title: Resilience in a Natural Model of Metabolic Dysfunction Through Changes in Longevity and Ageing-Related Metabolites

abstract:
Insights from extreme-adapted organisms, which have evolved natural strategies for promoting survivability under severe environmental pressures, may help guide future research into novel approaches for enhancing human longevity. The cave-adapted Mexican tetra,Astyanax mexicanus, has attracted interest as a model system for metabolic resilience, a term we use to denote the property of maintaining robust health and longevity under conditions that would have highly deleterious effects in other organisms (Fig 1). Cave-dwelling populations of Mexi-can tetra exhibit elevated blood glucose and possess a mutation in the insulin receptor that in humans has been linked to Rabson-Mendenhall syndrome, a condition characterized by severe insulin resistance that causes numerous developmental abnormalities, is highly associated with debilitating progression, and drastically reduces lifespan. In addition, cavefish develop large numbers of hypertrophic visceral adipocytes and possess vastly enriched stores of body fat compared to surface-dwelling counterparts. However, cavefish appear to avoid the progression of the respective pathologies typically associated with these conditions, such as accumulation of advanced glycation end products (AGEs), chronic tissue inflammation, impaired growth due to insulin dysregulation, and low survivability due to arterial disease. The metabolic strategies underlying the resilience properties of A. mexicanus cavefish, and how they relate to environmental challenges of the cave environment, are poorly understood. Here, we provide an untargeted metabolomics study of long- and short-term fasting in two A. mexicanus cave populations and one surface population. We find that, although cave-fish share many similarities with metabolic syndrome normally associated with the human state of obesity, important differences emerge, including a reduction in cholesteryl esters and intermediates of protein glycation, and an increase in antioxidants and metabolites associated with hypoxia and longevity. We find important overlaps between metabolic alterations in cave-dwelling Mexican tetra and other models of resilience and extreme longevity, such as naked mole-rats, including enhanced reliance on sugars as an energy source and a trend toward more potent antioxidant activity. This work suggests that certain metabolic features associated with human pathologies are not intrinsically harmful, but are rather consequences of suboptimal adaptation of humans to survival under adverse metabolic conditions, and suggests promising avenues for future investigation into the role of metabolic strategies in evolutionary adaptation and health. We provide a transparent pipeline for reproducing our analysis and a Shiny app for other researchers to explore and visualize our dataset.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/358077v3_fig1.gif" ALT="Figure 1">
View larger version (43K):
org.highwire.dtl.DTLVardef@bcaff5org.highwire.dtl.DTLVardef@1c40b89org.highwire.dtl.DTLVardef@1d17d69org.highwire.dtl.DTLVardef@80a483_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFig. 1.C_FLOATNO Metabolic resilience  survivability under a variety of extreme conditions. Certain populations of cavefish have adaptations that cause increased appetite (1) and increased fat accumulation (2) (in cases where nutrients are plentiful, such as in lab-raised populations). These same populations also exhibit robust health and longevity (2, 3) and do not suffer ill-effects due to high levels of visceral fat and hyper-glycemia, both of which are features of most cave populations. However, visceral fat accumulation in cave populations is highly dependent on nutrient availability and is not displayed in wild-caught specimens (4). Thus, cavefish paradoxically appear to tolerate both extremely low and extremely high levels of triglycerides, glucose, and other energy storage metabolites. We argue that these differences can be reconciled under a hypothesis whereby the cave environment selects not for resistance to nutrient deprivation per se, but rather resilience to a variety of nutrient availability states (such as seasonal floods). Survival under such challenging conditions ostensibly favors the ability to tolerate extreme metabolic states, including not only starvation but also high levels of potentially deleterious metabolites such as triglycerides and reactive oxygen species (ROS). We find evidence for elevated antioxidant levels and altered cholesterol / cholesteryl ester homeostasis in cavefish, suggesting that cavefish may use these mechanisms to offset potentially harmful metabolites and tolerate a broad range of metabolic conditions.

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title: Kinetic isotope tracing of glycerol and de novo proteogenic amino acids in Human Lung Carcinoma cells using glucose

abstract: 13C based Isotopic tracers of the media components can be used to kinetically track their contribution in the cell systems. A tracer ([U-13C6] glucose) was used to monitor its contribution into the central carbon metabolic pathways of human lung carcinoma (A549) cells by Gas chromatography-mass spectrometry (GC-MS) based mass isotopomer analysis. Calculated average 13C of methanolic extracts (glycerol: 5.46{+/-}3.53 % and lactate: 74.4{+/-}2.65 %), protein acid hydrolysates (serine: 4.51{+/-}0.21 %, glycine: 2.44{+/-}0.31 %, alanine: 24.56{+/-}0.59 %, glutamate: 8.81{+/-}0.85 %, proline: 6.96{+/-}0.53 % and aspartate: 10.72{+/-}0.95 %) and the culture filtrate (glycerol: 43.14{+/-}1.45 % and lactate: 81.67{+/-}0.91 %), showed significant contribution of 13C glucose. We observed the Warburg effect with higher levels of 13C lactate in the culture filtrate. 13C glycerol levels in culture supernatant showed significant increase with time and amino acids of glucogenic origin also contributed cellular protein biomass. The workflow adopted in this study for 13C analysis could be useful for the metabolic phenotyping of other mammalian cell systems under normal and perturbed (cancer and infection) conditions.

category: biochemistry


title: Design principles of acidic transcriptional activation domains

abstract: Acidic activation domains are intrinsically disordered regions of transcription factors that bind coactivators. The intrinsic disorder and low conservation of activation domains have made it difficult to identify general rules governing their function. To address this problem, we designed thousands of variants in seven acidic activation domains and measured their activities with a new high-throughput assay in human cell culture. From these data we identify three design principles of acidic activation domains: hydrophobic motifs must be balanced by acidic residues; acidic residues make large contributions to activity when they are adjacent to motifs; and motif composition reflects the structural constraints of coactivator interactions. These design principles motivated a simple predictor of activation domains in the human proteome: scanning for clusters of aromatic and leucine residues embedded in regions of high acidity identifies known activation domains and accurately predicts new ones. Our results support a flexible model in which acidic residues solubilize hydrophobic motifs so that they can interact with coactivators.

category: systems biology


title: Genetically-Modified Macrophages Accelerate Myelin Repair

abstract:
Preventing neurodegeneration-associated disability progression in patients with multiple sclerosis (MS) remains an unmet therapeutic need. As remyelination prevents degeneration of demyelinated axons, promoting this process in patients might halt the development of permanent disability. In demyelinating mouse lesions, local overexpression of Semaphorin 3F (Sema3F), an oligodendrocyte progenitor cell (OPC) attractant, increases OPC recruitment and remyelination. However, molecular targeting to MS lesions is a challenge because these are disseminated in the central nervous system.

We hypothesized that a clinically-relevant paradigm to deliver Sema3F to demyelinating lesions and increase OPC recruitment may be to use blood-derived macrophages as vehicles. Thus, we chose transplantation of genetically-modified hematopoietic stem cells (HSCs) as means of obtaining circulating monocytes that overexpress Sema3F. We first demonstrated that the supernatant from Sema3F-lentiviral vector transduced HSCs stimulates OPC migration in Neuropilin 2 (Nrp2, Sema3F receptor)-dependent fashion. We then investigated whether OPCs remain responsive to Sema3F with age. While Sema3F expression in the lesions of middle-aged and old mice (characterized by decreased efficiency of OPC recruitment and remyelination) was decreased, middle-aged OPCs retained Nrp2 expression and migrated in response to both recombinant Sema3F and Sema3F-transduced cell supernatant in vitro. We then investigated whether blood cells engineered to overexpress Sema3F can target demyelinating CNS lesions and improve remyelination. Thus, we transplanted Sema3F-transduced HSCs and obtained chimeric mice (with Sema3F overexpression in blood cells), in which we induced demyelinating spinal cord lesions. Transgene-carrying cells, predominantly macrophages, quickly infiltrated lesions in both control and Sema3F chimeras. While infiltration of Sema3F-expressing cells did not alter the inflammatory status of the lesions nor OPC survival, it increased OPC recruitment, which accelerated the onset of remyelination. Our results provide a proof-of-concept that blood cells, particularly monocyte-derived macrophages, can be used to deliver pro-remyelinating agents "at the right time and place", suggesting novel means for remyelination-promoting strategies.

category: neuroscience


title: COVID-19 Disease Map, a computational knowledge repository of SARS-CoV-2 virus-host interaction mechanisms

abstract: We describe a large-scale community effort to build an open-access, interoperable, and computable repository of COVID-19 molecular mechanisms - the COVID-19 Disease Map. We discuss the tools, platforms, and guidelines necessary for the distributed development of its contents by a multi-faceted community of biocurators, domain experts, bioinformaticians, and computational biologists. We highlight the role of relevant databases and text mining approaches in enrichment and validation of the curated mechanisms. We describe the contents of the Map and their relevance to the molecular pathophysiology of COVID-19 and the analytical and computational modelling approaches that can be applied for mechanistic data interpretation and predictions. We conclude by demonstrating concrete applications of our work through several use cases and highlight new testable hypotheses.


title: Phosphorylation triggers presynaptic phase separation of Liprin-α3 to control active zone structure

abstract: Liquid-liquid phase separation enables the assembly of membrane-less subcellular compartments, but testing its biological functions has been difficult. The presynaptic active zone, protein machinery in nerve terminals that defines sites for neurotransmitter release, may be organized through phase separation. Here, we discover that the active zone protein Liprin-3 rapidly and reversibly undergoes phase separation upon phosphorylation by PKC at a single site. RIM and Munc13 are co-recruited to membrane-attached condensates, and phospho-specific antibodies establish Liprin-3 phosphorylation in vivo. At synapses of newly generated Liprin-2/3 double knockout mice, RIM, Munc13 and the pool of releasable vesicles were reduced. Re-expression of Liprin-3 restored these defects, but mutating the Liprin-3 phosphorylation site to abolish phase condensation prevented rescue. Finally, PKC activation acutely increased RIM, Munc13 and neurotransmitter release, which depended on the presence of phosphorylatable Liprin-3. We conclude that Liprin-3 phosphorylation rapidly triggers presynaptic phase separation to modulate active zone structure and function.


title: A genome-wide CRISPR/Cas9 knock-out screen identifies the DEAD box RNA helicase DDX42 as a broad antiviral inhibitor

abstract: Genome-wide screens are powerful approaches to unravel new regulators of viral infections. Here, we used a CRISPR/Cas9 screen to reveal new HIV-1 inhibitors. This approach led us to identify the RNA helicase DDX42 as an intrinsic antiviral inhibitor. DDX42 was previously described as a non-processive helicase, able to bind RNA secondary structures such as G-quadruplexes, with no clearly defined function ascribed. Our data show that depletion of endogenous DDX42 significantly increased HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibited HIV-1, whereas a dominant-negative mutant increased infection. Importantly, DDX42 also restricted retrotransposition of LINE-1, infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 did not inhibit infection with three negative-strand RNA viruses, arguing against a general, unspecific effect on target cells, which was confirmed by RNA-seq analysis. DDX42 was found in the vicinity of viral elements by proximity ligation assays, and cross-linking RNA immunoprecipitation confirmed a specific interaction of DDX42 with RNAs from sensitive viruses. This strongly suggested a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Taken together, our results show for the first time a new and important role of DDX42 in intrinsic antiviral immunity.

category: microbiology


title: The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses


title: Waffle Method: A general and flexible approach for FIB-milling small and anisotropically oriented samples

abstract: Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens that do not vitrify well, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and cellular specimens that distribute poorly across grid squares. Here we present a general approach we call the  Waffle Method which leverages high-pressure freezing to address these challenges. We illustrate the mitigation of these challenges by applying the Waffle Method and cryo-ET to reveal the macrostructure of the polar tube in microsporidian spores in multiple complementary orientations, which was previously not possible due to preferred orientation of the spores on the grid. We demonstrate the broadness of the Waffle Method by applying it to three additional cellular samples and a single particle sample using a variety of cryo-FIB-milling hardware, with both manual and automated approaches. We also present a unique and critical stress-relief gap designed specifically for waffled lamellae. Additionally, we describe applications of the Waffle Method which are currently being explored. We propose the Waffle Method as a way to achieve many of the advantages of cryo-liftout on the specimen grid while avoiding the long, challenging, and technically-demanding process required for cryo-liftout.


title: The evolution of two distinct strategies of moth flight

abstract: Across insects, wing shape and size have undergone dramatic divergence even in closely related sister groups, but we do not yet know morphology changes in tandem with kinematics to support body weight within available aerodynamic power and how the specific force production patterns are linked to changes in behavior. Hawkmoths and wild silkmoths are two such diverse sister families with divergent wing morphology. Using 3d kinematics and quasi-steady aerodynamic modeling, we compare the aerodynamics and the contributions of wing shape, size, and kinematics in 10 moth species. We find that wing movement also diverges between the clades and underlies two distinct strategies for flight. Hawkmoths use wing kinematics, especially high frequencies, to enhance force, but wing morphologies that reduces power. Silkmoths use wing morphology to enhance force, and high amplitude wingstrokes to reduce power. Both strategies converge on similar aerodynamic power and can support similar body mass ranges, but their within-wingstroke force profiles are quite different and linked to the hovering flight of hawkmoths and the bobbing flight of silkmoths. These two groups of moths each fly more like other, distantly related insects than they do each other, demonstrating the convergence and diversity of flapping flight evolution.

category: physiology


title: Integrative transcriptomic analysis of tissue-specific metabolic crosstalk after myocardial infarction

abstract: Abstract/SummaryMyocardial infarction (MI) promotes a range of systemic effects, many of which are unknown. Here, we investigated the alterations associated with MI progression in heart and other metabolically active tissues (liver, skeletal muscle, and adipose) in a mouse model of MI (induced by ligating the left ascending coronary artery) and sham-operated mice. We performed a genome-wide transcriptomic analysis on tissue samples obtained 6- and 24-hours post MI or sham operation. By generating tissue-specific biological networks, we observed: (1) dysregulation in multiple biological processes (including immune system, mitochondrial dysfunction, fatty-acid beta-oxidation, and RNA and protein processing) across multiple tissues post MI; and (2) tissue-specific dysregulation in biological processes in liver and heart post MI. Finally, we validated our findings in two independent MI cohorts. Overall, our integrative analysis highlighted both common and specific biological responses to MI across a range of metabolically active tissues.


title: Markov Abstractions of Electrochemical Reaction-Diffusion in Synaptic Transmission for Neuromorphic Computing

abstract: Progress in computational neuroscience towards understanding brain function is challenged both by the complexity of molecular-scale electrochemical interactions at the level of individual neurons and synapses and the dimensionality of network dynamics across the brain covering a vast range of spatial and temporal scales. Our work abstracts an existing highly detailed, biophysically realistic 3D reaction-diffusion model of a chemical synapse to a compact internal state space representation that maps onto parallel neuromorphic hardware for efficient emulation at a very large scale and offers near-equivalence in input-output dynamics while preserving biologically interpretable tunable parameters.


title: Markov Chain Abstractions of Electrochemical Reaction-Diffusion in Synaptic Transmission for Neuromorphic Computing


title: Gamma-irradiated SARS-CoV-2 vaccine candidate, OZG-38.61.3, confers protection from SARS-CoV-2 challenge in human ACEII-transgenic mice

abstract: The SARS-CoV-2 virus caused the most severe pandemic around the world, and vaccine development for urgent use became a crucial issue. Inactivated virus formulated vaccines such as Hepatitis A, oral polio vaccine, and smallpox proved to be reliable approaches for immunization for prolonged periods. During the pandemic, we produced an inactivated SARS-CoV-2 vaccine candidate, having the advantages of being manufactured rapidly and tested easily in comparison with recombinant vaccines. In this study, an inactivated virus vaccine that includes a gamma irradiation process for the inactivation as an alternative to classical chemical inactivation methods so that there is no extra purification required has been optimized. The vaccine candidate (OZG-38.61.3) was then applied in mice by employing the intradermal route, which decreased the requirement of a higher concentration of inactivated virus for proper immunization, unlike most of the classical inactivated vaccine treatments. Hence, the novelty of our vaccine candidate (OZG-38.61.3) is that it is a non-adjuvant added, gamma-irradiated, and intradermally applied inactive viral vaccine. Efficiency and safety dose (either 1013 or 1014 viral copy per dose) of OZG-38.61.3 was initially determined in Balb/c mice. This was followed by testing the immunogenicity and protective efficacy of OZG-38.61.3. Human ACE2-encoding transgenic mice were immunized and then infected with a dose of infective SARS-CoV-2 virus for the challenge test. Findings of this study show that vaccinated mice have lower SARS-CoV-2 viral copy number in oropharyngeal specimens along with humoral and cellular immune responses against the SARS-CoV-2, including the neutralizing antibodies similar to those shown in Balb/c mice without substantial toxicity. Subsequently, plans are being made for the commencement of Phase 1 clinical trial of the OZG-38.61.3 vaccine for the COVID-19 pandemic.

category: immunology


title: Microtissue geometry and cell-generated forces drive patterning of liver progenitor cell differentiation in 3D

abstract: Towards understanding the impact of mechanical signaling on progenitor cell differentiation in three dimensional (3D) microenvironments, we implemented a hydrogel based microwell platform to produce arrays of multicellular microtissues in constrained geometries, which cause distinct profiles of mechanical signals. We applied this to a model liver development system to investigate the impact of geometry and stress on early liver progenitor cell fate. We fabricated 3D liver progenitor cell microtissues of varied geometries, including cylinder and toroid, and used image segmentation to track individual single cell fate. We observed patterning of hepatocytic makers to the outer shell of the microtissues, except at the inner diameter surface of the toroids. Biliary markers were distributed throughout the interior regions and was increased in toroid tissues compared to cylinder tissues. Finite element models of predicted stress distributions demonstrated that cell-cell tension correlated with hepatocytic fate, while compression correlated with decreased hepatocytic and increased biliary fate. This combined approach integrating microfabrication, imaging and analysis, and mechanical modeling demonstrate of how microtissue geometry can drive patterning of mechanical stresses that regulate cell differentiation trajectories. It also can serve as a platform for the further investigation of signaling mechanisms in the liver and other systems.

category: bioengineering


title: Pronounced proliferation of non-beta cells in response to beta-cell mitogens in isolated human islets of Langerhans

abstract: The potential to treat diabetes by increasing beta-cell mass is driving a major effort to identify beta-cell mitogens. Demonstration of mitogen activity in human beta cells is frequently performed in ex vivo assays. However, reported disparities in the efficacy of beta-cell mitogens led us to investigate the sources of this variability. We studied 35 male (23) and female (12) human islet batches covering a range of donor ages and BMI. Islets were kept intact or dispersed into single cells and cultured in the presence of harmine, glucose, or heparin-binding epidermal growth factor-like growth factor (HB-EGF), and subsequently analyzed by immunohistochemistry or flow cytometry. Proliferating cells were identified by double labelling with EdU and Ki67 and glucagon, c-peptide or Nkx6.1, and cytokeratin-19 to respectively label alpha, beta, and ductal cells. Harmine and HB-EGF stimulated human beta-cell proliferation, but the effect of glucose was dependent on the assay and the donor. Harmine potently stimulated alpha-cell proliferation and both harmine and HB-EGF increased proliferation of insulin- and glucagon-negative cells, including cytokeratin 19-positive cells. Given the abundance of non-beta cells in human islet preparations, our results suggest that assessment of beta-cell mitogens requires complementary approaches and rigorous identification of cell identity using multiple markers.

category: cell biology


title: Live-dead assay on unlabeled cells using phase imaging with computational specificity

abstract: Existing approaches to evaluate cell viability involve cell staining with chemical reagents. However, this step of exogenous staining makes these methods undesirable for rapid, nondestructive and long-term investigation. Here, we present instantaneous viability assessment of unlabeled cells using phase imaging with computation specificity (PICS). This new concept utilizes deep learning techniques to compute viability markers associated with the specimen measured by label-free quantitative phase imaging. Demonstrated on different live cell cultures, the proposed method reports approximately 95% accuracy in identifying live and dead cells. The evolution of the cell dry mass and projected area for the labelled and unlabeled populations reveal that the viability reagents decrease viability. The nondestructive approach presented here may find a broad range of applications, from monitoring the production of biopharmaceuticals, to assessing the effectiveness of cancer treatments.


title: Phosphorothioate Substitutions in RNA Structure Studied by Molecular Dynamics Simulations, QM/MM Calculations and NMR Experiments

abstract: Phosphorothioates (PTs) are important chemical modifications of the RNA backbone where a single non-bridging oxygen of the phosphate is replaced with a sulphur atom. PT can stabilize RNAs by protecting them from hydrolysis and is commonly used as tool to explore their function. It is, however, unclear what basic physical effects PT has on RNA stability and electronic structure. Here, we present Molecular Dynamics (MD) simulations, quantum mechanical (QM) calculations, and NMR spectroscopy measurements, exploring the effects of PT modifications in the structural context of the Neomycin-sensing riboswitch (NSR). The NSR is the smallest biologically functional riboswitch with a well-defined structure stabilized by a U-turn motif. Three of the signature interactions of the U-turn; an H-bond, an anion-{pi} interaction and a potassium binding site; are formed by RNA phosphates, making the NSR an ideal model for studying how PT affects RNA structure and dynamics. By comparing with high-level QM calculations, we reveal the distinct physical properties of the individual interactions facilitated by the PT. The sulphur substitution, besides weakening the direct H-bond interaction, reduces the directionality of H-bonding while increasing its dispersion and induction components. It also reduces the induction and increases dispersion component of the anion-{pi} stacking. The sulphur force-field parameters commonly employed in the literature do not reflect these distinctions, leading to unsatisfactory description of PT in simulations of the NSR. We show that it is not possible to accurately describe the PT interactions using one universal set of van der Waals sulphur parameters and provide suggestions for improving the force-field performance.

category: biophysics


title: Predicting modular functions and neural coding of behavior from a synaptic wiring diagram

abstract: How much can connectomes with synaptic resolution help us understand brain function? An optimistic view is that a connectome is a major determinant of brain function and a key substrate for simulating a brain. Here we investigate the explanatory power of connectomics using a wiring diagram reconstructed from a larval zebrafish brainstem. We identify modules of strongly connected neurons that turn out to be specialized for different behavioral functions, the control of eye and body movements. We then build a neural network model using a synaptic weight matrix based on the reconstructed wiring diagram. This leads to predictions that statistically match the neural coding of eye position as observed by calcium imaging. Our work shows the promise of connectome-based brain modeling to yield experimentally testable predictions of neural activity and behavior, as well as mechanistic explanations of low-dimensional neural dynamics, a widely observed phenomenon in nervous systems.


title: Inhibition of USP4 attenuates epithelial-mesenchymal transition of renal tubular epithelial cells by TβRI

abstract: The process of epithelial-mesenchymal transition (EMT) is required for the progression of renal interstitial fibrosis (RIF). Ubiquitin-specific protease 4 (USP4) can facilitate development of transforming growth factor, beta 1 (TGF-{beta}1) induced EMT in some cancer cells. However, the role of USP4 in EMT during RIF remains unknown. We aimed to explore the effect of USP4 on the EMT induced by TGF-{beta}1 of renal tubular epithelial cells and involved mechanism in RIF. In vivo, on the 7th and 14th day after unilateral ureteral obstruction (UUO), the expression of USP4 protein in the obstructed kidneys was detected by immunohistochemistry and Western blot assay. In vitro, NRK-52E cells were stimulated with TGF-{beta}1 10ng/ml. The protein expressions of USP4, E-cadherin and alpha smooth muscle actin (-SMA) were detected at different time points by Western blot. After transfected with USP4 siRNA, the cells were cultured with TGF-{beta}1 for additional 24 hours. The expressions of E-cadherin, -SMA, and TGF{beta} receptor type I (T{beta}RI) were detected by immunofluorescence. And the protein expressions of USP4, E-cadherin, -SMA and T{beta}RI were detected by Western blot assay. Compared with sham operation group, the expression of USP4 in UUO model group increased significantly with the prolongation of obstruction time. After NRK-52E was stimulated by TGF-{beta}1, the expression of USP4 protein increased gradually. At 6h, 12h, and 24h, the difference between the experimental group and the control group was statistically significant. At the same time, E-cadherin decreased significantly, while -SMA increased significantly. Compared with the TGF-{beta}1 group, the cells in USP4 siRNA transfection group restored E-cadherin and weakened -SMA expression. At the same time, protein expressions of USP4 and T{beta}RI were also significantly decreased. These data imply that USP4 is a harmful molecule induced by TGF-{beta}1, which plays an important role by upregulating the expression of T{beta}RI and promoting EMT of renal tubular epithelial cells, thereby facilitating renal interstitial fibrosis.

category: molecular biology


title: The effect of activity, energy use, and species identity on environmental DNA shedding of freshwater fish

abstract:
The quantitative measurement of environmental DNA (eDNA) from field-collected water samples is gaining importance for the monitoring of fish communities and populations. The interpretation of these signal strengths depends, among other factors, on the amount of target eDNA shed into the water. However, shedding rates are presumably associated with species-specific traits such as physiology and behavior. Although such differences between juvenile and adult fish have been previously detected, the general impact of movement and energy use in a resting state on eDNA release into the surrounding water remains hardly addressed.

In an aquarium experiment, we compared eDNA shedding between seven fish species occurring in European freshwaters. The investigated salmonids, cyprinids, and sculpin exhibit distinct adaptions to microhabitats, diets, and either solitary or schooling behavior. The fish were housed in aquaria with constant water flow and their activity was measured by snapshots taken every 30 s. Water samples for eDNA analysis were taken every 3 h and energy use was determined in an intermittent flow respirometer. After controlling for the effect of fish mass, our results demonstrate a positive correlation between target eDNA quantities as measured with digital PCR, fish activity, and energy use, as well as species-specific differences. For cyprinids, the model based on data from individual fish was only partly transferable to groups, which exhibited lower activity and higher energy use.

Our findings highlight the importance of fish physiology and behavior for the comparative interpretation of taxon-specific eDNA quantities. Species traits should therefore be incorporated into eDNA-based monitoring and conservation efforts.

category: ecology


title: Dynamical consequences of regional heterogeneity in the brains transcriptional landscape

abstract: Brain regions vary in their molecular and cellular composition, but how this heterogeneity shapes neuronal dynamics is unclear. Here, we investigate the dynamical consequences of regional heterogeneity using a biophysical model of whole-brain functional magnetic resonance imaging (MRI) dynamics in humans. We show that models in which transcriptional variations in excitatory and inhibitory receptor (E:I) gene expression constrain regional heterogeneity more accurately reproduce the spatiotemporal structure of empirical functional connectivity estimates than do models constrained by global gene expression profiles and MRI-derived estimates of myeloarchitecture. We further show that regional heterogeneity is essential for yielding both ignition-like dynamics, which are thought to support conscious processing, and a wide variance of regional activity timescales, which supports a broad dynamical range. We thus identify a key role for E:I heterogeneity in generating complex neuronal dynamics and demonstrate the viability of using transcriptional data to constrain models of large-scale brain function.


title: Magnetoencephalographic Signatures of Conscious Processing Before Birth

abstract: The concept of fetal consciousness is a widely discussed topic. In this study, we applied a hierarchical rule learning paradigm to investigate the possibility of fetal conscious processing during the last trimester of pregnancy. We used fetal magnetoencephalography, to assess fetal brain activity in 56 healthy fetuses between gestational week 25 and 40, during an auditory oddball paradigm containing first- and second-order regularities. The comparison of fetal brain responses towards standard and deviant tones revealed that the investigated fetuses show signs of hierarchical rule learning, and thus the formation of a memory trace for the second-order regularity. This ability develops over the course of the last trimester of gestation, in accordance with processes in physiological brain development and was only reliably present in fetuses older than week 35 of gestation. Analysis of fetal autonomic nervous system activity replicates findings in newborns, showing importance of activity state for cognitive processes. On the whole, our results support the assumption that fetuses in the last weeks of gestation are capable of consciously processing stimuli that reach them from outside the womb.


title: Characterization of spatial dynamics of fMRI data in white matter using diffusion-informed white matter harmonics

abstract: In this work, we leverage the Laplacian eigenbasis of voxel- wise white matter (WM) graphs derived from diffusion- weighted MRI data, dubbed WM harmonics, to characterize the spatial structure of WM fMRI data. Our motivation for such a characterization is based on studies that show WM fMRI data exhibit a spatial correlational anisotropy that coincides with underlying fiber patterns. By quantifying the energy content of WM fMRI data associated with subsets of WM harmonics across multiple spectral bands, we show that the data exhibits notable subtle spatial modulations under functional load that are not manifested during rest. WM har- monics provide a novel means to study the spatial dynamics of WM fMRI data, in such way that the analysis is informed by the underlying anatomical structure.


title: Propofol Anesthesia Concentration Rather Than Abrupt Behavioral Unresponsiveness Linearly Degrades Responses in the Rat Primary Auditory Cortex

abstract: Despite extensive knowledge of its molecular and cellular effects, how anesthesia affects sensory processing remains poorly understood. In particular, it remains unclear whether anesthesia modestly or robustly degrades activity in primary sensory regions, and whether such changes are linked to anesthesia drug concentration vs. behavioral unresponsiveness, since these are typically confounded. Here, we employed slow gradual intravenous propofol anesthesia induction together with auditory stimulation and intermittent assessment of behavioral responsiveness while recording epidural EEG, and neuronal spiking activity in the primary auditory cortex (PAC) of eight rats. We found that all main components of neuronal activity including spontaneous firing rates, onset response magnitudes, onset response latencies, post-onset neuronal silence duration, and late-locking to 40Hz click-trains, gradually deteriorated in a dose- dependent manner with increasing anesthesia levels without showing abrupt changes around loss of righting reflex or other time-points. Thus, the dominant factor affecting PAC responses is the anesthesia drug concentration rather than any sudden, dichotomous behavioral state changes. Our findings explain a wide array of seemingly conflicting results in the literature that, depending on the precise definition of wakefulness (vigilant vs. drowsy) and anesthesia (light vs. deep/surgical), report a spectrum of effects in primary regions ranging from minimal to dramatic differences.


title: Absolute measurement of cellular activities using photochromic single-fluorophore biosensors

abstract: Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a method that leverages the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity. We develop proof-of-concept photochromic variants of the popular GCaMP family of Ca2+ biosensors, and show that these can be used to resolve dynamic changes in the absolute Ca2+ concentration in live cells. We also show how our method can be expanded to fast imaging with reduced illumination intensities or to situations where the absolute illumination intensities are unknown. In principle, PEAQ biosensing can be applied to other biosensors with photochromic properties, thereby expanding the possibilities for fully quantitative measurements in complex and dynamic systems.


title: Engineering Gac/Rsm signaling cascade for optogenetic induction of pathogenicity switch in Pseudomonas aeruginosa

abstract: Bacterial pathogens operate by tightly controlling the pathogenicity to facilitate invasion and survival in host. While small molecule inducers can be designed to modulate pathogenicity to perform studies of pathogen-host interaction, these approaches, due to the diffusion property of chemicals, may have unintended, or pleiotropic effects that can impose limitations on their use. By contrast, light provides superior spatial and temporal resolution. Here, using optogenetics we reengineered GacS of the opportunistic pathogen Pseudomonas aeruginosa, signal transduction protein of the global Gac/Rsm cascade which is of central importance for regulation of infection factors. The resultant protein YGS24 displayed significant light-dependent activity of GacS kinases in Pseudomonas aeruginosa. When introduced in Caenorhabditis elegans host systems, YGS24 stimulated the pathogenicity of PAO1 in BHI and of PA14 in SK medium progressively upon blue-light exposure. This optogenetic system provides an accessible way to spatiotemporally control bacterial pathogenicity in defined host even specific tissues to develop new pathogenesis systems, which may in turn expedite development of innovative therapeutics.

category: synthetic biology


title: Towards end-to-end disease prediction from raw metagenomic data

abstract:
Analysis of the human microbiome using metagenomic sequencing data has demonstrated high ability in discriminating various human diseases. Raw metagenomic sequencing data require multiple complex and computationally heavy bioinformatics steps prior to data analysis. Such data contain millions of short sequences read from the fragmented DNA sequences and are stored as fastq files. Conventional processing pipelines consist in multiple steps including quality control, filtering, alignment of sequences against genomic catalogs (genes, species, taxonomic levels, functional pathways, etc.). These pipelines are complex to use, time consuming and rely on a large number of parameters that often provide variability and impact the estimation of the microbiome elements.

Training Deep Neural Networks directly from raw sequencing data is a promising approach to bypass some of the challenges associated with mainstream bioinformatics pipelines. Most of these methods use the concept of word and sentence embeddings that create a meaningful and numerical representation of DNA sequences, while extracting features and reducing the dimensionality of the data. In this paper we present an end-to-end approach that classifies patients into disease groups directly from raw metagenomic reads: metagenome2vec. This approach is composed of four steps (i) generating a vocabulary of k-mers and learning their numerical embeddings; (ii) learning DNA sequence (read) embeddings; (iii) identifying the genome from which the sequence is most likely to come and (iv) training a multiple instance learning classifier which predicts the phenotype based on the vector representation of the raw data. An attention mechanism is applied in the network so that the model can be interpreted, assigning a weight to the influence of the prediction for each genome. Using two public real-life data-sets as well a simulated one, we demonstrated that this original approach reaches high performance, comparable with the state-of-the-art methods applied directly on processed data though mainstream bioinformatics workflows. These results are encouraging for this proof of concept work. We believe that with further dedication, the DNN models have the potential to surpass mainstream bioinformatics workflows in disease classification tasks.

category: genomics


title: Low nitrogen availability inhibits the phosphorus starvation response in maize (Zea mays ssp. mays L.)

abstract:
BackgroundNitrogen (N) and phosphorus (P) are macronutrients essential for crop growth and productivity. In cultivated fields, N and P levels are rarely sufficient, contributing to the yield gap between realized and potential production. Fertilizer application increases nutrient availability, but not all farmers have access to fertilizers, nor are current rates of application sustainable or environmentally desirable. Transcriptomic studies of cereal crops have revealed dramatic responses to either low N or low P single stress treatments. In the field, however, levels of both N and P may be suboptimal. The interaction between N and P starvation responses remains to be fully characterized.

ResultsWe characterized growth and root and leaf transcriptomes of young maize plants under nutrient replete, low N, low P or combined low NP conditions. We identified 1,555 genes to respond to our nutrient treatments, in one or both tissues. A large group of genes, including many classical P starvation response genes, were regulated antagonistically between low N and P conditions. An additional experiment over a range of N availability indicated that a mild reduction in N levels was sufficient to repress the low P induction of P starvation genes. Although expression of P transporter genes was repressed under low N or low NP, we confirmed earlier reports of P hyper accumulation under N limitation.

ConclusionsTranscriptional responses to low N or P were distinct, with few genes responding in a similar way to the two single stress treatments. In combined NP stress, the low N response dominated, and the P starvation response was largely suppressed. A reduction in N availability was sufficient to repress the induction of P starvation associated genes. We conclude that activation of the transcriptional response to P starvation in maize is contingent on sufficient N availability.

category: plant biology


title: Dynamic influences on static measures of metacognition

abstract: Humans differ in their capability to judge the accuracy of their own choices via confidence judgments. Signal detection theory has been used to quantify the extent to which confidence tracks accuracy via M-ratio, often referred to as metacognitive efficiency. This measure, however, is static in that it does not consider the dynamics of decision making. This could be problematic because humans may shift their level of response caution to alter the tradeoff between speed and accuracy. Such shifts could induce unaccounted-for sources of variation in the assessment of metacognition. Instead, evidence accumulation frameworks consider decision making, including the computation of confidence, as a dynamic process unfolding over time. We draw on evidence accumulation frameworks to examine the influence of response caution on metacognition. Simulation results demonstrate that response caution has an influence on M-ratio. We then tested and confirmed that this was also the case in human participants who were explicitly instructed to either focus on speed or accuracy. We next demonstrated that this association between M-ratio and response caution was also present in an experiment without any reference towards speed. The latter finding was replicated in an independent dataset. In contrast, when data were analyzed with a novel dynamic measure of metacognition, which we refer to as v-ratio, in all of the three studies there was no effect of speed-accuracy tradeoff. These findings have important implications for research on metacognition, such as its measurement, domain-generality, individual differences, and neural correlates.


title: A phylogenomic perspective on gene tree conflict and character evolution in Caprifoliaceae using target enrichment data, with Zabelioideae recognized as a new subfamily

abstract: The use of diverse datasets in phylogenetic studies aiming for understanding evolutionary histories of species can yield conflicting inference. Phylogenetic conflicts observed in animal and plant systems have often been explained by hybridization, incomplete lineage sorting (ILS), or horizontal gene transfer. Here, we employed target enrichment data, species tree and species network approaches to infer the backbone phylogeny of the family Caprifoliaceae, while distinguishing among sources of incongruence. We used 713 nuclear loci and 46 complete plastome sequence data from 43 samples representing 38 species from all major clades to reconstruct the phylogeny of the family using concatenation and coalescence approaches. We found significant nuclear gene tree conflict as well as cytonuclear discordance. Additionally, coalescent simulations and phylogenetic species network analyses suggested putative ancient hybridization among subfamilies of Caprifoliaceae, which seems to be the main source of phylogenetic discordance. Ancestral state reconstruction of six morphological characters revealed some homoplasy for each character examined. By dating the branching events, we inferred the origin of Caprifoliaceae at approximately 66.65 Ma in the late Cretaceous. By integrating evidence from molecular phylogeny, divergence times, and morphology, we herein recognize Zabelioideae as a new subfamily in Caprifoliaceae. This work shows the necessity of using a combination of multiple approaches to identify the sources of gene tree discordance. Our study also highlights the importance of using data from both nuclear and chloroplast genomes to reconstruct deep and shallow phylogenies of plants.

category: evolutionary biology


title: BIO-LGCA: a cellular automaton modelling class for analysing collective cell migration

abstract:
1Collective dynamics in multicellular systems such as biological organs and tissues plays a key role in biological development, regeneration, and pathological conditions. Collective tissue dynamics - understood as population behaviour arising from the interplay of the constituting discrete cells - can be studied with on- and off-lattice agent-based models. However, classical on-lattice agent-based models, also known as cellular automata, fail to replicate key aspects of collective migration, which is a central instance of collective behaviour in multicellular systems.

To overcome drawbacks of classical on-lattice models, we introduce an on-lattice, agent-based modelling class for collective cell migration, which we call biological lattice-gas cellular automaton (BIO-LGCA). The BIO-LGCA is characterised by synchronous time updates, and the explicit consideration of individual cell velocities. While rules in classical cellular automata are typically chosen ad hoc, rules for cell-cell and cell-environment interactions in the BIO-LGCA can also be derived from experimental cell migration data or biophysical laws for individual cell migration. We introduce elementary BIO-LGCA models of fundamental cell interactions, which may be combined in a modular fashion to model complex multicellular phenomena. We exemplify the mathematical mean-field analysis of specific BIO-LGCA models, which allows to explain collective behaviour. The first example predicts the formation of clusters in adhesively interacting cells. The second example is based on a novel BIO-LGCA combining adhesive interactions and alignment. For this model, our analysis clarifies the nature of the recently discovered invasion plasticity of breast cancer cells in heterogeneous environments. A Python package which implements various interaction rules and visualisations of BIO-LGCA model simulations we have developed is available at https://github.com/sisyga/BIO-LGCA.

2 Author summaryPattern formation during embryonic development and pathological tissue dynamics, such as cancer invasion, emerge from individual inter-cellular interactions. In order to study the impact of single cell dynamics and cell-cell interactions on tissue behaviour, one needs to develop space-time-dependent on- or off-lattice agent-based models (ABMs), which consider the behaviour of individual cells. However, classical on-lattice agent-based models also known as cellular automata fail to replicate key aspects of collective migration, which is a central instance of collective behaviour in multicellular systems. Here, we present the rule- and lattice-based BIO-LGCA modelling class which allows for (i) rigorous derivation of rules from biophysical laws and/or experimental data, (ii) mathematical analysis of collective migration, and (iii) computationally efficient simulations.

category: bioinformatics


title: The adaptive benefit of evolved increases in hemoglobin-O2 affinity is contingent on tissue O2 diffusing capacity in high-altitude deer mice

abstract:
BackgroundComplex organismal traits are often the result of multiple interacting genes and sub-organismal phenotypes, but how these interactions shape the evolutionary trajectories of adaptive traits is poorly understood. We examined how functional interactions between cardiorespiratory traits contribute to adaptive increases in the capacity for aerobic thermogenesis (maximal O2 consumption, V{square}O2max, during acute cold exposure) in high-altitude deer mice (Peromyscus maniculatus). We crossed highland and lowland deer mice to produce F2 inter-population hybrids, which expressed genetically based variation in hemoglobin (Hb) O2 affinity on a mixed genetic background. We then combined physiological experiments and mathematical modeling of the O2 transport pathway to examine links between cardiorespiratory traits and V{square}O2max.

ResultsPhysiological experiments revealed that increases in Hb-O2 affinity of red blood cells improved blood oxygenation in hypoxia, but were not associated with an enhancement in V{square}O2max. Sensitivity analyses performed using mathematical modeling showed that the influence of Hb-O2 affinity on V{square}O2max in hypoxia was contingent on the capacity for O2 diffusion in active tissues.

ConclusionsThese results suggest that increases in Hb-O2 affinity would only have adaptive value in hypoxic conditions if concurrent with or preceded by increases in tissue O2 diffusing capacity. In high-altitude deer mice, the adaptive benefit of increasing Hb-O2 affinity is contingent on the capacity to extract O2 from the blood, which helps resolve controversies about the general role of hemoglobin function in hypoxia tolerance.


title: CK2 alpha prime and alpha-synuclein pathogenic functional interaction mediates inflammation and transcriptional dysregulation in Huntington's disease

abstract:
BackgroundHuntingtons Disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the HTT gene for which no therapies are available. This mutation causes HTT protein misfolding and aggregation, preferentially affecting medium spiny neurons (MSNs) of the basal ganglia. Transcriptional perturbations in synaptic genes and neuroinflammation are key processes that precede MSN dysfunction and motor symptom onset. Understanding the interplay between these processes is crucial to develop effective therapeutic strategies to treat HD. We investigated whether protein kinase CK2, a kinase upregulated in MSNs in HD and previously associated with Parkinsons disease (PD), participates in the regulation of neuroinflammation and synaptic function during HD progression.

MethodsWe used the heterozygous knock-in zQ175 HD mouse model and compared that to zQ175 mice lacking one allele of CK2. We performed neuropathological analyses using immunohistochemistry, cytokine proteome profiling, RNA-seq analyses in the striatum, electrophysiological recordings, and behavioral analyses. We also used the murine immortalized striatal cell lines STHdhQ7 and STHdhQ111 and studied the expression of various synaptic genes dysregulated by CK2.

ResultsWe showed that CK2 haploinsufficiency in zQ175 mice ameliorated neuroinflammation, HTT aggregation, transcriptional alterations, excitatory synaptic transmission, and motor coordination deficits. RNA-seq analyses also revealed a connection between -syn, a protein associated with PD, and the transcriptional perturbations mediated by CK2 in HD. We also found increased -syn serine 129 phosphorylation (pS129--syn), a post-translational modification linked to -synucleinopathy, in the nuclei of MSNs in zQ175 mice and in patients with HD. Levels of pS129--syn were ameliorated in zQ175 lacking one allele of CK2.

ConclusionsOur data demonstrated that CK2 contributes to transcriptional dysregulation of synaptic genes and neuroinflammation in zQ175 mice and its depletion improved several HD-like phenotypes in this mouse model. These effects were related to increased phosphorylation of S129--syn in the striatum of HD mice, suggesting that CK2 contributes to worsening HD by mediating synucleinopathy. Our study highlights a possible convergent mechanism of neurodegeneration between HD and PD and suggests targeting CK2 as a potential therapeutic strategy to ameliorate synaptic dysfunction in HD as well as other neurodegenerative diseases.


title: CK2 alpha prime and alpha-synuclein pathogenic functional interaction mediates synaptic dysregulation in Huntington's disease


title: Repulsive electrostatic interactions modulate dense and dilute phase properties of biomolecular condensates

abstract: Liquid-like membraneless organelles form via multiple, weak interactions between biomolecules. The resulting condensed states constitute novel solvent environments inside eukaryotic cells that partition biomolecules and may favour particular biochemical reactions. Here we demonstrate that, in addition to attractive interactions, repulsive electrostatic interactions modulate condensate properties. We find that net charge modulates the formation, morphology and solvent properties of model Ddx4 condensates in cells and in vitro and that a net negative charge is conserved across germ cell-specific Ddx4 orthologues. This conserved net charge provides a sensitivity to multivalent cations that is not observed in somatic paralogues. The disfavouring effect of a net negative charge in Ddx4 orthologues appears to be offset by increased charge patterning, indicating that fine tuning of both attractive and repulsive interactions can create responsive solvent environments inside biomolecular condensates.


title: A Library Of Induced Pluripotent Stem Cells From Clinically Well-Characterized, Diverse Healthy Human Individuals

abstract: A library of well-characterized human induced pluripotent stem cell (hiPSC) lines from clinically healthy human subjects could serve as a useful resource of normal controls for in vitro human development, disease modeling, genotype-phenotype association studies, and drug response evaluation. We report generation and extensive characterization of a gender-balanced, racially/ethnically diverse library of hiPSC lines from 40 clinically healthy human individuals who range in age from 22-61. The hiPSCs match the karyotype and short tandem repeat identity of their parental fibroblasts, and have a transcription profile characteristic of pluripotent stem cells. We provide whole genome sequencing data for one hiPSC clone from each individual, genomic ancestry determination, and analysis of Mendelian disease genes and risks. We document similar transcriptomic profiles, single-cell RNA-seq derived cell clusters and physiology of cardiomyocytes differentiated from multiple independent hiPSC lines. This extensive characterization makes this hiPSC library a valuable resource for many studies on human biology.


title: Multiple Mechanisms Underlie State-Independent Inhibitory Effects of Norfluoxetine on TREK-2 K2P Channels

abstract: The TREK subfamily of Two-Pore Domain (K2P) K+ channels are inhibited by fluoxetine and its metabolite, norfluoxetine (NFx). Although not the principal targets of this antidepressant, TREK channel inhibition by NFx has provided important insights into the conformational changes associated with channel gating and highlighted the role of the selectivity filter in this process. But despite the availability of TREK-2 crystal structures with NFx bound, the precise mechanisms underlying NFx inhibition remain elusive. NFx has previously been proposed to be a state-dependent inhibitor, but its binding site suggests many possible ways in which this positively charged drug might inhibit channel activity. Here we show that NFx exerts multiple effects on single channel behavior that influence both the open and closed states of the channel, and that the channel can become highly activated by 2-APB whilst remaining in the down conformation. We also show that that the inhibitory effects of NFx are unrelated to its positive charge, but can be influenced by agonists such as ML335 which alter filter stability, as well as by an intrinsic voltage-dependent gating process within the filter. NFx therefore not only inhibits channel activity by altering the equilibrium between up and down conformations, but can also directly influence filter gating. These results provide further insight into the complex allosteric mechanisms that modulate filter-gating in TREK K2P channels and highlight the different ways that filter gating can be regulated to permit polymodal regulation.


title: Dopamine D1 and D4 receptors contribute to light adaptation in ON-sustained retinal ganglion cells

abstract:
The adaptation of ganglion cells to increasing light levels is a crucial property of the retina. The retina must respond to light intensities that vary by 10-12 orders of magnitude, but the dynamic range of ganglion cell responses covers only [~]3 orders of magnitude. Dopamine is a crucial neuromodulator for light adaptation and activates receptors in the D1 and D2 families. D1Rs are expressed on horizontal cells and some bipolar, amacrine and ganglion cells. In the D2 family D2Rs are expressed on dopaminergic amacrine cells and D4Rs are primarily expressed on photoreceptors. However, the roles of activating these receptors to modulate the synaptic properties of the inputs to ganglion cells are not yet clear. Here we used single cell retinal patch-clamp recordings from the mouse retina to determine how activating D1Rs and D4Rs changed the light-evoked and spontaneous excitatory inputs to ON-sustained (ON-s) ganglion cells. We found that both D1R and D4R activation decrease the light-evoked excitatory inputs to ON-s ganglion cells, but that only the sum of the peak response decrease due to activating the two receptors was similar to the effect of light adaptation to a rod-saturating background. The largest effects on spontaneous excitatory activity of both D1R and D4R agonists was on the frequency of events, suggesting that both D1Rs and D4Rs are acting upstream of the ganglion cells.

New and NoteworthyDopamine by bright light conditions allows retinal neurons to reduce sensitivity to adapt to bright light conditions. It is not clear how and why dopamine receptors modulate retinal ganglion cell signaling. We found that both D1 and D4 dopamine receptors in photoreceptors and inner retinal neurons contribute significantly to the reduction in sensitivity of ganglion cells with light adaptation. However, light adaptation also requires dopamine independent mechanisms that could reflect inherent sensitivity changes in photoreceptors.


title: Early invasion of uropathogenic Escherichia coli into the bladder wall by solitary bacteria that are protected from antibiotics and neutrophil swarms in an organoid model

abstract: Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infections (UTIs) requiring antibiotic therapy. Recurrent infections, which occur in a quarter of treated individuals, may arise from "quiescent intracellular reservoirs" of bacteria that invade deeper layers of the bladder wall following infection and exfoliation of superficial umbrella cells. Here, we present a novel bladder organoid model of UPEC infection that recapitulates the stratified bladder architecture within a small volume suitable for live-cell imaging of host-pathogen dynamics with high spatiotemporal resolution. We confirm that bacteria injected into the organoid lumen rapidly enter superficial cells that resemble umbrella cells and proliferate to generate tightly packed colonies that resemble intracellular bacterial communities (IBCs), a hallmark of UPEC pathogenesis. Unexpectedly, at early stages of infection we detect individual "solitary" bacteria that penetrate deeper layers of the organoid wall, where they evade killing by antibiotics and neutrophils. Volumetric serial block face scanning electron microscopy of infected organoids reveals that solitary bacteria can be found throughout the bladder wall and may be intracellular or pericellular (sandwiched between uroepithelial cells). Unlike bacteria within IBCs, which are coccoid-shaped and non-flagellated, solitary bacteria within the bladder wall are rod-shaped and flagellated. We conclude that early invasion of deeper layers of the bladder wall, independent of IBC formation, results in the establishment of reservoirs of solitary bacteria that resist elimination by antibiotics and the host innate immune response.


title: Leveraging big data for classification of children who stutter from fluent peers

abstract:
IntroductionLarge datasets, consisting of hundreds or thousands of subjects, are becoming the new data standard within the neuroimaging community. While big data creates numerous benefits, such as detecting smaller effects, many of these big datasets have focused on non-clinical populations. The heterogeneity of clinical populations makes creating datasets of equal size and quality more challenging. There is a need for methods to connect these robust large datasets with the carefully curated clinical datasets collected over the past decades.

MethodsIn this study, resting-state fMRI data from the Adolescent Brain Cognitive Development study (N=1509) and the Human Connectome Project (N=910) is used to discover generalizable brain features for use in an out-of-sample (N=121) multivariate predictive model to classify young (3-10yrs) children who stutter from fluent peers.

ResultsAccuracy up to 72% classification is achieved using 10-fold cross validation. This study suggests that big data has the potential to yield generalizable biomarkers that are clinically meaningful. Specifically, this is the first study to demonstrate that big data-derived brain features can differentiate children who stutter from their fluent peers and provide novel information on brain networks relevant to stuttering pathophysiology.

DiscussionThe results provide a significant expansion to previous understanding of the neural bases of stuttering. In addition to auditory, somatomotor, and subcortical networks, the big data-based models highlight the importance of considering large scale brain networks supporting error sensitivity, attention, cognitive control, and emotion regulation/self-inspection in the neural bases of stuttering.


title: Forward variable selection improves the power of random forest for high- dimensional microbiome data

abstract:
BackgroundRandom forest (RF) captures complex feature patterns that differentiate groups of samples and is rapidly being adopted in microbiome studies. However, a major challenge is the high dimensionality of microbiome datasets. They include thousands of species or molecular functions of particular biological interest. This high dimensionality significantly reduces the power of random forest approaches for identifying true differences. The widely used Boruta algorithm iteratively removes features that are proved by a statistical test to be less relevant than random probes.

ResultWe developed a massively parallel forward variable selection algorithm and coupled it with the RF classifier to maximize the predictive performance. The forward variable selection algorithm adds new variable to a set of selected variables as far as the prespecified criterion of predictive power is improved. At each step, the parameters of random forest are optimized. We demonstrated the performance of the proposed approach, which we named RF-FVS, by analyzing two published datasets from large-scale case-control studies: (i) 16S rRNA gene amplicon data for Clostridioides difficile infection (CDI) and (ii) shotgun metagenomics data for human colorectal cancer (CRC). The RF-FVS approach further screened the variables that the Boruta algorithm left and improved the accuracy of the random forest classifier from 81% to 99.01% for CDI and from 75.14% to 90.17% for CRC.

ConclusionValid variable selection is essential for the analysis of high-dimensional microbiota data. By adopting the Boruta algorithm for pre-screening of the variables, our proposed RF-FVS approach improves the accuracy of random forest significantly with minimum increase of computational burden. The procedure can be used to identify the functional profiles that differentiate samples between different conditions.


title: Visual neuroscience methods for marmosets: efficient receptive field mapping and head-free eye tracking

abstract: The marmoset has emerged as a promising primate model system, in particular for visual neuroscience. Many common experimental paradigms rely on head fixation and an extended period of eye fixation during the presentation of salient visual stimuli. Both of these behavioral requirements can be challenging for marmosets. Here, we present two methodological developments, each addressing one of these difficulties. First, we show that it is possible to use a standard eye tracking system without head fixation to assess visual behavior in the marmoset. Eye tracking quality from head-free animals is sufficient to obtain precise psychometric functions from a visual acuity task. Secondly, we introduce a novel method for efficient receptive field mapping that does not rely on moving stimuli but uses fast flashing annuli and wedges. We present data recorded during head-fixation in areas V1 and V6 and show that receptive field locations are readily obtained within a short period of recording time. Thus, the methodological advancements presented in this work will contribute to establish the marmoset as a valuable model in neuroscience.


title: SurfStamp: 3D Printer Compatible Molecular Surface Representation

abstract:
SurfStamp is an application that is used to generate textures for surface models of proteins. The textures contain information about surface residues and the information is drawn directly on the 3D object of the models. This approach is more intuitive than the labeling functions that most three-dimensional (3D) structure viewers use to show residue information. Therefore, the use of this application enables researchers, readers, or audiences to easily determine which residues are contributing the surface they are focusing on.

AvailabilityThe application is provided under the open-source Apache License Version 2.0 (http://www.apache.org/licenses/LICENSE-2.0). The application and source code are available from https://github.com/yamule/SurfStamp-public/releases. The application is also available as a plugin for the recent versions of PyMOL (Schrodinger, 2015, https://github.com/schrodinger/pymol-open-source) from https://github.com/yamule/SurfStamp-pymol-plugin.


title: Impact of germline and somatic cell cycle checkpoint kinase mutations on breast cancer presentation and prognosis.

abstract: Cell cycle checkpoint kinases ATM/Chk2 and ATR/Chk1 are important tumor suppressors that are activated by DNA damage and other stressors to arrest replication. Germline variants in ATM and CHEK2 associate with increased incidence of estrogen receptor positive (ER+)/HER2- breast cancer and some evidence suggests that somatic inactivation of ATM/Chk2 induces therapy resistance in ER+/HER2- breast cancer patients. However, the relative contributions of germline and somatic inactivation of each of the four cell cycle checkpoint kinases to ER+/HER2- and ER-/PR-/HER2- (or triple negative breast cancer; TNBC) breast cancer incidence and outcomes has not yet been systematically assessed. Here, we comprehensively analyze associations between mutations in each cell cycle checkpoint kinase gene with age at diagnosis, tumor characteristics, and patient survival outcomes using six breast cancer datasets. Overall, we find that of all the cell cycle checkpoint kinases, CHEK2 (p=0.001) is the only gene that is preferentially mutated in ER+/HER2- breast cancer samples relative to TNBCs. CHEK2 mutations also associate with younger age at diagnosis of metastatic ER+/HER2- breast cancer (median age 41.5 years compared to 50 years for CHEK2 wildtype patients, p=0.03) that is highly PR positive (10% increase in PR positivity in metastatic vs primary CHEK2 mutant tumors, as opposed to a 25% decrease in PR positivity in CHEK2 wildtype tumors, p=0.03). These associations appear to be driven by germline variants, rather than somatic mutations. Whether germline or somatic, deleterious (truncating, frameshift or splice site) mutations in CHEK2 associate with rapid disease progression in response to standard first line endocrine therapy when compared to tumors with wildtype CHEK2 in two independent datasets (HR=6.15, p=0.01). Conversely, neither ATM nor ATR mutations are enriched in ER+/HER2- breast cancers relative to TNBC. However, co-incident mutation of ATR and TP53 is 2-fold enriched in this subset (p=0.002), and further enriched in metastatic (3.3%) vs primary (1%) ER+/HER2- breast cancers (p=0.002). This co-incident mutation of ATR and TP53 associates with decreased PR positivity in primary ER+/HER2- breast cancer diagnoses (p<0.0001) and significantly worse metastasis-free survival than mutations in either ATR or TP53 alone in two independent datasets (HR=2.01, p=0.007). This association is not specific to ER+/HER2- breast cancer but is observable in ER- breast cancer patients as well. These results provide the first systematic analysis of the contribution of germline and somatic mutations in cell cycle checkpoint kinases to tumor characteristics affecting patient prognosis and treatment response. They demonstrate a unique role for CHK2 in ER signaling and response to endocrine therapy, and a more universal role for ATR in combination with TP53 in metastatic progression across breast cancer subtypes. These findings provide rationale for streamlined testing of checkpoint kinase mutations as prognostic markers of metastatic potential and/or predictive markers of responsiveness to endocrine treatment in breast cancer patients.


title: Differential impact of mutations in cell cycle checkpoint kinases on breast cancer presentation and prognosis.

category: cancer biology


title: Combined fluorescence, optical diffraction tomography and Brillouin microscopy

abstract: Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples -- so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fuorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample -- a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.


title: Correlative all-optical quantification of mass density and mechanics of sub-cellular compartments with fluorescence specificity


title: Evolutionary rescue in one dimensional stepping stone models

abstract: Genetic variation and population sizes are critical factors for successful adaptation to novel environmental conditions. Gene flow between sub-populations is a potent mechanism to provide such variation and can hence facilitate adaptation, for instance by increasing genetic variation or via the introduction of beneficial variants. On the other hand, if gene flow between different habitats is too strong, locally beneficial alleles may not be able to establish permanently. In the context of evolutionary rescue, intermediate levels of gene flow are therefore often optimal for maximizing a species chance for survival in metapopulations without spatial structure. To which extent and under which conditions gene flow facilitates or hinders evolutionary rescue in spatially structured populations remains unresolved. We address this question by studying the differences between evolutionary rescue in the island model and in the stepping stone model in a gradually deteriorating habitat. We show that evolutionary rescue is modulated by the rate of gene flow between different habitats, which in turn depends strongly on the spatial structure and the pattern of environmental deterioration. We use these insights to show that in many cases spatially structured models can be translated into a simpler island model using an appropriately scaled effective migration rate.


title: Investigating spatiotemporal variation in the diet of Westland Petrel through metabarcoding, a non-invasive technique

abstract: As top predators, seabirds can be indirectly impacted by climate variability and commercial fishing activities through changes in marine communities. However, high mobility and foraging behaviour enables seabirds to exploit prey distributed patchily in time and space. This capacity to adapt to environmental change can be described through the study of their diet. Traditionally, the diet of seabirds is assessed through the morphological identification of prey remains in regurgitates. This sampling method is invasive for the bird and limited in terms of taxonomic resolution. However, the recent progress in DNA-based approaches is now providing a non-invasive means to more comprehensively and accurately characterize animal diets. Here, we used a non-invasive metabarcoding approach to characterize the diet of the Westland petrel (Procellaria westlandica), which is an endangered burrowing species, endemic to the South Island of New Zealand. We collected 99 fresh faecal samples at two different seasons and in two different sub-colonies. Our aims were to describe the diet of the Westland petrel, investigate seasonal and spatial variation in the petrels diet, and assess potential impacts of the New Zealand fishery industry. We found that amphipods were the most common prey, followed by cephalopods and fish. Our results could be the result of natural foraging behaviour, but also suggest a close link between the composition of prey items and New Zealands commercial fishing activities. In particular, the high abundance of amphipods could be the result of Westland petrels feeding on discarded fisheries waste (fish guts). Our results also showed significant differences in diet between seasons (before hatching vs chick-rearing season) and between sampling sites (two sub-colonies 1.5 km apart), indicating plasticity in the foraging strategy of the Westland petrel. Due to its non-invasive nature, metabarcoding of faecal samples can be applied to large numbers of samples to help describe dietary variation in seabirds and indicate their ecological requirements. In our example, dietary DNA (dDNA) provided valuable information regarding the dietary preferences of an iconic species in New Zealands unique biodiversity. dDNA can thus inform the conservation of endangered or at-risk species that have elusive foraging behaviours.


title: Metabarcoding faecal samples to investigate spatiotemporal variation in the diet of the endangered Westland petrel (Procellaria westlandica)


title: Determinants of different facets of beta diversity in Mediterranean marine amphipods

abstract: Relying on a purely taxonomic view of diversity may ignore the fact that ecological communities can be constituted of species having both distinct evolutionary histories and functional characteristics. Thus, considering how the multiple facets of diversity vary along environmental and geographic gradients may provide insights into the role of historic processes and current environmental conditions in determining the distribution of species, lineages and functions across space. By using distributional, taxonomic-distance and traits information, we explore the role of spatial/environmental gradients and of biogeographic subdivision of Mediterranean Sea on the different facets of beta diversity components in seagrass amphipods. Beta diversity partitioning and correlation analyses showed a nearly equal contribution of the replacement and richness components on total beta diversity for all facets, although the influence of environmental and geographic distance differs among components. While the replacement was mainly related to a pure spatial gradient, both the environmental and geographic distance were correlated with the richness component of beta diversities. Our results are in line with the complex paleobiogeographic history of the Mediterranean Sea, with the replacement component likely to be related to the progressive substitution of species of Atlantic origin with Mediterranean endemics along the west-east geographic gradient, and the richness component to the marked environmental difference between different basins. Moreover, the influence of biogeographic partition on the richness components suggests the role of spatially structured gradients at biogeographic level in determining the net loss/gain of species, lineages and functions, possibly influencing the assembly processes of passive dispersal organisms.


title: Are irrigated urban trees more sensitive to light and temperature than water?

abstract:
BackgroundReal-time monitoring of tree growth can provide novel information about trees in urban/suburban areas and the myriad ecosystem services they provide. By monitoring irrigated specimen trees we test the hypothesis that in trees with sufficient water, growth is governed by environmental factors regulating energy gain rather than by factors related to water use.

MethodsInternet enabled, high-resolution dendrometers were installed on three trees in Southampton, NY. The instruments, along with a weather station, streamed data to a project web page that was updated once an hour. (https://ecosensornetwork.com). Growing periods were determined using a Hidden Markov Model based on Zweifel et al.s (2016) zero-growth model. Linear models and conditional inference trees correlated environmental variables to growth magnitude and rate of growth.

ResultsGrowth was governed by the interacting environmental variables of air temperature, soil moisture, VPD and took place primarily at night. Radial growth of spruce began April 14 after the accumulation of 69.7 {degrees}C growing degrees days and ended September 7th. Cedar growth began later (April 26th), after the accumulation of 160.6 {degrees}C and ended later (November 3rd). During the observation period, these three modest suburban trees sequestered 108.3 kg of CO2.

ConclusionsThough irrigated, residential tree growth in our experiment was affected by environmental factors relating to both water use and energy gain through photosynthesis. Linking tree growth to fluctuations in environmental conditions facilitates the development of a predictive understanding useful for ecosystem management and growth forecasting across future altering climates.


title: Is the radial growth of irrigated urban trees more strongly correlated to light and temperature than water?


title: Increasing Grain Yield in Bread Wheat by Selection for High Spike Fertility Index

abstract: Spike fertility index (SF) has been proposed as a promising selection criterion for increasing grain yield (GY) in bread wheat. Here, changes in GY and related traits after simulated selection (10% intensity) for high SF or high GY per se were assessed in two RIL populations: (I) Avalon/Glupro and (II) Baguette 10/Klein Chaja, and in (III) advanced lines from a breeding program. Grain yield, SF, grain number per unit area (GN), grain weight (GW), test weight (TW) and grain protein content (GPC) were determined. Regardless of the environmental conditions, simulated selection for high SF always resulted in GN increases (between 1.6 and 27.4%). Average GY increase observed after selection for high SF (11.5%; N=10; SEM=5.8) did not differ (p=0.92) from the average GY increase observed after selection for GY per se (11.8%; N=10; SEM=4.9). Grain weight, GPC and TW tended to decrease with selection for high SF; however, these trade-offs might be avoided by concurrent selection. Our findings validate the use of SF as a selection criterion for increasing grain yield in bread wheat.


title: Value Shapes Abstraction During Learning

abstract: The human brain excels at constructing and using abstractions, such as rules, or concepts. Here, in two fMRI experiments, we demonstrate a mechanism of abstraction built upon the valuation of sensory features. Human volunteers learned novel association rules linking simple visual features. Mixture-of-experts reinforcement learning algorithms revealed that, with learning, high-value abstract representations increasingly guided participants behaviour, resulting in better choices and higher subjective confidence. We also found that the brain area computing value signals - the ventromedial prefrontal cortex - prioritized and selected latent task elements during abstraction, both locally and through its connection to the visual cortex. Such coding scheme predicts a causal role for valuation: in a second experiment, we used multivoxel neural reinforcement to test for the causality of feature valuation in the sensory cortex as a mechanism of abstraction. Tagging the neural representation of a tasks feature with rewards evoked abstraction-based decisions. Together, these findings provide a new interpretation of value as a goal-dependent, key factor in forging abstract representations.


title: Macrophages orchestrate the expansion of a pro-angiogenic perivascular niche during cancer progression

abstract: Tumor associated macrophages (TAMs) are a highly plastic stromal cell type which support cancer progression. Using single-cell RNA-sequencing of TAMs from a spontaneous murine model of mammary adenocarcinoma (MMTV-PyMT) we characterize a subset of these cells expressing lymphatic vessel endothelial hyaluronic acid receptor 1 (Lyve-1) which spatially reside proximal to blood vasculature. We demonstrate that Lyve-1+ TAMs support tumor growth and identify a pivotal role for these cells in maintaining a population of perivascular mesenchymal cells which express alpha-smooth muscle actin and phenotypically resemble pericytes. Using photolabeling techniques show that mesenchymal cells maintain their prevalence in the growing tumor through proliferation and uncover a role for Lyve-1+ TAMs in orchestrating a selective platelet-derived growth factor-CC-dependent expansion of the perivascular mesenchymal population, creating a pro-angiogenic niche. This study highlights the inter-reliance of the immune and non-immune stromal network which support cancer progression and provides therapeutic opportunities for tackling the disease.


title: Acetylation of conserved lysines fine-tune mitochondrial malate dehydrogenase activity in land plants

abstract:
Plants need to rapidly and flexibly adjust their metabolism to changes of their immediate environment. Since this necessity results from the sessile lifestyle of land plants, key mechanisms for orchestrating central metabolic acclimation are likely to have evolved early. Here, we explore the role of lysine acetylation as a posttranslational modification to directly modulate metabolic function. We generated a lysine acetylome of the moss Physcomitrium patens and identified 638 lysine acetylation sites, mostly found in mitochondrial and plastidial proteins. A comparison with available angiosperm data pinpointed lysine acetylation as a conserved regulatory strategy in land plants. Focusing on mitochondrial central metabolism, we functionally analyzed acetylation of malate dehydrogenase (mMDH), which acts as a hub of plant metabolic flexibility. In P. patens mMDH1, we detected a single acetylated lysine located next to one of the four acetylation sites detected in Arabidopsis thaliana mMDH1. We assessed the kinetic behavior of recombinant A. thaliana and P. patens mMDH1 with site-specifically incorporated acetyl-lysines. Acetylation of A. thaliana mMDH1 at K169, K170, and K334 decreases its oxaloacetate reduction activity, while acetylation of P. patens mMDH1 at K172 increases this activity. We found modulation of the malate oxidation activity only in A. thaliana mMDH1, where acetylation of K334 highly activated it. Comparative homology modelling of MDH proteins revealed that evolutionarily conserved lysines serve as hotspots of acetylation. Our combined analyses indicate lysine acetylation as a common strategy to fine-tune the activity of central metabolic enzymes with likely impact on plant acclimation capacity.

Significance statementWe explore the role of lysine acetylation as a mechanism to directly modulate mitochondrial metabolism in land plants by generating the lysine acetylome of the moss Physcomitrium patens and comparing with available angiosperm data. We found acetylation of evolutionarily conserved lysines as a strategy to fine-tune the activity of mitochondrial malate dehydrogenase in a species-dependent molecular context.


title: Enhancing lifespan of budding yeast by pharmacological lowering of amino acid pools

abstract: The increasing prevalence of age-related diseases and resulting healthcare insecurity and emotional burden require novel treatment approaches. Several promising strategies seek to limit nutrients and promote healthy aging. Unfortunately, the human desire to consume food means this strategy is not practical for most people but pharmacological approaches might be a viable alternative. We previously showed that myriocin, which impairs sphingolipid synthesis, increases lifespan in Saccharomyces cerevisiae by modulating signaling pathways including the target of rapamycin complex 1 (TORC1). Since TORC1 senses cellular amino acids, we analyses amino acid pools and identified 17 that are lowered by myriocin treatment. Studying the methionine transporter, Mup1, we found that newly synthesized Mup1 traffics to the plasma membrane and is stable for several hours but is inactive in drug-treated cells. Activity can be restored by adding phytosphingosine to culture medium thereby bypassing drug inhibition, thus confirming a sphingolipid requirement for Mup1 activity. Importantly, genetic analysis of myriocin-induced longevity revealed a requirement for the Gtr1/2 (mammalian Rags) and Vps34-Pib2 amino acid sensing pathways upstream of TORC1, consistent with a mechanism of action involving decreased amino acid availability. These studies demonstrate the feasibility of pharmacologically inducing a state resembling amino acid restriction to promote healthy aging.


title: Investigating epithelial-mesenchymal heterogeneity of tumors and circulating tumor cells with transcriptomic analysis and biophysical modeling

abstract:
IntroductionCellular heterogeneity along the Epithelial-Mesenchymal Plasticity (EMP) spectrum is a paramount feature observed in tumors and circulating tumor cells (CTCs). High-throughput techniques now offer unprecedented details on this variability at a single-cell resolution. Yet, there is no current consensus about how EMP in tumors propagates to that in CTCs. To investigate the relationship between EMP associated heterogeneity of tumors and that of CTCs, we integrated transcriptomic analysis and biophysical modeling.

MethodsWe apply three EMT (Epithelial-Mesenchymal Transition) scoring metrics to multiple tumor samples and CTC datasets from several cancer types. Moreover, we develop a biophysical model that couples EMT associated phenotypic switching in a primary tumor with cell migration. Finally, we integrate EMT transcriptomic analysis and in silico modeling to evaluate the predictive power of several measurements of tumor aggressiveness, including tumor EMT score, CTC EMT score, fraction of CTC clusters found in circulation, and CTC cluster size distribution.

ResultsAnalysis of high-throughput datasets reveals a pronounced heterogeneity without a well-defined relation between EMT traits in tumors and CTCs. Moreover, mathematical modeling predicts different phases where CTCs can be less, equally, or more mesenchymal than primary tumor depending on the dynamics of phenotypic transition and cell migration. Consistently, various datasets of CTC cluster size distribution from different cancer types are fitted onto different regimes of the model. By further constraining the model with experimental measurements of tumor EMT score, CTC EMT score, and fraction of CTC cluster in bloodstream, we show that none of these assays alone can provide sufficient information to predict the other variables.

ConclusionsBy integrating analysis of single cell gene expression and in silico modeling, we propose that the relationship between EMT progression in tumors and CTCs can be variable, and in general, predicting one from the other may not be as straightforward as tacitly assumed.


title: Open Science Discovery of Oral Non-Covalent SARS-CoV-2 Main Protease Inhibitor Therapeutics

abstract: The COVID-19 pandemic is a stark reminder that a barren global antiviral pipeline has grave humanitarian consequences. Future pandemics could be prevented by accessible, easily deployable broad-spectrum oral antivirals and open knowledge bases that derisk and accelerate novel antiviral discovery and development. Here, we report the results of the COVID Moonshot, a fully open-science structure-enabled drug discovery campaign targeting the SARS-CoV-2 main protease. We discovered a novel chemical scaffold that is differentiated from current clinical candidates in terms of toxicity, resistance, and pharmacokinetics liabilities, and developed it into noncovalent orally-bioavailable nanomolar inhibitors with clinical potential. Our approach leveraged crowdsourcing, high-throughput structural biology, machine learning, and exascale molecular simulations. In the process, we generated a detailed map of the structural plasticity of the main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. In a first for a structure-based drug discovery campaign, all compound designs (>18,000 designs), crystallographic data (>500 ligand-bound X-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2,400 compounds) for this campaign were shared rapidly and openly, creating a rich open and IP-free knowledgebase for future anti-coronavirus drug discovery.

category: biochemistry


title: Evidence for adaptive evolution in the receptor-binding domain of seasonal coronaviruses OC43 and 229E

abstract: Seasonal coronaviruses (OC43, 229E, NL63 and HKU1) are endemic to the human population, regularly infecting and reinfecting humans while typically causing asymptomatic to mild respiratory infections. It is not known to what extent reinfection by these viruses is due to waning immune memory or antigenic drift of the viruses. Here, we address the influence of antigenic drift on immune evasion of seasonal coronaviruses. We provide evidence that at least two of these viruses, OC43 and 229E, are undergoing adaptive evolution in regions of the viral spike protein that are exposed to human humoral immunity. This suggests that reinfection may be due, in part, to positively-selected genetic changes in these viruses that enable them to escape recognition by the immune system. It is possible that, as with seasonal influenza, these adaptive changes in antigenic regions of the virus would necessitate continual reformulation of a vaccine made against them.


title: Sea anemone genomes reveal ancestral metazoan chromosomal macrosynteny

abstract: Cnidaria (sea anemones, jellyfish, corals and hydra) form a close sister group to Bilateria. Within this clade, the sea anemone Nematostella vectensis has emerged as a slow evolving model for investigating characteristics of the cnidarian-bilaterian common ancestor, which diverged near the Cambrian explosion. Here, using long read sequencing and high throughput chromosome conformation capture, we generate high quality chromosome-level genome assemblies for N. vectensis and the closely related edwardsiid sea anemone, Scolanthus callimorphus. In both cases we find a robust set of 15 chromosomes comprising a stable linkage group detectable within all major clades of sequenced cnidarian genomes. Further, both genomes show remarkable chromosomal conservation with chordates. In contrast with Bilateria, we report that extended Hox and NK gene clusters are chromosomally linked but do not retain a tight spatial conservation. Accordingly, there is a lack of evidence for topologically associated domains, which have been implicated in the evolutionary pressure to retain tight microsyntenic gene clusters. We also uncover ultra-conserved noncoding elements at levels previously undetected in non-chordate lineages. Both genomes are accessible through an actively updated genome browser and database at https://simrbase.stowers.org


title: Antigenic Sensitivity of Membrane-Proximal Targeting Chimeric Antigen Receptors can be Fine-Tuned through Hinge Truncation

abstract:
BackgroundChimeric antigen receptor (CAR) technology has revolutionized the treatment of B-cell malignancies and steady progress is being made towards CAR-immunotherapies for solid tumours. Epidermal growth factor family receptors EGFR or HER2 are commonly overexpressed in cancer and represent proven targets for CAR-T therapy; given their expression in healthy tissues it is imperative that any targeting strategy consider the potential for on-target off-tumour toxicity.

MethodsHerein, we utilize high-throughput CAR screening to identify novel camelid single-domain antibody CARs (sdCARs) with high EGFR-specific CAR-T response. To optimize antigenic sensitivity of this EGFR-sdCAR, we performed progressive N-terminal truncation of the human CD8 hinge domain used as a spacer in many CAR constructs. Hinge truncation resulted in decreased CAR sensitivity to EGFR and improved selectivity for EGFR-overexpressing cells over EGFR-low target cells or healthy donor derived EGFR-positive fibroblasts. To investigate the molecular mechanism of hinge truncation, we test hinge-truncated scFv-based CARs targeting membrane proximal or membrane distal domains of EGFR-family proteins, HER2 and EGFRvIII. Finally, we proceed to test hinge variant EGFR-sdCAR functionality through in vitro and in vivo assessments in primary T cells derived from multiple donors.

ResultsFor CARs targeting membrane-proximal epitopes, hinge truncation by even a single amino acid provided fine control of the antigenic sensitivity, whereas CARs targeting membrane distal domains were not sensitive to even complete hinge domain removal. Hinge-modified EGFR-sdCARs showed consistent and predictable responses in Jurkat-CAR cells and primary human CAR-T cells in vitro and in vivo.

ConclusionsOverall, these results indicate that membrane-proximal epitope targeting CARs can be modified through hinge length tuning for programmable antigenic sensitivity and improved tumour selectivity.

O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/360925v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1f0db64org.highwire.dtl.DTLVardef@1d3ac43org.highwire.dtl.DTLVardef@1d270faorg.highwire.dtl.DTLVardef@f7614f_HPS_FORMAT_FIGEXP  M_FIG C_FIG O_LISingle amino acid truncations of CD8-hinge domain provide precise control of CAR antigen sensitivity
C_LIO_LITruncated hinge CARs show enhanced selectivity for antigen overexpressing tumour cells and decreased activity towards healthy antigen-expressing cells
C_LIO_LIEpitope location is a critical factor in determining hinge sensitivity for a CAR
C_LIO_LIHinge tuning can modulate CAR-T antigenic sensivity in vitro and in vivo
C_LI