanistic studies of normal and malignant hematopoiesis and the validation of novel therapies. Here, we describe a step-wise, directed-differentiation protocol in which organoids are generated from iPSCs committed to mesenchymal, endothelial and hematopoietic lineages. These 3-dimensional structures capture key features of human bone marrow - stroma, lumen-forming sinusoidal vessels and myeloid cells including pro-platelet forming megakaryocytes. The organoids supported the engraftment and survival of cells from patients with blood malignancies, including cancer types notoriously difficult to maintain ex vivo. Fibrosis of the organoid occurred following TGF{beta} stimulation and engraftment with myelofibrosis but not healthy donor-derived cells, validating this platform as a powerful tool for studies of malignant cells and their interactions within a human bone marrow-like milieu. This enabling technology is likely to accelerate discovery and prioritization of novel targets for bone marrow disorders and blood cancers.

Significance StatementWe present a 3D, vascularised human bone marrow organoid that supports growth of primary cells from patients with myeloid and lymphoid blood cancers. This model allows for mechanistic studies of blood cancers in the context of their microenvironment, and provides a much-needed, ex vivo tool for prioritization of new therapeutics.


title: High-fat diet promotes Acute Promyelocytic Leukemia through PPARδ-enhanced self-renewal of preleukemic progenitors

abstract:
Obesity is associated with a higher risk of developing many cancer types including acute promyelocytic leukaemia (APL), a subset of acute myeloid leukemias (AML) characterized by expression of the PML-RAR oncogene. The molecular mechanisms linking obesity and APL development are not known. To model clinical observations, we established a mouse model of diet-induced obesity using transgenic mice constitutively expressing PML-RARA  in the hematopoietic system (PML-RAR KI mice) fed either standard (SD) or high-fat (HFD) diets. HFD-fed PML-RAR KI mice developed leukaemia with reduced latency and increased penetrance, as compared to SD-fed mice. HFD leads to accumulation of DNA damage in hematopoietic stem cells (HSCs), but, surprisingly, this was not associated with mutational load gain, as shown by whole genome/exome sequencing of pre-leukemic and leukemic cells. Importantly, very few of the observed mutations were predicted to act as cancer drivers, suggesting the relevance of nongenetic mechanisms. HFD led to an expansion of hematopoietic progenitor cells with a concomitant reduction in long-term hematopoietic stem cells, and in the presence of PML-RAR this was also accompanied by an enhancement of in vitro and in vivo self-renewal. Interestingly, Linoleic Acid (LA), abundant in HFD, recapitulates the effect of HFD on the self-renewal of PML-RAR HPCs by activating the peroxisome proliferator-activated receptor delta (PPAR{delta}), a central regulator of fatty acid metabolism involved in the promotion of cancer progression. Our findings have implications for dietary or pharmacological interventions aimed at counteracting the cancer-promoting effect of obesity.

Key pointsO_LIhigh fat diet (HFD) promotes APL leukemogenesis in mouse models, reproducing the exquisite sensitivity to obesity observed in humans
C_LIO_LIalthough HFD leads to DNA damage and mutations, the molecular mechanism is nongenetic and linked to the transcription factor PPAR{delta}
C_LI


title: A Drosophila in vivo chemical screen reveals that combination drug treatment targeting MEK and DGKα mitigates Ras-driven polarity-impaired tumourigenesis.

abstract: The RAS oncogene and upregulation of the RAS signalling pathway is highly prevalent in human cancer, and therefore, therapeutically targeting the RAS pathway is a common treatment in cancer. However, RAS pathway upregulation is not sufficient to drive malignant cancer, since senescence mechanisms prevent cancer progression. Thus, additional mutations, such as mutations that prevent senescence or alter the tissue architecture (cell polarity), are required for RAS-driven tumour progression. Moreover, targeting RAS-driven cancers with RAS pathway inhibitors can often lead to undesirable side-effects and to drug resistance. Thus, identifying compounds that synergise with RAS-pathway inhibitors would enable lower doses of the RAS pathway inhibitors to be used and also decrease the acquisition of drug resistance. Here, in a boutique chemical screen using a Drosophila model of Ras-driven cell polarity-impaired cancer, we have identified compounds that reduce tumour burden by synergising with subtherapeutic doses of the RAS pathway inhibitor, Trametinib, which inhibits mitogen-activated kinase kinase (MEK). Analysis of one of the hits from the screen, Ritanserin, which targets serotonin receptors and diacy glycerol kinase alpha (DGK), revealed that DGK was the critical target in its synergism with Trametinib. We show that human mammary epithelial cells harbouring the H-RAS oncogene and knockdown of the cell polarity gene, SCRIB, are also sensitive to treatment with low doses of Trametinib and DGK inhibition. Mechanistically, DGK inhibition synergises with Trametinib by inhibiting MEK and mTOR activity. Altogether, our results provide evidence that targeting RAS-driven human cancers with RAS pathway and DGK inhibitors will be an effective combination therapy.


title: Induction of promoter methylation and transcriptional gene silencing upon high pressure spraying of 24-nt small RNAs in Nicotiana benthamiana

abstract: Exogenous RNA application is a promising transgene-free approach for crop protection and improvement. In almost all applications reported so far, exogenous RNA molecules (double stranded RNAs or small RNAs) are applied to plants in order to trigger degradation of a given mRNA (of plant, pest or pathogen origin), in a process termed post-transcriptional gene silencing (PTGS). However, whether exogenous RNAs can also trigger epigenetic modifications to plants such as RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) remains largely unaddressed. Here, we provide evidence that high pressure spraying of a 24-nt short interfering RNA (siRNA) designed to target the CaMV 35S promoter that drives the expression of a GFP transgene, resulted in promoter RdDM in N. benthamiana. Moreover, the methylation at the target site spreads both up and downstream neighboring sites. Importantly, GFP expression was reduced in the sprayed leaf tissues. Small RNA sequencing excluded the presence siRNAs mapping to the GFP gene body and documented substantial amount of secondary RNAs in the promoter, suggesting that the GFP silencing was transcriptional rather than post-transcriptional. Our study provides a proof of principle for spray induced epigenetic modification (SPIEM) that could be used in modern crop breeding platforms.

category: plant biology


title: Carbon dots boost dsRNA delivery in plants and increase local and systemic siRNA production

abstract: Carbon dots (CDs) have been proposed for nucleic acid delivery in many biological applications. In this work we have obtained carbon dots by a hydrothermal synthesis method for developing nanocomposites with dsRNA. These CDs were produced using glucose or saccharose as the nucleation source and passivated with branched polyethyleneimines for conferring positive charges. Hydrodynamic analyses and transmission electron microscopy TEM showed that they sized on average 4 and 5 nm, depending on the sugar. The CDs were fluorescent and showed a peak at 468 nm when excited with UV light. Physicochemical characteristics of their surfaces were revealed by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FT-IR). The {zeta} potential determined that both types of CDs had positive charges as well as good electrophoretic mobility and conductivity. Coating of the CDs to dsRNA was efficient but did not protect from nucleases. DsRNA naked or coated with the CDs were delivered to leaves of cucumber plants by spraying at 2.5 bar. Quantitation of the dsRNA that entered the leaves showed that when coated with the CDs, 50-fold more dsRNA was detected than when naked dsRNA was applied. Moreover, specific siRNAs derived from the sprayed dsRNAs were 130 times more abundant when the dsRNA was coated with the CDs. Systemic dsRNAs were determined in distal leaves showing a dramatic increase in concentration when delivered as a nanocomposite. Similarly, systemic siRNAs were significantly more abundant in distal leaves when spraying with the CD-dsRNA nanocomposite. Furthermore, FITC-labeled dsRNA was shown to accumulate in the apoplast and increase its entry in the plant when coated with CDs. These results indicate that CDs obtained by hydrothermal synthesis are suitable for dsRNA foliar delivery in RNAi plant applications and provide insight into CD-dsRNA translocations.


title: High salinity activates CEF and attenuates state transitions in both psychrophilic and mesophilic Chlamydomonas species

abstract: In the last decade, studies have revealed the importance of PSI-driven cyclic electron flow (CEF) in stress acclimation in model organisms like C. reinhardtii; however, these studies focused on transient, short-term stress. In addition, PSI-supercomplexes are associated with CEF during state transition response to short-term stress. On the other hand, the role of CEF during long-term stress acclimation is still largely unknown. In this study, we elucidate the involvement of CEF in acclimation response to long-term high salinity in three different Chlamydomonas species displaying varying salinity tolerance. We compared CEF rates, capacity for state transitions, and formation of supercomplexes after salinity acclimation in the model mesophile C. reinhardtii and two psychrophilic green algae C. priscuii (UWO241) and C. sp. ICE-MDV. CEF was activated under high salt in all three species, with the psychrophilic Chlamydomonas spp. exhibiting the highest CEF rates. High salt acclimation was also correlated with reduced state transition capacity and a PSI-supercomplex was associated with high CEF. We propose that under long-term stress, CEF is constitutively activated through assembly of a stable PSI-supercomplex. The proteomic composition of the long-term PSI-supercomplex is distinct from the supercomplex formed during state transitions, and its presence attenuates the state transition response.


title: Remorin proteins serve as membrane topology scaffolds in plants

abstract:
Organization of membrane topologies in plants has so far been mainly attributed to the cell wall and the cytoskeleton. Taking rhizobial infections of legume root cells, where plasma membranes undergo dynamic and large-scale topology changes, as an initial model, we challenged this paradigm and tested whether additional scaffolds such as plant-specific remorins that accumulate on highly curved and often wall-less plasma membrane domains, control local membrane dynamics. Indeed, loss-of-function mutants of the remorin protein SYMREM1 failed to develop stabilized membrane tubes as found in colonized cells in wild-type plants, but released empty membrane spheres instead. Expression of this and other remorins in wall-less protoplasts allowed engineering different membrane topologies ranging from membrane blebs to long membrane tubes. Reciprocally, mechanically induced membrane indentations were equally stabilized by SYMREM1. This function is likely supported by remorin oligomerization into antiparallel dimers and the formation of higher order membrane scaffolding structures. Taken together we describe an evolutionary confined mechanism that allows the stabilization of large-scale membrane conformations and curvatures in plants.

One-sentence summaryThe remorin SYMREM1 evolved as structural membrane scaffold that stabilizes membrane tubulation and curvature during symbiotic intracellular infections.


title: Epistatic Modifiers Influence the Expression of Continual Flowering in Strawberry

abstract:
O_LIPrevious work within the community led to the identification of a single dominant allele that controls the everbearing trait. However, frequent observations have indicated that crosses do not segregate in a Mendelian fashion, as would be expected for a trait controlled by a single dominant gene. Therefore, it was hypothesised that one or more unidentified epistatic alleles interact with the major gene.
C_LIO_LIA GWAS was conducted on 587 June-bearers and 207 everbearers to assess the genetic components associated with flowering habit. The segregation ratios of parental strawberry lines with known phenotypes were used to validate the identified alleles.
C_LIO_LIThree loci including the known, major FaPFRU locus and two epistatic modifiers were identified. These modifiers function as enhancers of the everbearing trait in individuals containing a single copy of the FaPFRU everbearing allele and appear to be functionally redundant. Principally, heterozygous individuals required the presence of two modifying alleles in order to allow expression of the everbearing trait.
C_LIO_LIInclusion of the epistatic alleles improved the prediction of everbearing segregation ratios, beyond that of a single allele model, however, a large proportion of the variation remained unexplained. Future work should identify the additional repressor and enhancer modifiers not identified here. Discovering the genetic components controlling the everbearing trait can enable genetic informed strawberry improvement.
C_LI

Society Impact StatementUntil the 1970s, the majority of commercial strawberry varieties produced a single bloom of flowers. However, continuously flowering, everbearing strawberries are now routinely cultivated and use is increasing. Indeed, introgression of the everbearing flowering trait can lead to economic benefits for growers through the production of a continual crop from the same plant. Utilising genetic guided improvement has the power to streamline everbearing generation. As such, utilising the genetic markers reported here, can help to identify everbearing individuals at an early time point in the breeding process. Furthermore, these markers can help to improve the predictions of progeny segregation ratios.


title: Increases in vein length compensate for leaf area lost to lobing in grapevine

abstract:
PremiseThere is considerable variation in leaf lobing and leaf size, including among grapevines, some of the most well-studied leaves. We examined the relationship between leaf lobing and leaf size across grapevine populations which varied in extent of leaf lobing.

MethodsWe used homologous landmarking techniques to measure 2,632 leaves across two years in 476 unique, genetically distinct grapevines from 5 biparental crosses which vary primarily in the extent of lobing. We determined to what extent leaf area could explain variation in lobing, vein length, and vein to blade ratio.

ResultsAlthough lobing was the primary source of variation in shape across the leaves we measured, leaf area varied only slightly as a function of lobing. Rather, leaf area increases as a function of total major vein length, total branching vein length, and decreases as a function of vein to blade ratio. These relationships are stronger for more highly lobed leaves, with the residuals for each model differing as a function of distal lobing.

ConclusionsFor a given leaf area, more highly lobed leaves have longer veins and higher vein to blade ratios, allowing them to maintain similar leaf areas despite increased lobing. These findings show how more highly lobed leaves may compensate for what would otherwise result in a reduced leaf area, allowing for increased photosynthetic capacity through similar leaf size.


title: Individualized Gaussian Process-based Prediction of Memory Performance and Biomarker Status in Ageing and Alzheimer's disease

abstract: Neuroimaging markers based on Magnetic Resonance Imaging (MRI) combined with various other measures (such as informative covariates, vascular risks, brain activity, neuropsychological test etc.,) might provide useful predictions of clinical outcomes during progression towards Alzheimers disease (AD). The Bayesian approach aims to provide a trade-off by employing relevant features combinations to build decision support systems in clinical settings where uncertainties are relevant. We tested the approach in the MRI data across 959 subjects, aged 59-89 years and 453 subjects with available neuropsychological test scores and CSF biomarker status (amyloid-beta (A{beta})42/40 & and phosphorylated tau (pTau)) from a large sample multi-centric observational cohort (DELCODE). In order to explore the beneficial combinations of information from different sources, we presented a MRI-based predictive modelling of memory performance and CSF biomarker status (positive or negative) in the healthy ageing group as well as subjects at risk of Alzheimers disease using a Gaussian process multikernel framework. Furthermore, we systematically evaluated predictive combinations of input feature sets and their model variations, i.e. (A) combinations of brain tissue classes and feature type (modulated vs. unmodulated), choices of filter size of smoothing (ranging from 0 to 15 mm full width at half maximum), and image resolution (1mm, 2mm, 4mm and 8mm); (B) incorporating demography and covariates (C) the impact of the size of the training data set (i.e., number of subjects); (D) the influence of reducing the dimensions of data and (E) choice of kernel types. Finally, the approach was tested to reveal individual cognitive scores at follow-up (up to 4 years) using the baseline features. The highest accuracy for memory performance prediction was obtained for a combination of neuroimaging markers, demographics, genetic information (ApoE4) and CSF-biomarkers explaining 57% of outcome variance in out of sample predictions. The best accuracy for A{beta}42/40 status classification was achieved for combination demographics, ApoE4 and memory score while usage of structural MRI improved the classification of individual patients pTau status.


title: Plakoglobin is a mechanoresponsive regulator of naïve pluripotency

abstract:
Biomechanical cues are instrumental in guiding embryonic development and cell differentiation. Understanding how these physical stimuli translate into transcriptional programs could provide insight into mechanisms underlying mammalian pre-implantation development. Here, we explore this by exerting microenvironmental control over mouse embryonic stem cells (ESCs). Microfluidic encapsulation of ESCs in agarose microgels stabilized the naive pluripotency network and specifically induced expression of Plakoglobin (Jup), a vertebrate homologue of {beta}-catenin. Indeed, overexpression of Plakoglobin was sufficient to fully re-establish the naive pluripotency gene regulatory network under metastable pluripotency conditions, as confirmed by single-cell transcriptome profiling. Finally, we found that in the epiblast, Plakoglobin was exclusively expressed at the blastocyst stage in human and mouse embryos - further strengthening the link between Plakoglobin and naive pluripotency in vivo. Our work reveals Plakoglobin as a mechanosensitive regulator of naive pluripotency and provides a paradigm to interrogate the effects of volumetric confinement on cell-fate transitions.

HighlightsO_LI3D agarose spheres stabilize the naive pluripotency network in mouse ESCs.
C_LIO_LIVolumetric confinement induces expression of Plakoglobin, a vertebrate homologue of {beta}-catenin.
C_LIO_LIPlakoglobin expression in the epiblast is specific to pre-implantation human and mouse embryos.
C_LIO_LIPlakoglobin overexpression maintains naive pluripotency independently of {beta}-catenin.
C_LI


title: Decomposing predictability to identify dominant causal drivers in complex ecosystems

abstract:
Ecosystems are complex systems of various physical, biological, and chemical processes. Since ecosystem datasets are composed of a mixture of different levels of stochasticity and nonlinearity, handling these data is a challenge for existing methods of time-series based causal inferences. Here we show that, by harnessing contemporary machine learning approaches, the concept of Granger causality can be effectively extended to the analysis of complex ecosystem time series and bridge the gap between dynamical and statistical approaches. The central idea is to use an ensemble of fast and highly predictive artificial neural networks to select a minimal set of variables that maximizes the prediction of a given variable. It enables decomposition of the relationship among variables through quantifying the contribution of an individual variable to the overall predictive performance. We show how our approach, EcohNet, can improve interaction network inference for a mesocosm experiment and simulated ecosystems. The application of the method to a long-term lake monitoring dataset yielded new but interpretable results on the drivers causing cyanobacteria blooms, which is a serious threat to ecological integrity and ecosystem services. Since performance of EcohNet is enhanced by its predictive capabilities, it also provides an optimized forecasting of overall components in complex ecosystems. EcohNet could be used to analyze complex and hybrid multivariate time series in many scientific areas not limited to ecosystems.

SignificanceEffective use of ecosystem monitoring data to resolve global environmental issues is a major challenge of ecology in the 21st century. A proposed solution to address this challenge is a time-series-based causal analysis, which can offer insight on the mechanical links between ecosystem components. In this work, a model-free framework named EcohNet is proposed that can robustly predict causal networks of ecosystem components. EcohNet utilizes ensemble predictions of echo state networks, which are known to be fast, accurate, and highly relevant for a variety of dynamical systems. It also can provide an optimized forecasting of overall components, and could be used to analyze complex and hybrid multivariate time series in many scientific areas, not only ecosystems.


title: What are you lookin' at?: A role for sexual conflict in coevolution of female display signals and male sensory adaptations?

abstract: The prevalence and diversity of female ornaments poses a challenge to evolutionary theory because males should prefer mates that spend resources on offspring rather than ornaments. Among dance flies, there is extraordinary variation in sexual dimorphism. Females of many species have conspicuous ornaments (leg scales and inflatable abdominal sacs). Meanwhile males of some species have regions of their eyes with larger ommatidial facets, common traits in many arthropod taxa that allow for regionally elevated photosensitivity and/or acuity. Here, we conduct a comparative study of these traits using both species descriptions available from the literature, as well as quantitative measures of eyes and ornaments from wild-caught flies. We show a conspicuous covariance between exaggerated male dorsal eye regions and the extent of female ornaments: species with highly ornamented females have males with more exaggerated eyes. We discuss this pattern in the context of competing hypotheses for their evolution.


title: MR-DoC2: bidirectional causal modeling with instrumental variables and data from relatives

abstract: Establishing causal associations is an essential step towards developing interventions for psychiatric, substance use and many other disorders. While randomized controlled trials (RCTs) are considered the gold standard for causal inference, they are unethical in many scenarios. Mendelian randomization (MR) can instead be used, but both methods focus on unidirectional causality. In this paper, we developed a new model, MRDoC2, based on the MRDoC model (Minic[a] et al 2018), which includes two polygenic risk scores. It handles bidirectional causation and confounding due to both familial and non-familial sources. OpenMx software was used to study its statistical power, which revealed that there was negligible effect of the difference in sources of variance in the exposure and outcome variables. This finding contrasts with the case for direction of causation modeling with twins but without genotype data, where no power exists when the proportions of trait variance are equal for the two variables.