title: Accurate self-assessment in 2D: protein contact map quality estimation by deep evolutionary reconciliation

abstract:
MotivationProtein contact maps have proven to be a valuable tool in the deep learning revolution of protein structure prediction, ushering in the recent breakthrough by AlphaFold2. However, self-assessment of the quality of predicted structures is typically performed at the granularity of 3D coordinates as opposed to directly exploiting the rotation- and translation-invariant 2D contact maps.

ResultsHere we present rrQNet, a deep learning method for self-assessment in 2D by contact map quality estimation. Our approach is based on the intuition that for a contact map to be of high quality, the residue pairs predicted to be in contact should be mutually consistent with the evolutionary context of the protein. The deep neural network architecture of rrQNet implements this intuition by cascading two deep modules--one encoding the evolutionary context and the other performing evolutionary reconciliation. The penultimate stage of rrQNet estimates the quality scores at the interacting residue-pair level, which are then aggregated for estimating the quality of a contact map. This design choice offers versatility at varied resolutions from individual residue pairs to full-fledged contact maps. Trained on multiple complementary sources of contact predictors, rrQNet facilitates generalizability across various contact maps. By rigorously testing using publicly available datasets and comparing against several in-house baseline approaches, we show that rrQNet accurately reproduces the true quality score of a predicted contact map and successfully distinguishes between accurate and inaccurate contact maps predicted by a wide variety of contact predictors.

AvailabilityThe open-source rrQNet software package is freely available at https://github.com/Bhattacharya-Lab/rrQNet.

Contactdbhattacharya@vt.edu


title: Uncovering hidden assembly artifacts: when unitigs are not safe and bidirected graphs are not helpful

abstract: Recent assemblies by the T2T and VGP consortia have achieved significant accuracy but required a tremendous amount of effort and resources. More typical assembly efforts, on the other hand, still suffer both from mis-assemblies (joining sequences that should not be adjacent) and from under-assemblies (not joining sequences that should be adjacent). To better understand the common algorithm-driven causes of these limitations, we investigated the unitig algorithm, which is a core algorithm at the heart of most assemblers. We prove that, contrary to popular belief, even when there are no sequencing errors, unitigs are not always safe (i.e. they are not guaranteed to be substrings of the sequenced genome). We also prove that the unitigs of a bidirected de Bruijn graph are different from those of a doubled de Bruijn graph and, contrary to our expectations, result in under-assembly. Using experimental simulations, we then confirm that these two artifacts exist not only in theory but also in the output of widely used assemblers. In particular, when coverage is low then even error-free data results in unsafe unitigs; also, unitigs may unnecessarily split palindromes in half if special care is not taken. To the best of our knowledge, this paper is the first to theoretically predict the existence of these assembler artifacts and confirm and measure the extent of their occurrence in practice.

category: bioinformatics


title: Computational modeling of the evolutionary transition from C3 to C4 photosynthesis

abstract:
C4 photosynthesis is an evolutionary adaptation that minimizes the adverse effects of the high photorespiration rate. Although it is widely accepted that the C4 plants are evolved from C3 ancestors, the knowledge about the details of this process is yet to be complete. One application of constraint-based metabolic network modeling is to simulate evolutionary trajectories that an organism endures under a certain selective pressure. However, this approach is barely used to predict the evolution of a complex trait in eukaryotes. Here, we utilized a genetic algorithm combined with a constraint-based metabolic network model of Arabidopsis thaliana to simulate the trajectories of C3 to C4 conversion under high photorespiration rate conditions combined with different environmental conditions. Our modeling predicted that the C3-C4 intermediates and C4 strategies are superior to C3 photosynthesis in these environmental conditions. Besides, resource scarcities drive different evolutionary trajectories toward the emergence of C4 photosynthesis.

AUTHOR SUMMARYIt is estimated that high photorespiration reduces C3 crops productivity up to 50% [1]. Carbon concentrating mechanisms have evolved in many plants to overcome adverse effects of high photorespiration rate in especial environments. C4 photosynthesis is one of these adaptations that separate carbon assimilation reactions spatially between mesophyll and bundle sheath cells of the leaves. It is reported that C4 photosynthesis is evolved more than 65 times in 19 different families of angiosperms independently from C3 ancestors. Therefore, it is hypothesized that the potential modules, at the level of metabolism, anatomy, and gene regulation, which are needed to perform C4 photosynthesis are present in C3 plants. Since C4 plants grow better than C3 plants in hot and dry environments, scientists are working on converting C3 crops such as rice to C4 plants. However, several challenges, including a lack of understanding of the evolutionary paths toward manifestation of C4 trait, have hampered the C3-to-C4 engineering ([2]). Here, we used a metabolic network model of Arabidopsis thaliana as a C3 model plant and attempted to understand the possible evolutionary events leading to emerging of C4 photosynthesis in various scenarios and examined the results.


title: Elucidating Collective Translocation of Nanoparticles Across the Skin Lipid Barrier: A Molecular Dynamics Study

abstract: The top layer of skin, the stratum corneum, provides a formidable barrier to the skin. Nanoparticles are utilized and further explored for personal and health care applications related to the skin. In past years several researchers have studied the translocation and permeation of nanoparticles of various shapes, sizes, and surface chemistry through the cell membranes. Most of these studies focused on a single nanoparticle and a simple bilayer system, whereas skin has a highly complex lipid membrane architecture. Moreover, it is highly unlikely that a nanoparticle formulation applied on the skin will not have multiple nanoparticle-nanoparticle and skin-nanoparticle interactions. In this study, we have utilized coarse-grained MARTINI molecular dynamics simulations to assess the interactions of two types (bare and dodecane-thiol coated) of nanoparticles with two models (single bilayer and double bilayer) of skin lipid membranes. The nanoparticles were found to be partitioned from the water layer to the lipid membrane as an individual entity as well as in the cluster form. It was discovered that each nanoparticle reached the interior of both single bilayer and double bilayer membrane irrespective of nanoparticle type and concentration, though coated particles were observed to efficiently traverse across bilayer when compared with bare particles. The coated nanoparticles also created a single large cluster inside the membrane, whereas bare nanoparticles were found in small clusters. Both the nanoparticles exhibited preferential interactions with cholesterol molecules present in the lipid membrane as compared to other lipid components of the membrane. We have also observed that the single membrane model exhibited unrealistic instability at moderate to the higher concentration of nanoparticles, and hence for translocation study, at minimum double bilayer model should be employed.

category: biophysics


title: Cytocapsular tube network-tumor system is an integrated physical target for the highly effective and efficient pharmacotherapy of solid cancers

abstract: Cancer is a leading cause of human lethality and cancer drug pan-resistant tumor metastasis (cdp-rtm) is a major source of cancer death. The integrated cytocapsular tube (CCT) networktumor system (CNTS) is essential for cancer evolution procedures including cancer cell proliferation, migration and dissemination in CCT networks, new tumor growth in CCT terminals, conventional cancer drug pan-resistance, and tumor relapse. The preclinical screening experimentations with CCTs and networks are necessary prerequisites for the discovery and development of cancer drug candidates for efficient clinical trials, and effective and precise clinical cancer pharmacotherapy. However, it is unknown whether the popularly employed conventional mouse experimentations for preclinical therapeutic development generate CCTs and networks. Here, we comprehensively investigated the cancer cell line derived xenografts (CDX) of 8 kinds of cancer cell lines, and patient cancer cells derived-xenografts (PDX) with 16 kinds of clinical primary and metastatic malignant tumor cells in multiple cancer stages by immunohistochemistry staining assays with CCT marker protein antibodies of anti-CM-01 antibodies. We found that there are no CCTs or CCT networks in all these examined 16 CDX and 22 PDX transplanted tumors. Our data evidenced that the conventional transplantation tumors for preclinical therapeutic development do not engender CCTs or CNTS, which is consistent with and provides an explanation of the poor clinical outcomes of the marketed cancer drugs. This study demonstrated that CCT network-tumor system (CNTS) is an integrated physical target for pharmacotherapy, and that preclinical experimentations engendering CNTS (such as CCT xenograft, CCTX) should be employed for the discovery and development of highly effective and efficient cancer drugs aimed for cure of solid cancers.

category: cancer biology


title: Genetic variation of human myokine signaling is dominated by biologic sex and sex hormones

abstract: Abstract/IntroductionProteins secreted from skeletal muscle, termed myokines, allow muscle to impact systemic physiology and disease. Myokines play critical roles in a variety of processes, including metabolic homeostasis, exercise improvements, inflammation, cancer and cognitive functions1-6. Despite the clear relevance of these factors in mediating a multitude of physiological outcomes, the genetic architecture, regulation and functions of myokines, as well as degree of conservation of these communication circuits remains inadequately understood. Given that biologic sex controls critical aspects of nearly every physiologic outcome, it is essential to consider when relating specific mechanisms to complex genetic and metabolic interactions. Specifically, many metabolic traits impacted by myokines show striking sex differences arising from hormonal7-10, genetic7,11 or gene-by-sex interactions12,13. In this study, we performed a genetic survey of myokine gene regulation and cross-tissue signaling in humans where sex as a biological variable was emphasized. While expression levels of a majority of myokines and cell proportions within skeletal muscle showed little differences between males and females, nearly all significant cross-tissue enrichments operated in a sex-specific or hormone-dependent fashion; in particular, with estrogens. These sex- and hormone-specific effects were consistent across key metabolic tissues: liver, pancreas, hypothalamus, intestine, heart, visceral and subcutaneous adipose tissue. Skeletal muscle estrogen receptor enrichments across metabolic tissues appeared stronger than androgen receptor and, surprisingly, ~3-fold higher in males compared to females. To define the causal roles of estrogen signaling on myokine gene expression and functions, we generated male and female mice which lack estrogen receptor  (Esr1) specifically in skeletal muscle and integrated global RNA-Sequencing with human data. These analyses highlighted mechanisms of sex-dependent myokine signaling conserved between species, such as myostatin enriched for divergent substrate utilization pathways between sexes. Several other sex-dependent mechanisms of myokine signaling were uncovered, such as muscle-derived TNF exerting stronger inflammatory signaling in females compared to males and GPX3 as a male-specific link between glycolytic fiber abundance and hepatic inflammation. Collectively, we provide the first genetic survey of human myokines and highlight sex and estrogen receptor signaling as critical variables when assaying myokine functions and how changes in cell composition impact other metabolic organs.

category: physiology


title: Development and validation of Arc nanobodies: new tools for probing Arc dynamics and function

abstract: Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N-and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc and analysis of capsid domain specific functions.

category: neuroscience


title: Tonic GluD1 channel current is independent of G protein activity in the dorsal raphe nucleus

abstract: AO_SCPLOWBSTRACTC_SCPLOWPreviously, using electrophysiological recordings from adult male and female mouse brain slices containing the dorsal raphe nucleus, we showed that GluD1R channels carry ionic current and are modulated via activation of Gq-coupled 1-adrenergic receptors (1-AR) in a GTP-dependent manner (Gantz et al., 2020). GluD1R channels also carry a tonic cation current, generally ~-20 pA at subthreshold membrane potentials (Gantz et al., 2020). The origin of tonic GluD1R channel current is unknown. Here, using the same preparation, we show there is no role of on-going G protein-coupled receptor activity in generating or sustaining tonic GluD1R channel current. Neither augmentation nor disruption of G protein activity had an effect on tonic GluD1R current. These results reveal that tonic GluD1R current arises from a mechanism separate from on-going activity of G protein-coupled receptors. Under current clamp, block of GluD1R channels hyperpolarized the membrane by ~10 mV at subthreshold potentials leading to reduced excitability. Thus, GluD1R channels carry a G protein-independent tonic current that contributes to subthreshold drive of action potential firing in the dorsal raphe nucleus.


title: ECS1 and ECS2 regulate polyspermy and suppress the formation of haploid plants by promoting double fertilization

abstract: In flowering plants, the number of pollen tubes that provide sperm cells to the female gametes is restricted by a pollen tube block. This safeguard mechanism is only activated after successful fertilization of both female gametes and involves the disintegration of pollen tube attracting synergid cells. Yu and coworkers previously reported that the endopeptidase ECS1 and ECS2, which are secreted by fertilized egg cells, prevent the attraction of supernumerary pollen tubes by cleaving the pollen tube attractant LURE11. Here we report on an earlier defect in ecs1 ecs2 mutants that is manifested by single rather than double fertilization of either egg or central cell. The defect is accompanied by a delay in synergid disintegration providing an alternative explanation for the extra pollen tubes observed in the double mutant. These results are corroborated by our finding that ecs1 ecs2 plants segregate both, haploid as well as polyspermy derived triploid plants.

category: plant biology


title: Core Transcription Programs Controlling Injury-Induced Neurodegeneration of Retinal Ganglion Cells

abstract: Neurodegenerative diseases are characterized by neuronal death and regenerative failure. However, gene regulatory programs governing how initial neuronal injuries lead to neuronal death remain poorly understood. In adult mice, optic nerve crush (ONC) injury, which severs all axons of retinal ganglion cells (RGCs), results in massive death of axotomized RGCs and regenerative failure of survivors. We performed an in vivo CRISPR/Cas9-based genome-wide screen of 1893 transcription factors (TFs) to seek repressors of RGC survival and axon regeneration following ONC. In parallel, we profiled the epigenetic and transcriptional landscapes of injured RGCs by ATAC-seq and RNA-seq to identify critical injury responsive TFs and their targets. Remarkably, these independent analyses converged on a set of four ATF/CEBP transcription factors - ATF3, ATF4, C/EBP{gamma} and CHOP (Ddit3) - as critical regulators of survival. Further studies indicate that these TFs contribute to two pro-death transcriptional programs: ATF3/CHOP preferentially regulate pathways activated by cytokines and innate immunity, whereas ATF4/C/EBP{gamma} regulate pathways engaged by intrinsic neuronal stressors. Manipulation of these TFs also protects RGCs in an experimental model of glaucoma, a prevalent disease in which RGCs die. Together, our results reveal core transcription programs that transform an initial axonal insult into a degenerative result and suggest novel strategies for treating neurodegenerative diseases.


title: Evolution of Daphnia population dynamics following invasion by a non-native predator

abstract:
O_LIPredators are frequently observed to cause evolutionary responses in prey phenotypes, which may, in turn, translate into evolutionary shifts in prey population dynamics. Although a link between predation and population growth has been demonstrated in experimental evolution studies, insights from natural populations are lacking.
C_LIO_LIHere we tested for evolutionary changes in the population dynamics of the herbivorous water flea Daphnia pulicaria in response to the invasion of the predatory spiny water flea (Bythotrephes longimanus) in the Great Lakes region, USA. Using a resurrection ecological approach and a 3-month population growth experiment (in the absence of predation) we compared population dynamics in daphnia from pre- and post-invasion time periods.
C_LIO_LIPost-invasion daphnia were able to maintain an overall higher population abundance throughout the growth experiment, both in terms of the number of individuals (28% higher) and total population biomass (33% higher). Estimation of population dynamics parameters from a theta-logistic model suggested that this was achieved through an increase in intrinsic population growth rate as well as increased carrying capacity.
C_LIO_LIThe observed difference in intrinsic rate of increase could not be predicted based on previous measurements of life-history traits in these clones. This indicates that care should be taken when extrapolating from a few life history traits measured in isolated individuals under controlled conditions to population dynamics.
C_LIO_LIWhereas previous experimental evolution studies of predator-prey interactions have demonstrated that genotypes that have evolved under predation have inferior population growth when the predator is absent, this was not the case for the Daphnia. We suggest that complexities in ecological interactions of natural ecosystems, such as the potential for spatial and temporal avoidance of predation, makes it challenging to provide general predictions about evolutionary responses in population dynamics to predators.
C_LI

category: evolutionary biology


title: Additive genetic effects in interacting species jointly determine the outcome of caterpillar herbivory

abstract:
Plant-insect interactions are common and important in basic and applied biology. Trait and genetic variation can affect the outcome and evolution of these interactions, but the relative contributions of plant and insect genetic variation and how these interact remain unclear and are rarely subject to assessment in the same experimental context. Here we address this knowledge gap using a recent host range expansion onto alfalfa by the Melissa blue butterfly. Common garden rearing experiments and genomic data show that caterpillar performance depends on plant and insect genetic variation, with insect genetics contributing to performance earlier in development and plant genetics later. Our models of performance based on caterpillar genetics retained predictive power when applied to a second common garden. Much of the plant genetic effect could be explained by heritable variation in plant phytochemicals, especially saponins, peptides, and phosphatidyl cholines, providing a mechanistic understanding of variation in the species interaction. We find evidence of polygenic, mostly additive effects within and between species, with consistent effects of plant genotype on growth and development across multiple butterfly species. Our results inform theories of plant-insect coevolution and the evolution of diet breadth in herbivorous insects and other host-specific parasites.

Teaser summaryThe combined, additive effects of plant and insect genetic variation explain Melissa blue caterpillar growth and development on alfalfa plants.

category: genomics


title: Poison frog dietary preference depends on prey type and alkaloid load

abstract: The ability to acquire chemical defenses through the diet has evolved across several major taxa, including insects, mollusks, birds and reptiles. Chemically defended organisms may need to balance chemical defense acquisition and nutritional quality of prey items. However, these dietary preferences and potential trade-offs are rarely considered in the framework of diet-derived defenses. Poison frogs (Family Dendrobatidae) acquire defensive alkaloids from their arthropod diet of ants and mites, although their dietary preferences have never been investigated. We conducted prey preference assays with the Dyeing Poison frog (Dendrobates tinctorius) to test the hypothesis that alkaloid load and prey traits would influence dietary preferences. We tested size preferences (big vs. small) within each of four prey groups (ants, beetles, flies, and larvae) and found that frogs generally preferred interacting with smaller prey items. Frog taxonomic prey preferences were also tested as we experimentally increased their chemical defense load by feeding frogs decahydroquinoline, an alkaloid compound similar to those naturally found in their diet. Contrary to our expectations, overall preferences did not change during alkaloid consumption, as frogs across groups preferred larvae over other prey. Finally, we assessed the protein and lipid content of prey items and found that small ants have the highest lipid content while large larvae have the highest protein content. Our results suggest that consideration of toxicity and prey nutritional value are important factors in understanding the evolution of acquired chemical defenses and niche partitioning as a whole.

category: animal behavior and cognition


title: Interaction of surface topography and taper mismatch on head-stem modular junction contact mechanics during assembly in modern total hip replacement

abstract: Implant failure due to fretting corrosion at the head-stem modular junction is an increasing problem in modular total hip arthroplasty. The effect of varying microgroove topography on modular junction contact mechanics has not been well characterized. The aim of this work was to employ a novel, microgrooved finite element (FEA) model of the hip taper interface and assess the role of microgroove geometry and taper mismatch angle on the modular junction mechanics during assembly. A two-dimensional, axisymmetric FEA model was created using a modern 12/14 taper design of a CoCrMo femoral head taper and Ti6Al4V stem taper. Microgrooves were modelled at the contacting interface of the tapers and varied based on height and spacing measurements obtained from a repository of measured retrievals. Additionally, taper angular mismatch between the head and stem was varied to simulate proximal- and distal-locked engagement. Forty simulations were conducted to parametrically evaluate the effects of microgroove surface topography and angular mismatch on predicted contact area, contact pressure, and equivalent plastic strain. Multiple linear regression analysis was highly significant (p < 0.001; R2 > 0.74) for all outcome variables. The regression analysis identified microgroove geometry on the head taper to have the greatest influence on modular junction contact mechanics. Additionally, there was a significant second order relationship between both peak contact pressure (p < 0.001) and plastic strain (p < 0.001) with taper mismatch angle. These modeling techniques will be used to identify the implant parameters that maximize taper interference strength via large in-silico parametric studies.

category: bioengineering


title: Comparison of Two Individual Identification Algorithms for Snow Leopards (Panthera uncia) after Automated Detection

abstract: 1. Photo-identification of individual snow leopards (Panthera uncia) is the primary technique for density estimation for the species. A high volume of images from multiple projects, combined with pre-existing historical catalogs, has made identifying snow leopard individuals within the images cost- and time-intensive. 2. To speed the classification among a high volume of photographs, we trained and evaluated image classification methods for PIE v2 (a triplet loss network), and we compared PIEs accuracy to the HotSpotter algorithm (a SIFT based algorithm). Analyzed data were collected from a curated catalog of free-ranging snow leopards photographed across years (2012-2019) in Afghanistan and from samples in captivity provided by zoos from Finland, Sweden, Germany, and the United States. 3. Results show that PIE outperforms HotSpotter. We also found weaknesses in the initial PIE model, like a minor amount of background matching, which was addressed, although likely not fully resolved, by applying background subtraction (BGS) and left-right mirroring (LR) methods. The PIE BGS LR model combined with Hotspotter showed a Rank-1: 85%, Rank-5: 95%, Rank-20: 99%. 4. Overall, our results recommend implementing PIE v2 simultaneously with HotSpotter on Whiskerbook.org.


title: Image-centric compression of protein structures improves space savings

abstract:
MotivationBecause of the rapid generation of data, the study of compression algorithms to reduce storage and transmission costs is important to bioinformaticians. Much of the focus has been on sequence data, including both genomes and protein amino acid sequences stored in FASTA files. However, there are few specialized compressors for structural protein data contained in PDB and mmCIF files. Current standard practice is to use an ordinary lossless compressors such as gZip on a sequential list of atomic coordinates, but this approach expends bits on saving an arbitrary ordering of atoms, and it also prevents reordering the atoms for compressibility. In this article, we demonstrate for the first time that image-based compression can significantly improve compression ratios. To this end, we implement a prototype compressor  PIC, specialized for point clouds of atom coordinates contained in PDB and mmCIF files. PIC maps the 3D data to a 2D 8-bit greyscale image and leverages the well developed PNG image compressor to minimize the size of the resulting image, forming the compressed file.

ResultsPIC outperforms gZip in terms of compression ratio on proteins over 20,000 atoms in size, with a savings over gZip of up to 37.4% on the proteins compressed. In addition, PICs compression ratio increases with protein size.

AvailabilityPrototype Python source code is available for download at https://github.com/lukestaniscia/PIC.

Contactluke.staniscia@mail.utoronto.ca, ywyu@math.toronto.edu


title: Triplet kinase-phosphatase targeting to overcome kinase inhibitor tolerance in brain tumor cells

abstract: Kinase inhibitor tolerance of human glioblastoma is an unmet clinical challenge and a mechanistic enigma. Here, we demonstrate that glioblastoma cell tolerance to multi-kinase inhibition can be reverted by reactivation of Protein Phosphatase 2A (PP2A). To characterize kinase targets of clinical stage multi-kinase inhibitor UCN-01 synergizing with PP2A reactivation, we established a strategy, named Actionable Targets of Multi-kinase Inhibitors (AToMI). AToMI revealed AKT and mitochondrial pyruvate dehydrogenase kinases (PDK1-4) as the co-targets for UCN-01-elicited synthetic lethality with PP2A reactivation. Notably, heterogeneous glioblastoma and medulloblastoma models were tolerant to AKT and PDK1-4 inhibitor monotherapies, and their combinations, but were effectively inhibited by triplet therapy including pharmacological PP2A reactivation. Mechanistically, overcoming the kinase therapy tolerance by the triplet therapy could be explained by combinatorial effects on signaling rewiring between AKT and PDK1-4, decrease in mitochondrial oxidative phosphorylation, and BH3-only protein mediated apoptosis priming. The brain-penetrant triplet combination had a significant in vivo efficacy in intracranial glioblastoma and medulloblastoma models. Collectively, we present a generalizable approach to identify actionable co-targets of multi-kinase inhibitors and demonstrate that overcoming of the kinase inhibitor tolerance in brain tumor cells requires triplet targeting of AKT, PDK1-4, and PP2A.


title: A new demethylase gene OsDML4 involved in high temperature induced floury endosperm formation in rice

abstract: High temperature (HT) can affect the accumulation of seed storage materials and cause adverse effects on the yield and quality in rice. DNA methylation plays an important role in plant growth and development. However, the temperature and DNA methylation interaction on rice seed development has not been studied yet. Here, we identified a new demethylase gene OsDML4 and discovered its function on cytosine demethylation to affect the endosperm formation during the grain filling. Knockout of OsDML4 induced floury endosperm only under HT, which resulted from dramatically reduced the transcription and accumulation of glutelins and 16-kDa prolamin. The expression of two important transcription factors RISBZ1 and RPBF was significantly declined in the osdml4 mutants. The absence of OsDML4 also caused adverse effects on the formation of protein bodies (PBs), the number of PB-II was greatly decreased and incomplete PB-II with empty space and abnormally shaped PB-II were observed in the osdml4 mutants. Whole-genome bisulfite sequencing analysis of seeds at 15 days after pollination revealed much higher global methylation levels of CG, CHG and CHH contexts in the osdml4 mutants compared to wild type (WT). Moreover, the methylation status of RISBZ1 promoter was hypermethylated but RPBF promoter was nearly unchanged. No significant difference was detected between WT and the osdml4 mutants under room temperature. In conclusion, our study demonstrates a novel OsDML4-mediated epigenetic regulatory mechanism involving in the formation of floury endosperm, which will provide a new perspective in regulating endosperm development and the accumulation of SSPs in rice.


title: Comprehensive Evaluation of ACE2-Fc Combination with Neutralization Antibody on Broad Protection against SARS-CoV-2 and Its Variants

abstract: Emerging SARS-CoV-2 variants are threatening the efficacy of antibody therapies. Combination treatments including ACE2-Fc have been developed to overcome the evasion of neutralizing antibodies (NAbs) in individual cases. Here we conducted a comprehensive evaluation of this strategy by combining ACE2-Fc with NAbs of diverse epitopes on the RBD. NAb+ACE2-Fc combinations efficiently neutralized HIV-based pseudovirus carrying the spike protein of the Delta or Omicron variants, achieving a balance between efficacy and breadth. In an antibody escape assay using replication-competent VSV-SARS-CoV-2-S, all the combinations had no escape after fifteen passages. By comparison, all the NAbs without combo with ACE2-Fc had escaped within six passages. Further, the VSV-S variants escaped from NAbs were neutralized by ACE2-Fc, revealing the mechanism of NAb+ACE2-Fc combinations survived after fifteen passages. We finally examined ACE2-Fc neutralization against pseudovirus variants that were resistant to the therapeutic antibodies currently in clinic. Our results suggest ACE2-Fc is a universal combination partner to combat SARS-CoV-2 variants including Delta and Omicron.

category: cell biology


title: In Silico Analysis Of The Effects Of Omicron Spike Amino Acid Changes On The Interactions With Human ACE2 Receptor And Structurally Characterized Complexes With Human Antibodies

abstract:
The new SARS-CoV-2 variant Omicron is characterised, among others, by more than 30 amino acid changes (including 4 deletions and 1 insertion) occurring on the spike glycoprotein.

We report a comprehensive analysis of the effects of the Omicron spike amino acid changes in the interaction with human ACE2 receptor or with human antibodies, obtained by analysing the publicly available resolved 3D structures. Our analysis predicts that amino acid changes occurring on amino acids interacting with the ACE2 receptor may increase Omicron transmissibility. The interactions of Omicron spike with human antibodies can be both negatively and positively affected by amino acid changes, with a predicted total loss of interactions only in few complexes. We believe that such an approach can be used to better understand SARS-CoV-2 transmissibility, detectability, and epidemiology, especially when extended to other than spike proteins.


title: Rapid prototyping of metabolites detection by bacterial biosensors in human fecal samples.

abstract: Gut metabolites are pivotal mediators of host-microbiome interactions and provide an important window on human physiology and disease. However, current methods to monitor gut metabolites rely on heavy and expensive technologies such as liquid chromatography-mass spectrometry (LCMS). In that context, robust, fast, field-deployable, and cost-effective strategies for monitoring fecal metabolites would support large-scale functional studies and routine monitoring of metabolites biomarkers associated with pathological conditions. Living cells are an attractive option to engineer biosensors due to their ability to detect and process many environmental signals and their self-replicating nature. Here we optimized a protocol for feces processing and gut metabolites detection using bacterial biosensors (bactosensors), enabling rapid evaluation of their operational capacity in these samples. We show that a simple filtration step is enough to remove host microbes and reproducibly obtain a physiological-derived media retaining important characteristics of human feces, such as matrix effects and endogenous metabolites. We measured how fecal samples affect the performance of biosensors for benzoate, lactate, anhydrotetracycline, and bile acids, and found that bactosensors are highly sensitive to fecal matrices. Sensitivity to the matrix is biosensor-dependent but also varies between individuals, highlighting the need for case-by-case optimization for bactosensors operation in feces. Finally, by detecting endogenous bile acids, we demonstrate that bactosensor can be used for metabolites monitoring in feces. This work lays the foundation for the optimization and use of bacterial biosensors for fecal metabolites monitoring. In the future, our method could also allow rapid pre-prototyping of engineered bacteria designed to operate in the gut, with applications to in situ diagnostics and therapeutics.

category: synthetic biology


title: Ex vivo and in vivo evidence that cigarette smoke-exposed T regulatory cells impair host immunity against Mycobacterium tuberculosis

abstract: A strong epidemiologic link exists between exposure to cigarette smoke (CS) and increased susceptibility to tuberculosis (TB). In vitro macrophage and in vivo murine studies showed that CS and nicotine impair host-protective immune cells against Mycobacterium tuberculosis (MTB) infection. However, little is known about how CS may affect immunosuppressive cells in the context of MTB infection. Thus, we investigated whether CS-exposed T regulatory cells (Tregs) could exacerbate MTB infection in co-culture with human macrophages and in the adoptive transfer of Tregs from air- and CS-exposed mice. We found that exposure of primary human Tregs to CS extract impaired the ability of human monocyte-derived macrophages to control an MTB infection by inhibiting phagosome-lysosome fusion and autophagosome formation. Neutralization of CTLA-4 on the CS extract-exposed Tregs abrogated the impaired control of MTB infection in macrophage and Treg co-cultures. In Foxp3+GFP+DTR+ (Thy1.2) mice depleted of endogenous Tregs, adoptive transfer of Tregs from donor CS-exposed B6.PL(Thy1.1) mice with subsequent MTB infection of the recipient Thy1.2 mice resulted in a greater burden of MTB in the lungs and spleens than those that received Tregs from airexposed mice. Mice that received Tregs from CS-exposed mice and then infected with MTB had modest but significantly reduced numbers of interleukin-12-positive dendritic cells and interferon-gamma-positive CD4+ T cells in the lungs and increased number of programmed cell death protein-1 positive CD4+ T cells in both the lungs and spleens.

category: immunology


title: Broadly-recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles

abstract: Sequence homology between SARS-CoV-2 and common-cold human coronaviruses (HCoVs) raises the possibility that memory responses to prior HCoV infection can impact the T cell response in COVID-19. We studied T cells recognizing SARS-CoV-2 and HCoVs in convalescent COVID-19 donors, and identified a highly conserved SARS-CoV-2 sequence S811-831, with two overlapping epitopes presented by common MHC-II proteins HLA-DQ5 and HLA-DP4. These epitopes were recognized by CD4+ T cells from convalescent COVID-19 donors, mRNA vaccine recipients, and by low-abundance CD4+ T cells in uninfected donors. TCR sequencing revealed a diverse repertoire with public TCRs. CD4+ T cell cross-reactivity was driven by the remarkably strong conservation of T cell contact residues in both HLA-DQ5 and HLA-DP4 binding frames, with distinct patterns of HCoV cross-reactivity explained by MHC-II binding preferences and substitutions at secondary TCR contact sites. These data highlight S811-831 as a highly-conserved CD4+ T cell epitope broadly recognized across human populations.


title: Metal cofactor stabilization by a partner protein is a widespread strategy employed for amidase activation

abstract:
Construction and remodeling of the bacterial peptidoglycan (PG) cell wall must be carefully coordinated with cell growth and division. Central to cell wall construction are hydrolases that cleave bonds in peptidoglycan. These enzymes also represent potential new antibiotic targets. One such hydrolase, the amidase LytH in Staphylococcus aureus, acts to remove stem peptides from PG, controlling where substrates are available for insertion of new PG strands and consequently regulating cell size. When it is absent, cells grow excessively large and have division defects. For activity, LytH requires a protein partner, ActH, that consists of an intracellular domain, a large rhomboid protease domain, and three extracellular tetratricopeptide repeats (TPRs). Here we demonstrate that the amidase-activating function of ActH is entirely contained in its extracellular TPRs. We show that ActH binding stabilizes metals in the LytH active site, and that LytH metal binding in turn is needed for stable complexation with ActH. We further present a structure of a complex of the extracellular domains of LytH and ActH. Our findings suggest that metal cofactor stabilization is a general strategy used by amidase activators and that ActH houses multiple functions within a single protein.

SIGNIFICANCE STATEMENTThe Gram-positive pathogen Staphylococcus aureus is a leading cause of antibiotic resistance-associated death in the United States. Many antibiotics used to treat S. aureus, including the beta-lactams, target biogenesis of the essential peptidoglycan (PG) cell wall. Some hydrolases play important roles in cell wall construction and are potential antibiotic targets. The amidase LytH, which requires a protein partner, ActH, for activity, is one such hydrolase. Here, we uncover how the extracellular domain of ActH binds to LytH to stabilize metals in the active site for catalysis. This work advances our understanding of how hydrolase activity is controlled to contribute productively to cell wall synthesis.

category: microbiology


title: Neutralization of Omicron SARS-CoV-2 by 2 or 3 doses of BNT162b2 vaccine

abstract: We report the antibody neutralization against Omicron SARS-CoV-2 after 2 and 3 doses of BNT162b2 mRNA vaccine. Vaccinated individuals were serially tested for their neutralization against wild-type SARS-CoV-2 (strain USA-WA1/2020) and an engineered USA-WA1/2020 bearing the Omicron spike glycoprotein. Plaque reduction neutralization results showed that at 2 or 4 weeks post-dose-2, the neutralization geometric mean titers (GMTs) were 511 and 20 against the wild-type and Omicron-spike viruses, respectively, suggesting that two doses of BNT162b2 were not sufficient to elicit robust neutralization against Omicron; at 1 month post-dose-3, the neutralization GMTs increased to 1342 and 336, respectively, indicating that three doses of vaccine increased the magnitude and breadth of neutralization against Omicron; at 4 months post-dose-3, the neutralization GMTs decreased to 820 and 171, respectively, suggesting similar neutralization decay kinetics for both variants. The data support a three-dose vaccine strategy and provide the first glimpse of the neutralization durability against Omicron.


title: Molecular basis for allosteric agonism and G protein subtype selectivity of galanin receptors

abstract: Peptide hormones and neuropeptides are complex signaling molecules that predominately function through G protein-coupled receptors (GPCRs). Two fundamental questions remained in the field of peptide-GPCR signaling systems are the basis for the diverse binding mode of peptide ligands and the specificity of G protein coupling. Here we report the structures of a neuropeptide, galanin, bound to its receptors, GAL1R and GAL2R, in complex with their primary G protein subtypes Gi and Gq, respectively. The structures reveal a unique binding pose of galanin, which almost  lay flat on the top of the receptor transmembrane domain pocket in an -helical conformation, and acts as an  allosteric-like agonist via a distinct signal transduction cascade. The structures also uncover the important features of intracellular loop 2 (ICL2) that mediate specific interactions with Gq, thus determining the selective coupling of Gq to GAL2R. ICL2 replacement in Gi-coupled GAL1R, OR, 5-HT1AR, and Gs-coupled b2AR and D1R with that of GAL2R promotes Gq coupling of these receptors, highlighting the dominant roles of ICL2 in Gq selectivity. Together our results provide important insights into peptide ligand recognition and allosteric activation of galanin receptors and uncover a general structural element for Gq coupling selectivity.

category: molecular biology


title: GLH/VASA helicases promote germ granule formation to ensure the fidelity of piRNA-mediated transcriptome surveillance

abstract: The ability to distinguish non-self from self is the key characteristic for any defense system. piRNAs function as guardians of the genome by silencing non- self nucleic acids and transposable elements in animals. Many piRNA factors are enriched in perinuclear germ granules, but whether their localization is required for piRNA biogenesis or function is not known. Here we show that GLH/VASA helicase mutants exhibit defects in forming perinuclear condensates containing PIWI and other small RNA cofactors. These mutant animals produce largely normal levels of piRNA but are defective in triggering piRNA silencing. Strikingly, while many piRNA targets are activated in GLH mutants, we observed that hundreds of endogenous genes are aberrantly silenced by piRNAs. This defect in self versus non-self recognition was also observed in other mutants where perinuclear P granules are disrupted. Together, our results argue that perinuclear germ granules function critically to promote the fidelity of piRNA- based transcriptome surveillance in C. elegans and preserve self versus non-self distinction.


title: Structural basis for Parkinson's Disease-linked LRRK2's binding to microtubules

abstract: Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinsons Disease (PD). Under some circumstances, LRRK2 co-localizes with microtubules in cells, an association enhanced by PD mutations. We report a cryo-electron microscopy structure of the catalytic half of LRRK2, containing its kinase, which is in a closed conformation, and GTPase domains, bound to microtubules. We also report a structure of the catalytic half of LRRK1, which is closely related to LRRK2, but is not linked to PD. LRRK1s structure is similar to LRRK2, but LRRK1 does not interact with microtubules. Guided by these structures, we identify amino acids in LRRK2s GTPase domain that mediate microtubule binding; mutating them disrupts microtubule binding in vitro and in cells, without affecting LRRK2s kinase activity. Our results have implications for the design of therapeutic LRRK2 kinase inhibitors.


title: A Genome-wide CRISPR Screen Identifies WDFY3 as a Novel Regulator of Macrophage Efferocytosis

abstract:
AbstractsPhagocytic clearance of dying cells, termed efferocytosis, must occur efficiently to maintain homeostasis and prevent disease. Yet, our understanding of this important biological process remains incomplete. To search for novel regulators of efferocytosis, we performed a FACS-based genome-wide CRISPR knockout screen in primary murine macrophages. We identified a novel role for WDFY3 in efferocytosis by macrophages. WDFY3 deficiency in macrophages specifically impaired uptake, not binding, of apoptotic cells due to defective actin depolymerization. We further revealed that WDFY3 directly interacts with GABARAP, thus facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components. Although the C-terminus of WDFY3 was sufficient to rescue impaired degradation, full-length WDFY3 is still required for regulating uptake. Finally, WDFY3 is required for efficient efferocytosis in vivo in mice and in primary human macrophages. The work expands our knowledge of the mechanisms of macrophage efferocytosis, and more broadly, provides a general strategy for genome-wide CRISPR screen to interrogate complex functional phenotypes in primary macrophages.

HighlightsO_LIFunctional readout for pooled genome-wide CRISPR screen in primary macrophages.
C_LIO_LIWDFY3 is discovered as a regulator of macrophage efferocytosis in vitro and in vivo.
C_LIO_LIWDFY3 deficiency led to impaired uptake, as opposed to binding, of apoptotic cells due to defective actin depolymerization.
C_LIO_LIWDFY3 directly interacts with GABARAP, facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components.
C_LIO_LIC-terminal WDFY3 is sufficient to regulate the degradation of engulfed apoptotic cells while full-length WDFY is required for regulating uptake.
C_LI


title: ESCPE-1 Mediates Retrograde Endosomal Sorting of the SARS-CoV-2 Host Factor Neuropilin-1

abstract: Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed  cargoes) from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify novel retrograde cargo proteins of the Endosomal SNX-BAR Sorting Complex Promoting Exit-1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterised host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalised with the NRP1-interacting peptide of the SARS-CoV-2 Spike protein. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.


title: VCF2Prot: An Efficient and Parallel Tool for Generating Personalized Proteomes from VCF Files

abstract:
MotivationThe ability to generate sample-specific protein sequences is a crucial step in neo-antigen discovery, cancer vaccine development, and proteogenomics. The revolutionary increase in the throughput of sequencers has fueled large-scale genomic and transcriptomic studies, holding great promises for the emerging field of personalized medicine. However, most sequencing projects store their sequencing data in an abbreviated variant calling format (VCF) that is not immediately amenable to subsequent proteomic and peptidomic analyses. Furthermore, data processing of such increasingly massive genome-scale datasets calls for parallel and concurrent programming, and consequently refactoring of existing algorithms and/or the development of new parallel algorithms.

ResultsHere, we introduce sequence intermediate representation (SIR), a novel and generic algorithm for generating personalized or sample-specific protein sequences from a consequence-called VCF file and the corresponding reference proteome. An implementation of SIR, named VCF2Prot, was developed to aid personalized medicine and proteogenomics by generating personalized proteomes in FASTA format from a collection of consequence-called genomic alterations stored in a VCF file. Benchmarking VCF2Prot against the recently published PrecisionProDB showed an ~1000-fold improvement in runtime (depending on the input size). Furthermore, in a scale-up study VCF2Prot processed a VCF file containing 99,254 variants observed across 8,192 patients in ~ 11 minutes, demonstrating the massive improvement in the execution speed and the utility of SIR and VCF2prot in bridging large-scale genomic and proteomic studies.

Availability and ImplementationVCF2Prot comes with a permissive MIT-license, enabling the commercial and non-commercial utilization of the tool. The source code along with precompiled versions for Linux/Mac OS are available at https://github.com/ikmb/vcf2prot. The modular units used for building VCF2Prot are available as a Rust crate at https://crates.io/crates/ppgg with documentations and examples at https://docs.rs/ppgg/0.1.4/ppgg/ under the same MIT-license.


title: Development and characterization of a human arteriovenous malformation (AVM)-on-a-chip model

abstract: Brain arteriovenous malformations (AVMs) are a disorder wherein abnormal, enlarged blood vessels connect arteries directly to veins, without an intervening capillary bed. AVMs are one of the leading causes of hemorrhagic stroke in children and young adults. Most human sporadic brain AVMs are associated with genetic activating mutations in the KRAS gene. Our goal was to develop an in vitro model that would allow for simultaneous morphological and functional phenotypic data capture in real time during AVM disease progression. By generating human endothelial cells harboring a clinically relevant mutation found in most human patients (activating mutations within the small GTPase KRAS) and seeding them in a dynamic microfluidic cell culture system that enables vessel formation and perfusion, we demonstrate that vessels formed by KRAS4AG12V mutant endothelial cells (ECs) were significantly wider and more leaky than vascular beds formed by wild-type ECs, recapitulating key structural and functional hallmarks of human AVM pathogenesis. Immunofluorescence staining revealed a breakdown of adherens junctions in mutant KRAS vessels only, leading to increased vascular permeability, a hallmark of hemorrhagic stroke. Finally, pharmacological blockade of MEK kinase activity, but not PI3K inhibition, improved endothelial barrier function (decreased permeability) without affecting vessel diameter. Collectively, our studies describe the creation of human KRAS-dependent AVM-like vessels in vitro in a self-assembling microvessel platform that is amenable to phenotypic observation and drug delivery.


title: Pairwise and higher-order epistatic effects among somatic cancer mutations across oncogenesis

abstract: Cancer occurs as a consequence of multiple somatic mutations that lead to uncontrolled cell growth. Mutual exclusivity and co-occurrence of mutations imply--but do not prove--that they can exert synergistic or antagonistic epistatic effects on oncogenesis. Knowledge of these interactions, and the consequent trajectories of mutation and selection that lead to cancer has been a longstanding goal within the cancer research community. Recent research has revealed mutation rates and scaled selection coefficients for specific recurrent variants across many cancer types. However, estimation of pairwise and higher-order effects--essential to estimation of the trajectory of likely cancer genotoypes--has been a challenge. Therefore, we have developed a continuous-time Markov chain model that enables the estimation of mutation origination and fixation (flux), dependent on somatic cancer genotype. Coupling the continuous-time Markov chain model with a deconvolution approach provides estimates of underlying rates of mutation and selection across the trajectory of oncogenesis. We demonstrate computation of fluxes and selection coefficients in a somatic evolutionary model for the four most frequently variant driver genes (TP53, LRP1B, KRAS and STK11) from 585 cases of lung adenocarcinoma. Our analysis reveals multiple antagonistic epistatic effects that reduce the possible routes of oncogenesis, and inform cancer research regarding viable trajectories of somatic evolution whose progression could be forestalled by precision medicine. Synergistic epistatic effects are also identified, most notably in the somatic genotype TP53+LRP1B for mutations in the KRAS gene, and in somatic genotypes containing KRAS or TP53 mutations for mutations in the STK11 gene. Large positive fluxes of KRAS variants were driven by large selection coefficients, whereas the flux toward LRP1B mutations was substantially aided by a large mutation rate for this gene. The approach enables inference of the most likely routes of site-specific variant evolution and estimation of the strength of selection operating on each step along the route, a key component of what we need to know to develop and implement personalized cancer therapies.


title: Virus-assisted directed evolution of enhanced suppressor tRNAs in mammalian cells

abstract: Site-specific incorporation of unnatural amino acids (Uaas) in living cells relies on engineered aminoacyl-tRNA synthetase/tRNA pairs borrowed from a distant domain of life. Such heterologous suppressor tRNAs often show poor intrinsic activity, presumably due to the failure to optimally interact with a non-native translation system. This limitation can be addressed in E. coli using directed evolution. However, no suitable selection system is currently available to do the same in mammalian cells. Here we report virus-assisted directed evolution of tRNAs (VADER) in mammalian cells, which employs a double-sieve selection scheme to facilitate single-step enrichment of active-yet-orthogonal tRNA mutants from naive libraries. Using VADER, we developed improved mutants of M. mazei pyrrolysyl-tRNA, the most popular Uaa mutagenesis platform in eukaryotes. We also show that the higher activity of the most efficient mutants is specific for mammalian cells, alluding to an improved interaction with the unique mammalian translation system.


title: Macrophage activation of cGAS and TRIM5 distinguish pandemic and non-pandemic HIV

abstract: Pandemic viruses remain a global threat to health and economics but how they adapt to become pandemic remains poorly understood. Here we compare pandemic HIV-1(M) and non-pandemic HIV-(O) and HIV-2 strains finding that non-pandemic HIV replicate poorly in myeloid cell models due to activation of cGAS and TRIM5, and ensuing antiviral responses. We use phylogenetics and viral capsid structural biology to define specific differences between pandemic and non-pandemic HIV capsids and demonstrate that their genetic reversal in HIV-1(M) mutants causes TRIM5, cGAS and innate immune activation. We propose a model in which the parental lineage of pandemic HIV-1(M) has uniquely evolved a dynamic capsid that avoids activation of cGAS and TRIM5 to establish cloaked replication in myeloid cells. The unique adaptations of the pandemic virus lineage suggests a role in effective human-to-human transmissibility and highlight the importance of avoiding innate immune activation during pandemic human-to-human viral transmission.


title: IL-21 lowers the B cell receptor affinity threshold for participation in a T cell dependent immune response.

abstract:
The expansion of antigen-specific T and B cells and generation of germinal centers (GC) are prerequisites for long-lasting, high-affinity antibody-mediated immune protection. Affinity for antigen is fundamental to GC B cell recruitment as it determines access to T cell help and thus competitiveness within the response. However, how T-cell derived signals contribute to such recruitment is incompletely understood. Here we report how the signature cytokine of follicular helper T cells, IL-21, acts as a key regulator of the initial B cell response. By activating AKT and S6, IL-21 accelerates cell cycle progression and the rate of cycle entry of B cells, increasing their contribution to the ensuing GC. This effect is most pronounced on B cells receiving weak T cell co-stimulation in vitro and on low-affinity B cells in vivo, which require IL-21 receptor signaling for their recruitment and persistence in the early response. Thus, IL-21 determines the composition of the T-dependent B cell response by regulating the breadth of BCR affinities that can participate.

SummaryThe cytokine IL-21 is a regulator of B cell responses, increasing antibody quantity and quality. Here, we report IL-21s role during germinal center initiation, where it increases the response magnitude and lowers the B cell receptor affinity threshold for participation.


title: Metatranscriptomics reveals a shift in microbial community composition and function during summer months in a coastal marine environment

abstract: Temperate coastal marine waters are often thermally stratified from spring through fall, but can be dynamic and disrupted by tidal currents and wind-driven upwelling. These mixing events introduce deeper, cooler water with a higher partial pressure of CO2 (pCO2), and its associated microbial communities to the surface. Anecdotally, there have been concerns that these changes in water quality as well as in microbial composition and activity may be involved in mass mortality events of Pacific oysters (Crassostrea gigas) on the East Coast of Vancouver Island, British Columbia. Therefore, improved understanding of the composition and microbial activity of marine waters associated with seasons and abiotic variables may be useful in managing these mortality events. To characterize both compositional and functional changes associated with abiotic factors, here we generate a reference metatranscriptome from the Strait of Georgia over the representative seasons and analyze metatranscriptomic profiles of the microorganisms present within intake water containing different pCO2 levels at a shellfish hatchery in British Columbia from June through October. Abiotic factors studied include pH, temperature, alkalinity, aragonite, calcite and pCO2. Community composition changes were observed to occur at broad taxonomic levels, and most notably to vary with temperature and pCO2. Functional gene expression profiles indicated a strong difference between early (June-July) and late summer (August-October) associated with viral activity. The taxonomic data suggests this could be due to the termination of cyanobacteria and phytoplankton blooms by viral lysis in the late season. Functional analysis indicated fewer differentially expressed transcripts associated with abiotic variables (e.g., pCO2) than with the temporal effect. Microbial composition and activity in these waters varies with both short-term effects observed alongside abiotic variation as well as long-term effects observed across seasonal changes, as captured in the samples analyzed here. The analysis of both taxonomy and functional gene expression simultaneously in the same samples (i.e., metatranscriptomics) provided a more comprehensive view for monitoring water bodies than either would in isolation.

category: genetics


title: Overestimated Polygenic Prediction due to Overlapping Subjects in Genetic Datasets

abstract: Recently, polygenic risk score (PRS) has gained significant attention in studies involving complex genetic diseases and traits. PRS is often derived from summary statistics, from which the independence between discovery and replication sets cannot be monitored. Prior studies, in which the independence is strictly observed, report a relatively low gain from PRS in predictive models of binary traits. We hypothesize that the independence assumption may be compromised when using the summary statistics, and suspect an overestimation bias in the predictive accuracy. To demonstrate the overestimation bias in the replication dataset, prediction performances of PRS models are compared when overlapping subjects are either present or removed. We consider the task of Alzheimers disease (AD) prediction across genetics datasets, including the International Genomics of Alzheimers Project (IGAP), AD Sequencing Project (ADSP), and Accelerating Medicine Partnership - Alzheimers Disease (AMP-AD). PRS is computed from either sequencing studies for ADSP and AMP-AD (denoted as rPRS) or the summary statistics for IGAP (sPRS). Two variables with the high heritability in UK Biobank, hypertension, and height, are used to derive an exemplary scale effect of PRS. Based on the scale effect, the expected performance of sPRS is computed for AD prediction. Using ADSP as a discovery set for rPRS on AMP-AD, {Delta}AUC and {Delta}R2 (performance gains in AUC and R2 by PRS) record 0.069 and 0.11, respectively. Both drop to 0.0017 and 0.0041 once overlapping subjects are removed from AMP-AD. sPRS is derived from IGAP, which records {Delta}AUC and {Delta}R2 of 0.051{+/-}0.013 and 0.063{+/-}0.015 for ADSP and 0.060 and 0.086 for AMP-AD, respectively. On UK Biobank, rPRS performances for hypertension assuming a similar size of discovery and replication sets are 0.0036{+/-}0.0027 ({Delta}AUC) and 0.0032{+/-}0.0028 ({Delta}R2). For height, {Delta}R2 is 0.029{+/-}0.0037. Considering the high heritability of hypertension and height of UK Biobank, we conclude that sPRS results from AD databases are inflated. The higher performances relative to the size of the discovery set were observed in PRS studies of several diseases. PRS performances for binary traits, such as AD and hypertension, turned out unexpectedly low. This may, along with the difference in linkage disequilibrium, explain the high variability of PRS performances in cross-nation or cross-ethnicity applications, i.e., when there are no overlapping subjects. Hence, for sPRS, potential duplications should be carefully considered within the same ethnic group.


title: Spatial positioning and matrix programs of cancer-associated fibroblasts promote T cell exclusion in human lung tumors

abstract:
It is currently accepted that activated cancer-associated fibroblasts (CAF) participate in T cell exclusion from tumor nests, but it remains unclear how they promote barrier phenotypes, and whether specific subsets are involved. Here, using single-cell RNA sequencing coupled with multiplex imaging on a large cohort of lung tumors, we identify four main CAF populations, of which only two are associated with T cell exclusion: (i) MYH11+SMA+ CAF, which are present in early-stage tumors and form a single-cell layer lining cancer aggregates, and (ii) FAP+SMA+ CAF, which appear in more advanced tumors and organize in patches within the stroma or in multiple layers around tumor nests. Both CAF populations show a contractility phenotype together with dense and aligned matrix fiber deposition compared to the T cell-permissive CAF. Yet they express distinct matrix genes, including COL4A1/COL9A1 (MYH11+SMA+ CAF) and COL11A1/COL12A1 (FAP+SMA+ CAF). Hereby, we uncovered unique molecular programs of CAF driving T cell marginalization, whose targeting should increase immunotherapy efficacy in patients bearing T cell-excluded tumors.

SIGNIFICANCEThe cellular and molecular programs driving T cell marginalization in solid tumors remain unclear. Here, we describe two CAF populations associated with T cell exclusion in human lung tumors. We demonstrate the importance of pairing molecular and spatial analysis of the tumor microenvironment, a prerequisite to develop new strategies targeting T cell-excluding CAF.


title: The Androgen Receptor: A Therapeutic Target in Desmoplastic Small Round Cell Sarcoma

abstract:
Desmoplastic small round cell tumor (DSRCT) is an aggressive, usually incurable sarcoma subtype that predominantly occurs in post-pubertal young males. Recent evidence suggests that the androgen receptor (AR) can promote tumor progression in DSRCTs. However, the mechanism of AR-induced oncogenic stimulation remains undetermined. Herein, we demonstrate that enzalutamide and AR-directed antisense oligonucleotides (AR-ASO) block 5-dihydrotestosterone (DHT)-induced DSRCT cell proliferation and reduce xenograft tumor burden. Gene expression analysis and chromatin immunoprecipitation sequencing (ChIP-seq) were performed to elucidate how AR signaling regulates cellular epigenetic programs. Remarkably, ChIP-seq revealed novel DSRCT-specific AR DNA binding sites adjacent to key oncogenic regulators, including WT1 (the C-terminal partner of the pathognomonic fusion protein) and FOXF1. Additionally, AR occupied enhancer sites that regulate the Wnt pathway, neural differentiation, and embryonic organ development, implicating AR in dysfunctional cell lineage commitment. Our findings have immediate clinical implications given the widespread availability of FDA-approved androgen-targeted agents used for prostate cancer.

ONE SENTENCE SUMMARYWe demonstrate that DSRCT, an aggressive pediatric cancer, is an AR-driven malignancy capable of responding to androgen deprivation therapy.


title: Bacteria-mediated stabilization of murine norovirus

abstract:
Enteric viruses encounter various bacteria in the host which can impact infection outcomes. The interactions between noroviruses and enteric bacteria are not well understood. Previous work determined that murine norovirus (MNV), a model norovirus, had decreased replication in antibiotic-treated mice compared with conventional mice. Although this suggests that microbiota promote MNV infection, the mechanisms are not completely understood. Additionally, prior work with other enteric viruses such as poliovirus and coxsackievirus B3 demonstrated that virions bind bacteria, and exposure to bacteria stabilizes viral particles and limits premature RNA release. Therefore, we examined interactions between MNV and specific bacteria and the consequences of these interactions. We found that the majority of Gram-positive bacteria tested stabilized MNV, while Gram-negative bacteria did not stabilize MNV. Both Gram-positive and Gram-negative bacteria bound to MNV. However, bacterial binding alone was not sufficient for virion stabilization since Gram-negative bacteria bound MNV but did not stabilize virions. Additionally, we found that bacterial conditioned media also stabilized MNV and this stabilization may be due to a small heat stable molecule. Overall this work identifies specific bacteria and bacterial components that stabilize MNV and may impact virion stability in the environment.

ImportanceEnteric viruses are exposed to a wide variety of bacteria in the intestine, but effects of bacteria on viral particles are incompletely understood. We found that murine norovirus (MNV) virion stability is enhanced in the presence of several Gram-positive bacterial strains. Virion stabilizing activity was also present in bacterial culture medium, and activity was retained upon heat or protease treatment. These results suggest that certain bacteria and bacterial products may promote MNV stability in the environment, which could influence viral transmission.


title: Predicting evolution in experimental range expansions of an aquatic model system

abstract: Predicting range expansion dynamics is a challenge for both fundamental and applied research in conservation and global change biology. However, if ecological and evolutionary processes occur on the same time scale, predictions are challenging to make. Combining experimental evolution and mathematical modelling, we assessed the predictability of independent realisations of range expansions in a laboratory model system, the freshwater protozoan Paramecium caudatum. We followed ecological dynamics and evolutionary change in range core and front populations in the experiment. These settings were recreated in a predictive mathematical model, parametrized with dispersal and growth data of the of the 20 founder strains in the experiment. We find that short-term evolution was driven by selection for increased dispersal at the front and general selection for higher growth rates in all treatments. There was a good quantitative match of predicted and observed trait changes. Phenotypic divergence was mirrored by a complete genotypic divergence, indicating the highly repeatable fixation of strains that also were the most likely winners in our model. Long-term evolution in the experimental range front lines resulted in the emergence of a dispersal syndrome, namely a competition - colonisation trade-off. Altogether, both model and experiment highlight the importance of dispersal evolution as a driver of range expansions. Our study suggests that evolution at range fronts may follow predictable trajectories, at least for simple scenarios, and that predicting these dynamics may be possible from knowledge of few key parameters.


title: GGDB: A Grameneae Genome Alignment Database of Homologous Genes Hierarchically Related to Evolutionary Events

abstract:
Owing to their economic values, Gramineae plants have been preferentially sequenced their genomes. These genomes are often quite complex, e.g., harboring many duplicated genes, which were the main source of genetic innovation and often the results of recurrent polyploidization. Deciphering the complex genome structure and linking duplicated genes to specific polyploidization events are important to understand the biology and evolution of plants. However, the effort has been held back due to its high complexity in analyzing these genomes. Here, by hierarchically relating duplicated genes in colinearity to each polyploidization or speciation event, we analyzed 29 well-assembled and up-to-date Gramineae genome sequences, separated duplicated genes produced by each event, established lists of paralogous and orthologous genes, and eventually constructed an on-line database, GGDB (http://www.grassgenome.com/). Homologous gene lists from each plant and between them can be displayed, searched, and downloaded from the database. Interactive comparison tools were deployed to demonstrate homology among user-selected plants, to draw genome-scale or local alignment figures, phylogenetic trees of genes corrected by exploiting gene colinearity, etc. Using these tools and figures, users can easily observe genome structural changes, and explore the effects of paleo-polyploidy on crop genome structure and function. The GGDB will be a useful platform to improve understanding the genome changes and functional innovation of Gramineae plants.

Key pointsO_LIGGDB is the only portal hosting Grameneae colinear homologous genes hierarchically related to evolutionary events, especially polyploidization, which have occurred recursively.
C_LIO_LIAllows systematic analysis of colinear gene relationships and function origination and/or divergence across Grameneae plants.
C_LIO_LIServing the Grameneae research community, with new genomes, modules, tools, and analysis.
C_LI


title: Single cell genotyping of matched bone marrow and peripheral blood cells in treatment naive and AZA-treated MDS and CMML

abstract:
Progressively acquired somatic mutations in hematopoietic stem cells are central to pathogenesis in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). They can lead to proliferative advantages, impaired differentiation and progressive cytopenias. MDS or CMML patients with high-risk disease are treated with hypomethylating agents including 5-azacytidine (AZA). Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated hematopoietic stem and progenitor cells (HSPCs). However, the contribution of mutated HSPCs to steadystate hematopoiesis in MDS and CMML is unclear. To address this, we characterised the somatic mutations of individual stem, progenitor (common myeloid progenitor, granulocyte monocyte progenitor, megakaryocyte erythroid progenitor), and matched circulating (monocyte, neutrophil, naive B cell) haematopoietic cells in treatment naive and AZA-treated MDS and CMML via high-throughput single cell genotyping. The mutational burden was similar across multiple hematopoietic cell types, and even the most mutated stem and progenitor clones maintained their capacity to differentiate to mature myeloid and, in some cases, lymphoid cell types in vivo. Our data show that even highly mutated HSPCs contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment.

Key points* Highly mutated HSPCs contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment.
* The mutational burden in matched bone marrow and peripheral blood cells in MDS and CMML was similar throughout myelopoiesis.


title: The Intestine is a Major Contributor to Circulating TCA Cycle Intermediates in Mice

abstract: The tricarboxylic acid (TCA) cycle is the epicenter of cellular aerobic metabolism. TCA cycle intermediates facilitate energy production and provide anabolic precursors, but also function as intra- and extracellular metabolic signals regulating pleiotropic biological processes. Despite the importance of circulating TCA cycle metabolites as signaling molecules, the source of circulating TCA cycle intermediates remains uncertain. We observe that in mice, the concentration of TCA cycle intermediates in the portal blood exceeds that in tail blood indicating that the gut is a major contributor to circulating TCA cycle metabolites. With a focus on succinate as a representative of TCA cycle intermediate with signaling activities and using a combination of germ-free mice and isotopomer tracing, we demonstrate that intestinal microbiota are not major contributors to circulating TCA cycle metabolites. Moreover, we demonstrate that the endogenous succinate production is markedly higher than intestinal succinate absorption in normal physiological conditions. Altogether, these results indicate that endogenous succinate production within the intestinal tissue is a major physiological source of circulating succinate. These results provide a foundation for investigation into the role of intestine in regulating circulating TCA cycle metabolites and related signaling effects in health and disease.


title: The Retinal Basis of Photoaversion in Neonatal Mice

abstract: Aversive responses to bright light (photoaversion) require signaling from the eye to the brain. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) encode absolute light intensity and are thought to provide the light signals for photoaversion. Consistent with this, neonatal mice exhibit photoaversion prior to the developmental onset of image vision, and melanopsin deletion abolishes photoaversion in neonates. It is not well understood how the population of ipRGCs, which constitutes multiple physiologically distinct types (denoted M1-M6 in mouse) encodes light stimuli to produce an aversive response. Here, we provide several lines of evidence that M1 ipRGCs that lack the Brn3b transcription factor drive photoaversion in neonatal mice. First, neonatal mice lacking TRPC6 and TRPC7 ion channels failed to turn away from bright light, while two photon Ca2+ imaging of their acutely isolated retinas revealed reduced photosensitivity in M1 ipRGCs, but not other ipRGC types. Second, mice in which all ipRGC types except for Brn3b-negative M1 ipRGCs are ablated, exhibited normal photoaversion. Third, pharmacological blockade or genetic knockout of gap junction channels expressed by ipRGCs, which reduces the light sensitivity of M2-M6 ipRGCs in the neonatal retina, had small effects on photoaversion only at the brightest light intensities. Finally, M1s were not strongly depolarized by spontaneous retinal waves, a robust source of activity in the developing retina that depolarizes all other ipRGC types. M1s therefore constitute a separate information channel between the neonatal retina and brain that could ensure behavioral responses to light but not spontaneous retinal waves.


title: Multiscale computer modeling of spreading depolarization in brain slices

abstract:
Spreading depolarization (SD) is a slow-moving wave of neuronal depolarization accompanied by a breakdown of ion concentration homeostasis, followed by long periods of neuronal silence (spreading depression), and associated with several neurological conditions. We developed multiscale (ions to tissue slice) computer models of SD in brain slices using the NEURON simulator: 36,000 neurons (2 voltage-gated ion channels; 3 leak channels; 3 ion exchangers/pumps) in the extracellular space (ECS) of a slice (1 mm sides, varying thickness) with ion (K+, Cl-, Na+) and O2 diffusion and equilibration with a surrounding bath. Glia and neurons cleared K+ from the ECS via Na+/K+pumps. SD propagated through the slices at realistic speeds of 2-5 mm/min, which was augmented by 25-30% in models incorporating the effects of hypoxia or of propionate. In both cases, the speedup was mediated principally by ECS shrinkage. Our model allows us to make the following testable predictions: 1. SD can be inhibited by enlarging ECS volume; 2. SD velocity will be greater in areas with greater neuronal density, total neuronal volume, or larger/more dendrites; 3. SD is all-or-none: initiating K+ bolus properties have little impact on SD speed; 4. Slice thickness influences SD due to relative hypoxia in the slice core, exacerbated by SD in a pathological cycle; 5. Higher neuronal spike rates and faster spread will be observed in the core than the periphery of perfused slice during SD.

SignificanceSpreading depolarization (SD) is a slow moving wave of electrical and ionic imbalances in brain tissue and is a hallmark of several neurological disorders. We developed a multiscale computer model of brain slices with realistic neuronal densities, ions, and oxygenation. Our model shows that SD is exacerbated by and causes hypoxia, resulting in strong SD dependence on slice thickness. Our model also predicts that the velocity of SD propagation is not dependent on its initiation, but instead on tissue properties, including the amount of extracellular space and the total area of neuronal membrane, suggesting faster SD following ischemic stroke or traumatic brain injury.


title: Prospective Motion Correction and Automatic Segmentation of Penetrating Arteries in Phase Contrast MRI at 7 T

abstract:
PurposeTo develop a prospective motion correction (MC) method for phase contrast (PC) MRI of penetrating arteries (PA) in centrum semiovale at 7 T and evaluate its performance using automatic PA segmentation.

MethodsHead motion was monitored and corrected during the scan based on fat navigator images. Two convolutional neural networks (CNN) were developed to automatically segment PAs and exclude surface vessels. Real-life scans with MC and without MC (NoMC) were performed to evaluate the MC performance. Motion score was calculated from the range of translational and rotational motion parameters. MC vs NoMC pairs were divided according to their score differences into groups with similar, less, or more motions during MC. Data reacquisition was also performed to evaluate whether it can further improve PA visualization.

ResultsIn the group with similar motion, more PA counts (NPA) were obtained with MC in 9 (60%) cases, significantly more than the number of cases (1) with less PAs (p = 0.011; binomial test). In the group with less motion during MC, MC images had more or similar NPA in all cases, while in the group with more motion during MC, the numbers of cases with less and more NPA during MC were not significantly different (3 vs 0). Data reacquisition did not further increase NPA. CNNs had higher sensitivity (0.85) and accuracy (Dice coefficient 0.85) of detecting PAs than a threshold based method.

ConclusionsProspective MC and CNN based segmentation improved the visualization and delineation of PAs in PC MRI at 7 T.


title: Wildlife and Marine Mammal Spatial Observatory: Observation and automated detection of Southern Right Whales in multispectral satellite imagery

abstract:
The Wildlife and Marine Mammal Spatial Observatory is a joint research effort for the census of wildlife and particularly of marine mammals in satellite imagery. In that context, this paper illustrates the development of a high accuracy algorithm for the detection of right whales in sub-meter resolution multispectral satellite imagery with the constraint of a relatively small sample support of 580 southern right whale images. A significant space is devoted to exploratory data analysis to describe the statistical structure of right whale pixels and ocean surface pixels across multispectral bands.

Observations of southern right whale in satellite imagery are divided into typical and atypical right whale forms and the first observations of right whale mother and calf pairs in satellite imagery are presented. Measurements of whales are furthermore automatically extracted from whale observations (major axis length, minor axis length, etc). A significant space is also devoted to statistical data exploration, a step frequently overlooked in machine learning solutions, that yet offers interesting insight into the structure of animal detection in satellite imagery. The extracted statistics can readily be used by researchers to develop detection solutions even with low sample support. The adopted solution for detection consists of feature extraction with a convolutional neural network followed by classification with a support vector machine. 20 different convolutional neural networks were tested for feature extraction. Biostatistics parameters (accuracy, sensitivity, specificity and precision) were measured for comparison. Most architectures generally achieved high performance with low false positive and false negative rates. 100% accuracy is achieved in the case of 2 convolutional neural networks, Nasnet Large and Inception V3, and only with a specific selection of multispectral bands.

NB: This is a preprint that does not include satellite imagery due recent reviews


title: Characterizing Variability of Audiovisual Speech Perception Based on Periodic and Aperiodic Features of Prestimulus Brain Activity

abstract: Burgeoning pieces of evidence support the idea that differences in the properties of large-scale cortical networks underlie perceptual variability. Here, employing psychophysical experiments in tandem with electroencephalogram (EEG) recordings, we explored the inter-individual and inter-trial differences in McGurk percept to probe the underlying brain dynamics before (prestimulus) and after (poststimulus) the stimulus presentation. Initially, based on our participants (n = 18) overall susceptibility to the McGurk effect, we categorized them into two groups - rare and frequent groups of perceivers. Subsequently, we investigated the differences in their neuronal dynamics manifested in the oscillatory properties of the prestimulus and compared it with the poststimulus EEG signals during the McGurk perception (audio- /pa/, video- /ka/) and, also during the perception of the congruent audiovisual (AV) stimuli (syllables /pa/, /ta/, and /ka/). We primarily focused on the power spectrum components (periodic oscillations and aperiodic components) and global coherence. At the inter-individual and inter-trial level, we observed that for the prestimulus duration, the rare perceivers aperiodic (or "background" 1/f) component elicited a lower beta band (15-30 Hz) and gamma band (31-45 Hz) activity than the frequent group of perceivers. Moreover, the periodic oscillations for the rare group showed significance in the alpha (8-12 Hz) and beta bands during the prestimulus time window. Rare perceivers also showed an increased global coherence in the theta (4-7 Hz), alpha, and beta bands but a lower gamma band activity than the frequent perceivers. Finally, rare perceivers elicited higher cross-hemispheric connectivity (imaginary coherence) than the frequent group at the source across different frequency bands, supporting the notion that the fluctuations in the distributed large-scale network underlie the perceptual variability in speech across individuals.


title: A snapshot of brain and cognition in healthy mid-life and older adults

abstract: Deficits in memory are seen as a canonical sign of ageing and a prodrome to dementia in older adults. However, the nature of cognitive and brain changes across a wider aperture of adulthood is not well known. We quantify the relationship between cognitive function and brain morphology from mid-life to older adulthood, and the influence of age, sex, amyloid and genetic risk for dementia. We analyzed three observational cohorts (PISA, AIBL, ADNI) with cognitive, genetic and neuroimaging measures comprising a total of 1570 healthy mid-life and older adults (mean age 72, range 49-90 years, 1330 males) and 1365 age- and sex-matched adults with mild cognitive impairment or Alzheimers disease. Among healthy adults, we find robust modes of co-variation between regional sulcal width and multidomain cognitive function that change from mid-life to the older age range. The most prominent cortical changes in mid-life are predominantly associated with changes in executive functions, whereas they are most strongly associated with poorer memory function in older age. These cognitive changes are accompanied by an age-dependent pattern of sulcal widening. Amyloid exerts a weak, but significant, influence on cognition, but not on sulcal width. The APOE {varepsilon}4 allele also exerts a weak influence on cognition, but only significantly in the (larger and older) AIBL cohort. These findings provide new insights into brain and cognition in mid-life and older adults, suggesting that cognitive screening in mid-life cohorts should encompass executive functions as well as memory.


title: Hsa-miR-31-5p controls a metabolic switch in psoriatic keratinocytes that identifies therapeutic intervention

abstract: Psoriasis is characterized by a combination of keratinocyte hyperproliferation and immune cell activation. Immune cell activation requires increased glucose consumption, consequently limiting glucose availability for other cell types like keratinocytes. In psoriasis Hsa-microRNA-31-5p (miR-31) is highly expressed in keratinocytes. Here we show that miR-31 expression in keratinocytes is induced by limited glucose availability and increases survival under limiting glucose conditions, by increasing glutamine metabolism. In addition, miR-31 induced glutamine metabolism results in secretion of specific metabolites (aspartate and glutamate) but also immuno-modulatory factors. We show that this miR-31-induced secretory phenotype is sufficient to induce Th17 cell differentiation, a hallmark of psoriasis. Inhibition of glutaminase (GLS) using CB-839 impedes miR31-induced metabolic rewiring and secretion of immuno-modulatory factors. Concordantly, pharmacological targeting of GLS alleviated psoriasis pathology in a mouse model of psoriasis. Together our data illustrate an emerging concept of metabolic interaction across cell compartments that characterizes disease development, which can be employed to design effective treatment options for disease, as shown here for psoriasis.


title: A monotone single index model for missing-at-random longitudinal proportion data

abstract: Beta distributions are commonly used to model proportion valued response variables, commonly encountered in longitudinal studies. In this article, we develop semi-parametric Beta regression models for proportion valued responses, where the aggregate covariate effect is summarized and flexibly modeled, using a interpretable monotone time-varying single index transform of a linear combination of the potential covariates. We utilize the potential of single index models, which are effective dimension reduction tools and accommodate link function misspecification in generalized linear mixed models. Our Bayesian methodology incorporates the missing-at-random feature of the proportion response, and utilize Hamiltonian Monte Carlo sampling to conduct inference. We explore finite-sample frequentist properties of our estimates, and assess the robustness via detailed simulation studies. Finally, we illustrate our methodology via application to a motivating longitudinal dataset on obesity research recording proportion body fat.

category: developmental biology


title: Butterfly wings exhibit spatial variation in chromatin accessibility

abstract: Butterfly wings exhibit a diversity of patterns which can vary between forewings and hindwings and spatially across the same wing. Regulation of morphological variation involves changes in how genes are expressed across different spatial scales which is driven by chromatin dynamics during development. How patterns of chromatin dynamics correspond to morphological variation remains unclear. Here we compared the chromatin landscape between forewings and hindwings and also across the proximal and distal regions of the hindwings in two butterfly species, Bicyclus anynana and Danaus plexippus. We found that the chromatin profile varied significantly between the different wing regions, however, there was no clear correspondence between the chromatin profile and the wing patterns. In some cases, wing regions with different phenotypes shared the same chromatin profile whereas those with a similar phenotype had a different profile. We also found that in the forewing, open chromatin regions (OCRs) were AT rich whereas those in the hindwing were GC rich. GC content of the OCRs also varied between the proximal and hindwing regions. These differences in GC content were also reflected in the transcription factor binding motifs that were differentially enriched between the wings and wing regions. Our results suggest that distinct wing patterns may result from the interaction of pioneer factors, including Hox genes, differentially opening chromatin in different wings and wing regions and cooperating with other transcriptions factors, that show preferences for specific GC content, to function either as activator or repressors of nearby genes.


title: Found in translation: Microproteins are a new class of potential host cell impurity in mAb drug products

abstract:
Mass spectrometry (MS) has emerged as a powerful approach for the detection of Chinese hamster ovary (CHO) cell protein impurities in antibody drug products. The incomplete annotation of the Chinese hamster genome, however, limits the coverage of MS-based host cell protein (HCP) analysis. In this study, we performed ribosome footprint profiling (Ribo-seq) of translation initiation and elongation to refine the Chinese hamster genome annotation. Analysis of these data resulted in the identification of thousands of previously uncharacterised non-canonical proteoforms in CHO cells, such as N-terminally extended proteins and short open reading frames (sORFs) predicted to encode for microproteins. MS- based HCP analysis of adalimumab with the extended protein sequence database, resulted in the detection of CHO cell microprotein impurities in a mAb drug product for the first time. Further analysis revealed that the CHO cell microprotein population is altered over the course of cell culture and in response to a change in cell culture temperature. The annotation of non-canonical Chinese hamster proteoforms permits a more comprehensive characterisation of HCPs in antibody drug products using MS.

HighlightsO_LIAnalysis of translation initiation and elongation using ribosome footprint profiling provides a refined annotation of the Chinese hamster genome.
C_LIO_LI7,769 novel Chinese proteoforms were identified including those initiating at near cognate start codons.
C_LIO_LI941 N-terminal extensions of annotated genes were identified.
C_LIO_LI5,553 short open reading frames (sORFs) predicted to encode microproteins (i.e., proteins < 100 aa) were also characterised.
C_LIO_LIThe annotation of non-canonical proteins increases the coverage of MS-based host-cell protein analysis in monoclonal antibody drug products.
C_LIO_LI8 microproteins were found in adalimumab drug product.
C_LIO_LITranscripts annotated as non-coding can contain short open reading frames (sORFs) predicted to encode peptides (or microproteins) which are found to undergo changes in expression and translational regulation at reduced cell culture temperature.
C_LIO_LI95 of the novel proteoforms of which 79 were microproteins were subsequently identified in a second CHO K1 cell line using LC-MS/MS based proteomics. A comparison of protein abundance revealed that 13 microproteins were found to be differentially expressed between the exponential growth and stationary phases of cell culture.
C_LI


title: scFeatures: Multi-view representations of single-cell and spatial data for disease outcome prediction

abstract: Recent advances in single-cell technologies enable scientists to measure molecular data at high-resolutions and hold the promise to substantially improve clinical outcomes through personalised medicine. However, due to a lack of tools specifically designed to represent each sample (e.g. patient) from the collection of cells sequenced, disease outcome prediction on the sample level remains a challenging task. Here, we present scFeatures, a tool that creates interpretable molecular representation of single-cell and spatial data using 17 types of features motivated by current literature. The feature types span across six distinct categories including cell type proportions, cell type specific gene expressions, cell type specific pathway scores, cell type specific cell-cell interaction scores, overall aggregated gene expressions and spatial metrics. By generating molecular representation using scFeatures for single-cell RNA-seq, spatial proteomic and spatial transcriptomic data, we demonstrate that different types of features are important for predicting different disease outcomes in different datasets and the downstream analysis of features uncover novel biological discoveries.

category: bioinformatics


title: Iron Deficiency Induces Heart Failure with Ectopic Cardiac Calcification in Mice with Metabolic Syndrome

abstract: Iron deficiency is linked to worse clinical status and outcomes in heart failure. Although metabolic syndrome contributes to the development of heart failure, the impact of iron deficiency in heart failure complicated with metabolic syndrome remains obscure. KKAy mice were used as a model of metabolic syndrome. Four-week-old male C57BL/6J and KKAy mice were fed either a normal diet or iron-restricted (IR) diet for 12 weeks. During the experiment, 40% of mice died due to pulmonary congestion in KKAy mice with IR diet (KKAy-IR), while no mice died in other groups. Necropsy showed the presence of multiple white lesions on the cardiac surface in those KKAy-IR mice. Echocardiography and histological analyses revealed that KKAy-IR mice exhibited cardiac hypertrophy and cardiac dysfunction with cardiac calcification. Cardiac mRNA of ectonucleotide pyrophosphatase/phosphodiesterase-1 (Enpp1), a key enzyme for bone mineralization, was highly abundant in KKAy-IR mice. Of note, iron restriction-induced cardiac calcification and dysfunction were attenuated by etidronate, an inhibitor of bone mineralization, with decreased cardiac Enpp1 mRNA abundance in KKAy-IR mice. In conclusion, iron deficiency leads to ectopic cardiac calcification and dysfunction with the increase of Enpp1 in metabolic syndrome model mice.

category: pathology