title: Computational Modeling of Aerosol Particle Transport through Lung Mucosa

abstract: Delivery of aerosols to the lungs have great potential for the treatment of various lung diseases. However, the lungs are coated by a protective mucus layer whose complex properties make this form of delivery difficult. Mucus is non-Newtonian and cleared from the lungs over time by ciliated cells. Further, its gel-like structure hinders the diffusion of particles through it. Any aerosolized lung disease treatment must have certain properties to circumvent this barrier, and these properties may vary between diseases, drugs, and patients. Using Computational Fluid Dynamics (CFD) modeling, a model of this mucus layer was constructed such that the diffusion of an impacted aerosol might be studied. The model predicts what amount of a particle of a certain size might be expected to penetrate the mucus and reach the underlying tissue, as well as the distance downstream of the dosage site where concentration is maximized. Using this information, a personalized treatment plan may be designed. The model maintains modularity so that various lung regions and patient health states may be simulated.


title: A method for independent estimation of false localisation rate for phosphoproteomics

abstract:
Phosphoproteomics methods are commonly employed in labs to identify and quantify the sites of phosphorylation on proteins. In recent years, various software tools have been developed, incorporating scores or statistics related to whether a given phosphosite has been correctly identified, or to estimate the global false localisation rate (FLR) within a given data set for all sites reported. These scores have generally been calibrated using synthetic data sets, and their statistical reliability on real datasets is largely unknown. As a result, there is considerable problem in the field of reporting incorrectly localised phosphosites, due to inadequate statistical control.

In this work, we develop the concept of using scoring and ranking modifications on a decoy amino acid, i.e. one that cannot be modified, to allow for independent estimation of global FLR. We test a variety of different amino acids to act as the decoy, on both synthetic and real data sets, demonstrating that the amino acid selection can make a substantial difference to the estimated global FLR. We conclude that while several different amino acids might be appropriate, the most reliable FLR results were achieved using alanine and leucine as decoys, although we have a preference for alanine due to the risk of potential confusion between leucine and isoleucine amino acids. We propose that the phosphoproteomics field should adopt the use of a decoy amino acid, so that there is better control of false reporting in the literature, and in public databases that re-distribute the data. Data are available via ProteomeXchange with identifier PXD028840.

category: bioinformatics


title: The biomechanical context influences the output signaling, independently of PIK3CA mutations in breast cancer cells.

abstract: Mechanical stresses including tensile and compressive stresses are ubiquitous in nature, and are now well-recognized as being inherent to the development of most cancers. They are integrated by mechanotransduction in cells. Tensile stress is largely associated with YAP/TAZ pathway activation. However, less is known about signaling induced by compressive stress, the latter arising from extracellular matrix remodeling and local tumor growth. In the present study, we used transcriptomic data obtained after unidirectional compression of wildtype and mutant PIK3CA breast cancer cells from Kim et al., 2019. We analyzed in an unbiased manner signatures of cell signaling activation including phosphoinositide 3-kinases (PI3Ks) activity pathway in response to compressive stress. Because we found that PI3K activation occurred upon compression, we studied PI3K isoform-specific pathways using known transcriptional targets of PI3K inhibitor (BYL719) or of PI3K{beta} inhibitor (AZD8186). Our study provides transcriptomic evidences for the role of PI3K pathway in compression-induced mechanotransduction, through the roles of isoform-specific class I PI3Ks and independently of PIK3CA alterations. In a compressive environment, the canonical pathways (YAP/TAZ and Piezo) was not increased, while other targetable mechanisms, such as PI3K signal or autophagy, may provide a proliferative advantage and increased cell resistance to chemotherapies.


title: Trivial Excitation Energy Transfer to Carotenoids is an Unlikely Mechanism for Non-Photochemical Quenching in LHCII

abstract: Higher plants defend themselves from bursts of intense light via the mechanism of Non-Photochemical Quenching (NPQ). It involves the Photosystem II (PSII) antenna protein (LHCII) adopting a conformation that favours excitation quenching. In recent years several structural models have suggested that quenching proceeds via energy transfer to the optically forbidden and short-lived S1 states of a carotenoid. It was proposed that this pathway was controlled by subtle changes in the relative orientation of a small number of pigments. However, quantum chemical calculations of S1 properties are not trivial and therefore its energy, oscillator strength and lifetime are treated as rather loose parameters. Moreover, the models were based either on a single LHCII crystal structure or Molecular Dynamics (MD trajectories) about a single minimum. Here we try and address these limitations by parameterizing the vibronic structure and relaxation dynamics of lutein in terms of observable quantities, namely linear absorption (LA) transient absorption (TA) and two-photon excitation (TPE) spectra. We also analyze a number of minima taken from an exhaustive meta-dynamical search of the LHCII potential energy surface. We show that trivial, Coulomb-mediated energy transfer to S1 is an unlikely quenching mechanism. Pigment movements are insufficient to switch the system between quenched and unquenched states. Modulation of S1 energy level as a quenching switch is similarly unlikely. Moreover, the quenching predicted by previous models is likely an artefact of quantum chemical over-estimation of S1 oscillator strength and the real mechanism likely involves non-trivial inter-molecular states.

category: biophysics


title: Environmental variability and longevity predict the speed of the acute glucocorticoid response across birds.

abstract: The acute glucocorticoid response is a key mediator of the coordinated vertebrate response to unpredictable challenges. Rapid glucocorticoid increases initiate changes that allow animals to cope with stressors. The scope of the glucocorticoid response -- defined here as the absolute increase in glucocorticoids -- is associated with individual differences in performance and varies across species with environment and life history. In addition to varying in scope, responses can differ enormously in speed; however, relatively little is known about whether speed and absolute glucocorticoid levels covary, how selection shapes speed, or what aspects of speed are important. We used corticosterone samples collected at 5 time points from 1,750 individuals of 60 species of birds to ask i) how the speed and scope of the glucocorticoid response covary and ii) whether variation in absolute or relative speed is predicted by environmental context or life history. As predicted by a recent model, faster absolute glucocorticoid responses were strongly associated with a larger scope. Despite this covariation, the relative speed of the glucocorticoid response (standardized within species) varied independently of absolute scope, suggesting that selection could operate on both features independently. Species with faster relative glucocorticoid responses lived in locations with more variable temperature and had shorter lifespans. Our results suggest that rapid changes associated with the speed of the glucocorticoid response, such as those occurring through non-genomic receptors, might be an important determinant of coping ability and we emphasize the need for studies designed to measure speed independently of absolute glucocorticoid levels.

category: physiology


title: The relative speed of the glucorticoid stress response varies independently of scope and is predicted by environmental variability and longevity across birds.


title: Estimating null and potent modes of feedforward communication in a computational model of cortical activity

abstract: Communication across anatomical areas of the brain is key to both sensory and motor processes. Dimensionality reduction approaches have shown that the covariation of activity across cortical areas follows well-delimited patterns. Some of these patterns fall within the "potent space" of neural interactions and generate downstream responses; other patterns fall within the "null space" and prevent the feedforward propagation of synaptic inputs. Despite growing evidence for the role of null space activity in visual processing as well as preparatory motor control, a mechanistic understanding of its neural origins is lacking. Here, we developed a mean-rate model that allowed for the systematic control of feedforward propagation by potent and null modes of interaction. In this model, altering the number of null modes led to no systematic changes in firing rates, correlations, or mean synaptic strengths across areas, making it difficult to characterize feedforward communication with common measures of functional connectivity. A novel measure termed the null ratio captured the proportion of null modes relayed from one area to another. Applied to simultaneous recordings of primate cortical areas V1 and V2 during image viewing, the null ratio revealed that feedforward interactions have a broad null space that may reflect properties of visual stimuli.

category: neuroscience


title: Acute silencing uncovers multiple forms of activity-dependent neuronal survival in the mature entorhinal cortex

abstract: Neurodegenerative diseases are characterized by selective vulnerability of distinct cell populations; however, the cause for this specificity remains elusive. Many circuits that degenerate in disease are shaped by neural activity during development, raising the possibility that mechanisms governing early cell loss may be misused when activity is compromised in the mature brain. Here we show that electrical activity and synaptic transmission are both required for neuronal survival in the adult entorhinal cortex, but these silencing methods trigger distinct means of degeneration in the same neuronal population. Competition between active and inactive cells drives axonal disintegration caused by synaptic inhibition, but not axon retraction due to electrical suppression. These findings suggest that activity-dependence may persist in some areas of the adult brain long after developmental critical periods have closed. We speculate that lifelong plasticity required to support memory may render entorhinal neurons vulnerable to prolonged activity changes in disease.


title: Increased Functional Connectivity Characterises the Prodromal Phase of the Migraine Cycle

abstract:
Episodic migraine is reflected by cyclic changes in behaviour and cortical processing. In this study, we aimed to identify how functional connectivities change over the entire migraine cycle.

By using longitudinal neuroimaging and a whole-brain analysis approach, we tested 12 episodic migraine patients across 82 fMRI recordings during spontaneous headache attacks, with follow-up measurements over the pain-free interval without any external stimulation.

We focussed on two different trajectories with a linear increase of connectivity and a peak either during the prodromal phase or during the headache attack. We found cycle-related connectivity changes for the anterior insula and the posterior orbitofrontal cortex. Both regions showed the highest connections with sensory, motor, and cingulate areas in the prodromal phase. The connectivity between the hypothalamus and the locus coeruleus was strongest during the headache attack.

The peak connectivity during the prodromal phase and its collapse during the headache can be regarded as a mechanism of normalising cortical processing. The increase of synchronicity of functional connections during the ictal phase of the migraine cycle may contribute to the variety of symptoms of migraine attacks, such as headache, hypersensitivity to sensory modalities, and autonomous symptoms. We speculate about a malfunction at the molecular level in agranular frontal and insular brain regions, which needs to be followed up upon in subsequent studies.

Abbreviated SummaryEpisodic migraine is a cyclic disease. We investigated functional connectivities over an entire migraine cycle. We found cycle-related connectivity changes for two proximate agranular regions. The prodromal increase and the collapse of connectivity during the headache may reflect normalising cortical processing.


title: Microglia remodel the synaptic signaling required for context-dependent cognitive performance

abstract: Microglial homeostatic functions are fundamental to regulate the central nervous system microenvironment. We use conditional cell-specific gene targeting, RNA-seq profiling, high-throughput proteomics, phosphoproteomics, systems biology, and animal behavior to report a critical role for the RhoGTPase Rac1 in regulating adult microglia physiology. Ablation of Rac1 in adult microglia impaired their ability to sense and interpret the brain microenvironment and affected their capacity to communicate with synapses to drive cognitive performance, both at the steady-state and during experience-dependent plasticity. Overall, our results reveal a novel and central role for Rac1 as a regulator of microglia homeostasis and a molecular driver of the microglia-synapse crosstalk required for context-dependent sociability and learning related to memory.


title: Rac1 signaling in microglia is essential for synaptic proteome plasticity and experience-dependent cognitive performance


title: Characterization of red fluorescent reporters for dual-color in vivo three-photon microscopy

abstract:
SignificanceThree-photon (3P) microscopy significantly increases the depth and resolution of in vivo imaging due to decreased scattering and nonlinear optical sectioning. Simultaneous excitation of multiple fluorescent proteins is essential to study multicellular interactions and dynamics in the intact brain.

AimWe characterized the excitation laser pulses at a range of wavelengths for 3P microscopy, and then explored the application of tdTomato or mScarlet and EGFP for dual-color single-excitation structural 3P imaging deep in the living mouse brain.

ApproachWe used frequency-resolved optical gating to measure the spectral intensity, phase, and retrieved pulse widths at a range of wavelengths. Then, we performed in vivo single wavelength-excitation 3P imaging in the 1225 - 1360 nm range deep in the mouse cerebral cortex to evaluate the performance of tdTomato or mScarlet in combination with EGFP.

ResultsWe find that tdTomato and mScarlet, expressed in oligodendrocytes and neurons respectively, have high signal-to-background in the 1300-1360 nm range, consistent with enhanced 3P cross sections.

ConclusionsThese results suggest that a single excitation wavelength source is advantageous for multiple applications of dual-color brain imaging and highlight the importance of empirical characterization of individual fluorophores for 3P microscopy.


title: Human placental bed transcriptomic profiling reveals inflammatory activation of endothelial cells in preeclampsia

abstract:
RationaleFunctional characteristics of endothelial cells (ECs) within the human placental bed are unknown and may provide insight into the adaptive biology of ECs in disorders of vascular remodelling like preeclampsia.

ObjectiveTo determine transcriptional profiles of human placental bed ECs and systemic biomarker profiles in women with normal pregnancy, and women with preeclampsia, a condition characterized by extensive EC dysfunction, poor development of spiral arteries underlying the placenta and long-term susceptibility to atherosclerosis and hypertension.

Methods & resultsWe obtained biopsy samples from the uterine placental bed, of five women with preeclampsia with fetal growth restriction (FGR) due to impaired spiral artery development and four controls undergoing Caesarean section. CD31+CD146+ ECs were isolated and sorted by flow cytometry for RNA-sequencing using CEL-Seq2 protocol. Data were analyzed by unsupervised clustering, gene set enrichment (GSEA) and pathway analysis. 67 circulating biomarkers of EC function and inflammation were measured in 20 women with preeclampsia with FGR and 20 controls by multiplex immunoassay. Transcriptional profiling showed various differentially expressed genes (FDR<0.05) in placental bed ECs of preeclampsia patients, with enhanced activity of pathways associated with vasoconstriction, platelet activation and innate immunity. GSEA was suggestive of a VEGF- and PlGF deprived state of preeclampsia-derived ECs. Moreover, the transcriptomic profile was similar to that of human umbilical vein endothelial cells (HUVECs) treated with plasma from preeclampsia patients, pointing towards a central role for circulating factors in EC dysfunction. Unsupervised clustering of subjects by EC-related circulating factors identified distinct profiles for healthy pregnancy and preeclampsia, in particular for those women with low platelets and elevated liver enzymes, which was predominantly driven by sFLT-1, endoglin, PlGF, leptin, SAA-1 and sICAM-1.

ConclusionsWe revealed inflammatory activation of EC and a key role for systemic factors in EC dysfunction in women with preeclampsia associated with impaired spiral artery development.

category: immunology


title: Metabolic Potential of Microbial Communities in the Hypersaline Sediments of the Bonneville Salt Flats

abstract: The Bonneville Salt Flats (BSF) appear to be entirely desolate when viewed from above, but in reality they host rich microbial communities just below the surface salt crust. In this study, we investigate the metabolic potential of the BSF microbial ecosystem. The predicted and measured metabolic activities provide new insights into the ecosystem functions of evaporite landscapes and are an important analog for potential subsurface microbial ecosystems on ancient and modern Mars. Hypersaline and evaporite systems have been investigated previously as astrobiological analogs for Mars and other salty celestial bodies. Still, these studies have generally focused on aquatic systems and cultivation-dependent approaches. Here, we present an ecosystem-level examination of metabolic pathways within the shallow subsurface of evaporites. We detected aerobic and anaerobic respiration as well as methanogenesis in BSF sediments. Metagenome-assembled genomes (MAGs) of diverse bacteria and archaea encoded a remarkable diversity of metabolic pathways, including those associated with carbon fixation, carbon monoxide oxidation, acetogenesis, methanogenesis, sulfide oxidation, denitrification, and nitrogen fixation. These results demonstrate the potential for multiple energy sources and metabolic pathways in BSF and highlight the possibility for vibrant microbial ecosystems in the shallow subsurface of evaporites.

category: microbiology


title: Displacement of PKA catalytic subunit from AKAP signaling islands drives pathology in Cushing's syndrome

abstract: Mutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushings syndrome. Here we define mechanisms of action for the PKAc-L205R and W196R variants. Both Cushings mutants are excluded from A kinase anchoring protein (AKAP) signaling islands and consequently diffuse throughout the cell. Kinase-dead experiments show that PKA activity is required for cortisol hypersecretion. However, kinase activation is not sufficient, as only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate mutant PKAc into AKAP signaling islands abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Phosphoproteomics show that PKAc-L205R and W196R engage different mitogenic signaling pathways. ERK activity is elevated in adrenal-specific PKAc-W196R knock-in mice. Conversely, PKAc-L205R attenuates Hippo signaling, thereby upregulating the YAP/TAZ transcriptional co-activators. Thus, aberrant localization of each Cushings variant promotes the transmission of a distinct downstream pathogenic signal.

category: molecular biology


title: Mod3D: A Low-Cost, Flexible Modular System of Live-Cell Microscopy Chambers and Holders

abstract: Live-cell microscopy imaging typically involves the use of high-quality glass-bottom chambers that allow cell culture, gaseous buffer exchange and optical properties suitable for microscopy applications. However, commercial sources of these chambers can add significant annual costs to cell biology laboratories. Consumer products in three-dimensional printing technology, for both Filament Deposition Modeling (FDM) and Masked Stereo Lithography (MSLA), have resulted in more biomedical research labs adopting the use of these devices for prototyping and manufacturing of lab plastic-based items, but rarely consumables. Here we describe a modular, live-cell chamber with multiple design options that can be mixed per experiment. Single reusable carriers and the use of biodegradable plastics, in a hybrid of FDM and MSLA manufacturing methods, reduce plastic waste. The system is easy to adapt to bespoke designs, with concept-to-prototype in a single day, offers significant cost savings to the users over commercial sources, and no loss in dimensional quality or reliability.

category: cell biology


title: Novel genetic sex markers reveal unexpected lack of, and similar susceptibility to, sex reversal in free-living common toads in both natural and anthropogenic habitats

abstract: Anthropogenic environmental changes are affecting biodiversity and microevolution worldwide. Ectothermic vertebrates are especially vulnerable, since their sexual development can be disrupted by environmental changes, which can cause sex reversal, a mismatch between genetic and phenotypic sex, potentially leading to sex-ratio distortion and population decline. Despite these implications, we have scarce empirical knowledge on the incidence of sex reversal in nature. Populations in anthropogenic environments may experience sex reversal more frequently, or alternatively, they may adapt to resist sex reversal. To test these alternative hypotheses, we developed PCR-based genetic sex markers for the common toad (Bufo bufo). We assessed the prevalence of sex reversal in wild populations living in natural, agricultural and urban habitats, and the susceptibility of the same populations to two ubiquitous estrogenic pollutants in a common-garden experiment. We found negligible sex-reversal frequency in free-living adults despite the presence of various endocrine-disrupting pollutants in their breeding ponds. Individuals from different habitat types showed similar susceptibility to sex reversal in the laboratory: all genetic males developed female phenotype when exposed to 1 {micro}g/L 17- ethinylestradiol (EE2) during larval development, whereas no sex reversal occurred in response to 1 ng/L EE2 and a glyphosate-based herbicide with 3 {micro}g/L or 3 mg/L glyphosate. The latter results do not support that populations in anthropogenic habitats would have either increased propensity for or higher tolerance to chemically induced sex reversal. Thus, the surprisingly low sex-reversal frequency in wild toads compared to other ectothermic vertebrates studied before might indicate idiosyncratic, potentially species-specific resistance to sex reversal.

category: ecology


title: Does timing of birth affect juvenile and mare survival in wild plains zebra?

abstract: In large herbivores, the timing of births is mainly driven by the seasonal availability of their food resource. Population dynamics is strongly influenced by juvenile survival and recruitment, which highly depend on whether individuals are born during a favourable period or not. If births often occur during the most suitable season in northern cyclical environments for many large herbivore species, zebra give birth year-round at Hwange National Park, Zimbabwe, a tropical bushland characterized by the succession of a favourable wet season and a less favourable dry season. We used capture-recapture models for analysing long term observation data collected between 2008 and 2019 in this zebra population. We investigated the effect of the season (as a categorical variable) and the time spent in dry season on three categories of juveniles (younger foals of less than six months old, older foals between six and twelve months old, and yearlings between one and two years old) and mares survival, according to their reproductive state. The season had no effect on any survival. Younger foals annual survival was not affected by the time spent in dry season, whereas older foals and yearlings annual survival decreased with an increasing exposure to the dry season. Mares annual survival also decreased with an increasing time spent in dry season, whatever the reproductive status, but to a large extend when non-reproducing. The timing of birth, by determining the external conditions experienced by the offspring and their mothers during critical phases of their life cycle, plays a determinant role in their survival. As climate change is expected to lead to more frequent droughts, longer and harsher dry seasons in tropical ecosystems, we hypothesize a detrimental effect on zebra population dynamics in the future.


title: Inferring copy number variation from gene expression data: methods, comparisons, and applications to oncology

abstract: Copy number variations (CNVs) are genomic events where the number of copies of a particular gene varies from cell to cell. Cancer cells are associated with somatic CNV changes resulting in gene amplifications and gene deletions. However, short of single-cell whole-genome sequencing, it is difficult to detect and quantify CNV events in single cells. In contrast, the rapid development of single-cell RNA sequencing (scRNA-seq) technologies has enabled easy acquisition of single-cell gene expression data. In this work, we employ three methods to infer CNV events from scRNA-seq data and provide a statistical comparison of the methods results. In addition, we combine the analysis of scRNA-seq and inferred CNV data to visualize and determine subpopulations and heterogeneity in tumor cell populations.


title: Identification of Significant Gene Expression Changes in Multiple Perturbation Experiments using Knockoffs

abstract:
MotivationLarge-scale multiple perturbation experiments have the potential to reveal a more detailed understanding of the molecular pathways that respond to genetic and environmental changes. A key question in these studies is which gene expression changes are important for the response to the perturbation.

ResultsWe present here a method based on the model-X knockoffs framework to identify significant gene expression changes in multiple perturbation experiments. This approach makes no assumptions on the functional form of the dependence between the responses and the perturbations and provides finite sample false discovery rate control for the set of important gene expression responses. In a large-scale multiple perturbation gene expression data set from the Library of Integrated Network-Based Cellular Signature (LINCS) NIH program, we identified important genes whose expression is modulated in response to perturbation with anthracycline, vorinostat, trichostatin-a, geldanamycin, and sirolimus. Furthermore, we compared the set of important genes that respond to these small molecules to identify co-responsive pathways.

Availability and Implementationhttps://github.com/flahertylab/deepYknockoff

Contactpflaherty@umass.edu and zhaott0416@gmail.com

Supplementary informationSupplementary data are available at Bioinformatics online.


title: Preventing ovariectomy-induced weight gain decreases tumor burden in rodent models of obesity and postmenopausal breast cancer.

abstract: Obesity and adult weight gain are linked to increased breast cancer risk and poorer clinical outcomes in postmenopausal women, particularly for hormone-dependent tumors. Menopause is a time when significant weight gain occurs in many women, and clinical and preclinical studies have identified menopause (or ovariectomy) as a period of vulnerability for breast cancer development and promotion. We hypothesized that preventing weight gain after ovariectomy (OVX) may be sufficient to prevent the formation of new tumors and decrease growth of existing mammary tumors. Here, we tested this hypothesis in a rat model of obesity and carcinogen-induced postmenopausal mammary cancer and validated our findings in a murine xenograft model with implanted human tumors. In both models, preventing weight gain after OVX significantly decreased obesity-associated tumor development and growth. Importantly, we did not induce weight loss in these animals, but simply prevented weight gain. In both lean and obese rats, preventing weight gain reduced visceral fat accumulation and associated insulin resistance. Similarly, the intervention decreased circulating tumor-promoting growth factors and inflammatory cytokines (ie, BNDF, TNF, FGF2), with greater effects in obese compared to lean rats. In obese rats, preventing weight gain decreased adipocyte size, adipose tissue macrophage infiltration, reduced expression of the tumor-promoting growth factor FGF-1, and reduced phosphorylated FGFR in tumors. Together, these findings suggest that the underlying mechanisms associated with the anti-tumor effects of weight maintenance are multifactorial, and that weight maintenance during the peri-/post-menopausal period may be a viable strategy for reducing obesity-associated breast cancer risk and progression in women.

category: cancer biology


title: Photosynthetic induction upon transfer from low to high light is affected by leaf nitrogen content in tomato

abstract: The response of photosynthetic CO2 assimilation to changes of illumination affects plant growth and crop productivity under natural fluctuating light conditions. However, the effects of nitrogen (N) supply on photosynthetic physiology after transition from low to high light are seldom studied. To elucidate this, we measured gas exchange and chlorophyll fluorescence under fluctuating light in tomato (Solanum lycopersicum) seedlings grown with different N conditions. After transition from low to high light, the induction speeds of net CO2 assimilation (AN), stomatal conductance (gs) and mesophyll conductance (gm) delayed with the decline in leaf N content. The times to reach 90% of maximum AN, gs and gm were negatively correlated to leaf N content. This delayed photosynthetic induction in plants grown under low N concentration was mainly caused by the slow induction response of gm rather than that of gs. Furthermore, the photosynthetic induction upon transfer from low to high light was hardly limited by photosynthetic electron flow. These results indicate that decreased leaf N content declines carbon gain under fluctuating light in tomato. Increasing the induction kinetics of gm has the potential to enhance the carbon gain of field crops grown in infertile soil.

category: plant biology


title: MIR822 modulates monosporic female gametogenesis through an ARGONAUTE9-dependent pathway in Arabidopsis thaliana

abstract: In the ovule of flowering plants, the establishment of the haploid generation occurs when a somatic subepidermal cell specified as the gametophytic precursor differentiates into a Megaspore Mother Cell (MMC) and initiates meiosis. As most flowering plants, Arabidopsis thaliana (Arabidopsis) undergoes a monosporic type of gametogenesis as three meiotically derived cells degenerate without further division, and a single one - the functional megaspore (FM) - divides mitotically to form the female gametophyte. The genetic basis and molecular mechanisms that control monosporic gametogenesis remain largely unknown. In Arabidopsis, ARGONAUTE proteins are involved the control of megasporogenesis. In particular, mutations in ARGONAUTE9 (AGO9) lead to the ectopic differentiation of gametic precursors that can give rise to apomeiotically derived female gametophytes. Here, we show that Arabidopsis plants carrying loss-of-function mutations in the AGO9-interacting microRNA miR822a give rise to extranumerary surviving megaspores that acquire a FM identity and divide without giving rise to differentiated female gametophytes. The overexpression of three miR822a target genes encoding Cysteine/Histidine-Rich C1 domain proteins (At5g02350, At5g02330 and At2g13900) results in defects equivalent to those found in mir822 plants. All three miR822a targets are overexpressed in ago9 mutant ovules, confirming that miR822a acts through an AGO9-dependent pathway to negatively regulate Cysteine/Histidine-Rich C1 domain proteins and restricts the survival of meiotically derived cells to a single megaspore. Our results identify a microRNA-dependent mechanism that is involved in the control of megaspore degeneration and the most prevalent form of female gametogenesis in flowering plants.


title: Cryo-EM Structures of Amyloid-β 42 Filaments from Human Brain

abstract: Filament assembly of amyloid-{beta} peptides ending at residue 42 (A{beta}42) is a central event in Alzheimers disease. We report the cryo-EM structures of A{beta}42 filaments from brain. Two structurally related S-shaped protofilament folds give rise to two types of filaments. Type I filaments were found mostly in the brains of individuals with sporadic Alzheimers disease and Type II filaments in individuals with familial Alzheimers disease and other conditions. The structures of A{beta}42 filaments from brain differ from those of filaments assembled in vitro. By contrast, in AppNL-F knock-in mice, A{beta}42 deposits were made of Type II filaments. Knowledge of A{beta}42 filament structures from human brain may lead to the development of inhibitors of assembly and improved imaging agents.


title: Structural network alterations in focal and generalized epilepsy follow axes of epilepsy risk gene expression: An ENIGMA study

abstract: Epilepsy is associated with genetic risk factors and cortico-subcortical network alterations, but associations between neurobiological mechanisms and macroscale connectomics remain unclear. This multisite ENIGMA-Epilepsy study examined whole-brain structural covariance networks in patients with epilepsy and related findings to postmortem co-expression patterns of epilepsy risk genes. Brain network analysis included 578 adults with temporal lobe epilepsy (TLE), 288 adults with idiopathic generalized epilepsy (IGE), and 1,328 healthy controls from 18 centres worldwide. Graph theoretical analysis of structural covariance networks revealed increased clustering and path length in orbitofrontal and temporal regions in TLE, suggesting a shift towards network regularization. Conversely, people with IGE showed decreased clustering and path length in fronto-temporo-parietal cortices, indicating a random network configuration. Syndrome-specific topological alterations reflected expression patterns of risk genes for hippocampal sclerosis in TLE and for generalized epilepsy in IGE. These imaging-genetic signatures could guide diagnosis, and ultimately, tailor therapeutic approaches to specific epilepsy syndromes.


title: Molecular atlas of the human brain vasculature at the single-cell level

abstract: A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains poorly understood. Here, we performed single-cell RNA sequencing of 599,215 freshly isolated endothelial, perivascular and other tissue-derived cells from 47 fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We uncover extensive molecular heterogeneity of healthy fetal and adult human brains and across eight vascular-dependent CNS pathologies including brain tumors and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict numerous endothelial-to-perivascular cell ligand-receptor crosstalk including immune-related and angiogenic pathways, thereby unraveling a central role for the endothelium within brain neurovascular unit signaling networks. Our single-cell brain atlas provides insight into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.


title: Ochratoxin A induces behavioral and neurochemical changes in adult zebrafish

abstract: Ochratoxin A (OTA) is a mycotoxin produced by species of filamentous fungi widely found as a contaminant in food and with high toxic potential. Studies have shown that this toxin cause kidney and liver damage, however, data on the effects of exposure to OTA on the central nervous system are still scarce. Zebrafish (Danio rerio) is a teleost often used in translational research due to its physiological, genetic, and behavioral homology with mammals, in addition to being useful as an environmental bioindicator. Thus, this study aimed to investigate the effects of exposure to OTA on behavioral and neurochemical parameters in adult zebrafish. The animals were treated with different doses of OTA (1.38, 2.77, and 5.53 mg/kg) and submitted to behavioral evaluations in the open tank and social interaction tests. Subsequently, they were euthanized, and the brains were used to assess markers associated with oxidative status. In the open tank test OTA induced changes in distance, absolute turn angle, mean speed, and time-freezing. However, no significant effects were observed in the social interaction test. Moreover, OTA also induced alterations in neurochemical parameters with changes in non-protein thiols (NPSH), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR). This study showed that OTA can affect neurobiological aspects in zebrafish even at low doses.


title: Chronic alcohol drinking persistently suppresses thalamostriatal excitation of cholinergic neurons to impair cognitive flexibility

abstract: Exposure to addictive substances impairs flexible decision-making. Cognitive flexibility is mediated by striatal cholinergic interneurons (CINs). However, how chronic alcohol drinking alters cognitive flexibility through CINs remains unclear. Here, we report that chronic alcohol consumption and withdrawal impaired reversal of instrumental learning. Chronic alcohol consumption and withdrawal also caused a long-lasting (21 d) reduction of excitatory thalamic inputs onto CINs and reduced pause response of CINs in the dorsomedial striatum (DMS). CINs are known to inhibit glutamatergic transmission in dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) but facilitate this transmission in D2-MSNs, which may contribute to flexible behavior. We discovered that chronic alcohol drinking impaired CIN-mediated inhibition in D1-MSNs and facilitation in D2-MSNs. Importantly, in vivo optogenetic induction of long-term potentiation of thalamostriatal transmission in DMS CINs rescued alcohol-induced reversal learning deficits. These results demonstrate that chronic alcohol drinking reduces thalamic excitation of DMS CINs, compromising their regulation of glutamatergic transmission in MSNs, which may contribute to alcohol-induced impairment of cognitive flexibility. These findings provide a neural mechanism underlying inflexible drinking in alcohol use disorder.


title: Performance Scaling for Structural MRI Surface Parcellations: A Machine Learning Analysis in the ABCD Study

abstract: The use of pre-defined parcellations on surface-based representations of the brain as a method for data reduction is common across neuroimaging studies. In particular, prediction-based studies typically employ parcellation-driven summaries of brain measures as input to predictive algorithms, but the choice of parcellation and its influence on performance is often ignored. Here we employed pre-processed structural magnetic resonance imaging data (sMRI) from the ABCD Study(R) to examine the relationship between 220 parcellations and out-of-sample predictive performance across 45 phenotypic measures in a large sample of 9-10-year-old children (N=9,432). Choice of Machine Learning (ML) pipeline and use of alternative multiple parcellation-based strategies were also assessed. Relative parcellation performance was dependent on the spatial resolution of the parcellation, with larger numbers of parcels (up to [~]4000) outperforming coarser parcellations, according to a power-law scaling of between 1/4 and 1/3. Performance was further influenced by the type of parcellation, ML pipeline, and general strategy, with existing literature-based parcellations, a support vector based pipeline, and ensembling across multiple parcellations, respectively, as the highest performing. These findings highlight the choice of parcellation as an important influence on downstream predictive performance, showing in some cases that switching to a higher resolution parcellation can yield a relatively large boost to performance.


title: Zapline-plus: a Zapline extension for automatic and adaptive removal of frequency-specific noise artifacts in M/EEG

abstract: Removing power line noise and other frequency-specific artifacts from electrophysiological data without affecting neural signals remains a challenging task. Recently, an approach was introduced that combines spectral and spatial filtering to effectively remove line noise: Zapline (de Cheveigne, 2020). This algorithm, however, requires manual selection of the noise frequency and the number of spatial components to remove during spatial filtering. Moreover, it assumes that noise frequency and spatial topography are stable over time, which is often not warranted. To overcome these issues, we introduce Zapline-plus, which allows adaptive and automatic removal of frequency-specific noise artifacts from M/EEG and LFP data. To achieve this, our extension first segments the data into periods (chunks) in which the noise is spatially stable. Then, for each chunk, it searches for peaks in the power spectrum, and finally applies Zapline. The exact noise frequency around the found target frequency is also determined separately for every chunk to allow fluctuations of the peak noise frequency over time. The number of to-be-removed components by Zapline is automatically determined using an outlier detection algorithm. Finally, the frequency spectrum after cleaning is analyzed for suboptimal cleaning and parameters are adapted accordingly if necessary before re-running the process. The software creates a detailed plot for monitoring the cleaning. We showcase the efficacy of the different features of our algorithm by applying it to four openly available data sets, two EEG sets containing both stationary and mobile task conditions, and two MEG sets containing strong line noise.


title: Precision localization of cellular proteins with fluorescent Fab-based probes

abstract: Currently, a major technical limitation of microscopy based image analysis is linkage error - a visualization error that is measured by the distance between the cellular protein to be detected and the fluorescence emitter used for detection. With continuously improving resolution of todays (super-resolution) microscopes, the linkage errors can severely hamper the correct interpretation of images and is usually introduced in experiments by the use of standard intracellular staining reagents such as fluorescently labelled antibodies. The linkage error of standard labelled antibodies is caused by the size of the antibody and the random distribution of fluorescent emitters on the antibody surface. Together, these two factors account for a fluorescence displacement of ~40nm when staining proteins by indirect immunofluorescence; and ~20nm when staining with fluorescently coupled primary antibodies. In this study, we describe a class of staining reagents that effectively reduce the linkage error by more than five-fold when compared to conventional staining techniques. These reagents, called Fluo-N-Fabs, consist of an antigen binding fragment of a full-length antibody (Fab/ fragment antigen binding) that is selectively conjugated at the N-terminal amino group with fluorescent organic molecules, thereby reducing the distance between the fluorescent emitter and the protein target of the analysis. Fluo-N-Fabs also exhibit the capability to penetrate tissues and highly crowded cell compartments, thus allowing for the efficient detection of cellular epitopes of interest in a wide range of fixed samples. We believe this class of reagents realize an unmet need in cell biological super resolution imaging studies where the precise localization of the target of interest is crucial for the understanding of complex biological phenomena.


title: Myogenetic oligodeoxynucleotides as anti-nucleolin aptamers inhibit the growth of embryonal rhabdomyosarcoma cells

abstract:
BackgroundEmbryonal rhabdomyosarcoma (ERMS) is the muscle-derived tumor retaining myogenic ability. iSN04 and AS1411, which are myogenetic oligodeoxynucleotides (myoDNs) serving as anti-nucleolin aptamers, have been reported to inhibit the proliferation and induce the differentiation of myoblasts. The present study investigated the effects of iSN04 and AS1411 on the growth of multiple ERMS1 cell lines in vitro.

MethodsThree patient-derived ERMS cell lines, ERMS1, KYM1, and RD, were used. Nucleolin expression and localization in these cells was confirmed by immunostaining. The effects of iSN04 or AS1411 on the growth of ERMS cells were examined by cell counting, EdU staining, quantitative RT-PCR (qPCR), and three-dimensional culture of tumorspheres.

ResultsIn all ERMS cell lines, nucleolin was abundantly expressed, and localized and concentrated in nucleoli, similar to myoblasts. Both iSN04 and AS1411 (10-30 M) significantly decreased the number of all ERMS cells; however, their optimal conditions were different among the cell lines. iSN04 (10 M) markedly reduced the ratio of EdU+ cells, indicating the inhibition of cell proliferation. qPCR demonstrated that iSN04 suppressed the cell cycle, partially promoted myogenesis, but did not induce apoptosis. Finally, both iSN04 and AS1411 (10-30 M) disrupted the formation and outgrowth of RD tumorspheres mimicking in vivo tumorigenesis.

ConclusionsERMS cells expressed nucleolin, and their growth was inhibited by the anti-nucleolin aptamers, iSN04 and AS1411. The present study provides the first evidence that anti-nucleolin aptamers can be used as nucleic acid drugs for chemotherapy against ERMS.


title: Nuclear DNA-encoded fragments of mitochondrial DNA (mtDNA) confound analysis of selection of mtDNA mutations in human primordial germ cells.

abstract: The resilience of the mitochondrial genome to a high mutational pressure depends, in part, on purifying selection against detrimental mutations in the germline. It is crucial to understand the mechanisms of this process. Recently, Floros et al. concluded that much of the purifying selection takes place during the proliferation of primordial germ cells (PGCs) because, according to their analysis, the synonymity of mutations in late PGCs was seemingly increased compared to those in early PGCs. We re-analyzed the Floros et al. mutational data and discovered a high proportion of sequence variants that are not true mutations, but originate from NUMTs, the latter of which are segments of mitochondrial DNA (mtDNA) inserted into nuclear DNA, up to millions of years ago. This is a well-known artifact in mtDNA mutational analysis. Removal of these artifacts from the Floros et al. dataset abolishes the reported effect of purifying selection in PGCs. We therefore conclude that the mechanism of germline selection of mtDNA mutations remains open for debate, and more research is needed to fully elucidate the timing and nature of this process.

category: genomics


title: Adjusting for Common Variant Polygenic Scores Improves Yield in Rare Variant Association Analyses

abstract: With the emergence of large-scale sequencing data, methods for improving power in rare variant analyses (RVAT) are needed. Here, we show that adjusting for common variant polygenic scores improves the yield in gene-based RVAT across 65 quantitative traits in the UK Biobank (up to 20% increase at =2.6x10-6), without a marked increase in false-positive rates or genomic inflation. Our results illustrate how adjusting for common variant effects can aid in rare variant association discovery.


title: Simplifying MS1 and MS2 spectra to achieve lower mass error, more dynamic range, and higher peptide identification confidence on the Bruker timsTOF Pro

abstract:
1For bottom-up proteomic analysis, the goal of analytical pipelines that process the raw output of mass spectrometers is to detect, characterise, identify, and quantify peptides. The initial steps of detecting and characterising features in raw data must overcome some considerable challenges. The data presents as a sparse array, sometimes containing billions of intensity readings over time. These points represent both signal and chemical or electrical noise. Depending on the biological samples complexity, tens to hundreds of thousands of peptides may be present in this vast data landscape. For ion mobility-based LC-MS analysis, each peptide is comprised of a grouping of hundreds of single intensity readings in three dimensions: mass-over-charge (m/z), mobility, and retention time. There is no inherent information about any associations between individual points; whether they represent a peptide or noise must be inferred from their structure. Peptides each have multiple isotopes, different charge states, and a dynamic range of intensity of over six orders of magnitude. Due to the high complexity of most biological samples, peptides often overlap in time and mobility, making it very difficult to tease apart isotopic peaks, to apportion the intensity of each and the contribution of each isotope to the determination of the peptides monoisotopic mass, which is critical for the peptides identification.

Here we describe four algorithms for the Bruker timsTOF Pro that each play an important role in finding peptide features and determining their characteristics. These algorithms focus on separate characteristics that determine how candidate features are detected in the raw data. The first two algorithms deal with the complexity of the raw data, rapidly clustering raw data into spectra that allows isotopic peaks to be resolved. The third algorithm compensates for saturation of the instruments detector thereby recovering lost dynamic range, and lastly, the fourth algorithm increases confidence of peptide identifications by simplification of the fragment spectra. These algorithms are effective in processing raw data to detect features and extracting the attributes required for peptide identification, and make an important contribution to an analytical pipeline by detecting features that are higher quality and better segmented from other peptides in close proximity. The software has been developed in Python using Numpy and Pandas and made freely available with an open-source MIT license to facilitate experimentation and further improvement (DOI 10.5281/zenodo.6513126). Data are available via ProteomeXchange with identifier PXD030706.

2 Author SummaryThe primary goal of mass spectrometry data processing pipelines in the proteomic analysis of complex biological samples is to identify peptides accurately and comprehensively with abundance across a broad dynamic range. It has been reported that detection of low-abundance peptides for early-disease biomarkers in complex fluids is limited by the sensitivity of biomarker discovery platforms (1), the dynamic range of plasma abundance, which can exceed ten orders of magnitude (2), and the fact that lower abundance proteins provide the most insight in disease processes (3). As mass spectrometry hardware improves, the corresponding increase in amounts of data for analysis pushes legacy software analysis methods out of their designed specification. Additionally, experimentation with new algorithms to analyse raw data produced by instruments such as the Bruker timsTOF Pro has been hampered by the lack of modular, open-source software pipelines written in languages accessible by the large community of data scientists. Here we present several algorithms for simplifying MS1 and MS2 spectra that are written in Python. We show that these algorithms are effective to help improve the quality and accuracy of peptide identifications.


title: Heptanol-mediated phase separation determines phase preference of molecules in live cell membranes

abstract: Plasma membranes contain diverse nanoscale assemblies of lipid and protein domains. Specific localization of lipids and proteins in these domains is often essential for membrane function and integrity. Due to the nanoscale size and dynamic nature of membrane domains, identification of molecules residing in domains either is not possible with modern imaging techniques or requires advanced methods with high spatiotemporal resolution. Such methods need expensive equipment making these approaches inaccessible and thus difficult to implement at large scale. Here, we present a novel membrane fluidizer-induced clustering (MFIC) approach to identify the phase-preference of molecules in intact cell membranes. Experiments in phase-separated bilayers and live cells on molecules with known phase preference demonstrate that heptanol hyperfluidizes the membrane and stabilizes phase separation in cell membranes. The domain stabilization results in a transition of nano-to micron-sized clusters of associated molecules and allows identification of molecules localized in domains using routine microscopy techniques. This assay can be carried out on both genetically and extrinsically labelled molecules in live cell membranes, does not require any invasive sample preparation and can be carried out in 10-15 minutes. This inexpensive and easy to implement assay can be conducted at large-scale and will allow easy identification of molecules partitioning into domains.


title: Single-molecule analysis of the entire perfringolysin O poreformation pathway

abstract: The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings ([~]35-mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.


title: Optimal Precision and Accuracy in 4Pi-STORM using Dynamic Spline PSF Models

abstract: Dual-objective 4Pi fluorescence detection enables single molecule localization microscopy, e.g. PALM and STORM, with sub-10 nanometer spatial resolution in 3D. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation requirements and the challenging nature of the data analysis. The point spread function (PSF) of the 4Pi optical system is difficult to model, leading to periodic image artifacts and compromised resolution. In this work we report the development of a 4Pi-STORM microscope which obtains improved resolution and accuracy by modeling the 4Pi PSF dynamically, while using a simpler optical design. We introduce dynamic spline PSF models, which incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal dynamics of the optical system. Our method reaches the theoretical limits for localization precision while largely eliminating phase-wrapping artifacts, by making full use of the information content of the data. With a 3D precision as high as 2 - 3 nanometers, 4Pi-STORM achieves new levels of image detail, and extends the range of biological questions that can be addressed by fluorescence nanoscopy, as we demonstrate by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.


title: High survival following bleaching highlights the resilience of a highly disturbed region of the Great Barrier Reef

abstract:
Natural bleaching events provide an opportunity to examine how local scale environmental variation influences bleaching severity and recovery. During the 2020 marine heatwave, we documented widespread and severe coral bleaching (75 - 98% of coral cover) throughout the Keppel Islands in the Southern inshore Great Barrier Reef. Acropora, Pocillopora and Porites were the most severely affected genera, while Montipora was comparatively less susceptible. Site-specific heat-exposure metrics were not correlated with Acropora bleaching severity, but recovery was faster at sites that experienced lower heat exposure. Despite severe bleaching and exposure to accumulated heat that often results in coral mortality (degree heating weeks [~] 4 - 8), cover remained stable. Approximately 94% of fate-tracked Acropora millepora colonies survived, perhaps owing to reduced irradiance stress from high turbidity, heterotrophic feeding, and large tidal flows that can increase mass transfer. Severe bleaching followed by rapid recovery, and the continuing dominance of Acropora populations in the Keppel Islands is indicative of high resilience. These coral communities have survived an 0.8 {degrees}C increase in average temperatures over the last 150 years. However, recovery following the 2020 bleaching was driven by the easing of thermal stress, which may challenge their recovery potential under further warming.

Open Research StatementData are not yet provided but are being compiled. Upon acceptance data will be archived on GitHub.


title: Spatial and temporal control of expression with light-gated LOV-LexA

abstract:
The ability to drive expression of exogenous genes in different tissues and cell types, under control of specific enhancers, has catapulted discovery in biology. While many enhancers drive expression broadly, several genetic tricks have been developed to obtain access to isolated cell types. However, studies of topographically organized neuropiles, such as the optic lobe in fruit flies, have raised the need for a system that can access subsets of cells within a single neuron type, a feat currently dependent on stochastic flip-out methods. To access the same subsets of cells consistently across flies, we developed LOV-LexA, a light-gated expression system based on the bacterial LexA transcription factor and the plant-derived LOV photosensitive domain. Expression of LOV-Lex in larval fat body as well as pupal and adult neurons enables spatial and temporal control of expression of transgenes under LexAop sequences with blue light. The LOV-LexA tool thus provides another layer of intersectional genetics, allowing for light-controlled genetic access to the same subsets of cells within an expression pattern across individual flies.

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category: genetics


title: BioThings SDK: a toolkit for building high-performance data APIs in biomedical research

abstract:
SummaryTo meet the increased need of making biomedical resources more accessible and reusable, Web APIs or web services have become a common way to disseminate knowledge sources. The BioThings APIs are a collection of high-performance, scalable, annotation as a service APIs that automate the integration of biological annotations from disparate data sources. This collection of APIs currently includes MyGene.info, MyVariant.info, and MyChem.info for integrating annotations on genes, variants, and chemical compounds, respectively. These APIs are used by both individual researchers and application developers to simplify the process of annotation retrieval and identifier mapping. Here, we describe the BioThings Software Development Kit (SDK), a generalizable and reusable toolkit for integrating data from multiple disparate data sources and creating high-performance APIs. This toolkit allows users to easily create their own BioThings APIs for any data type of interest to them, as well as keep APIs up-to-date with their underlying data sources.

Availability and implementationThe BioThings SDK is built in Python and released via PyPI (https://pypi.org/project/biothings/).

Its source code is hosted at its github repository (https://github.com/biothings/biothings.api).


title: Mitochondrial Haplogroup Association with Fasting Glucose Response in African Americans Treated with a Thiazide Diuretic

abstract: Hypertensive African Americans have ~50% response rate to thiazide diuretic treatment. This contributes to a high prevalence of uncontrolled high blood pressure. Here, we examine the role the mitochondrial genome has on thiazide diuretic treatment response in hypertensive African Americans enrolled in a clinical trial. Participants from the Antihypertensive and Lipid Lowering Treatment to Prevent Heart Attack Trial (ALLHAT, n= 4279) were genotyped using the Illumina Infinium Multi-Ethnic Beadchip. Haplotype groups were called using HaploGrep. We used linear regression analysis to examine the association between mitochondrial haplogroups (L, M, and N) and change in blood pressure and change in fasting glucose over 6 months and two years, respectively. The analysis revealed a null association between mitochondrial haplogroups M and N vs. L for each of the outcomes. In subgroup analysis, the L subclades L1, L2, and L3/L4 (vs. L0) were each inversely associated with fasting glucose response (p < 0.05). This discovery analysis suggests the mitochondrial genome has a small effect on fasting glucose but not blood pressure response to thiazide diuretic treatment in African Americans.

category: pharmacology and toxicology


title: Genetic architecture underlying variation in floral meristem termination in Aquilegia

abstract: Floral organs are produced by floral meristems (FMs), which harbor stem cells in their centers. Since each flower only has a finite number of organs, the stem cell activity of a FM will always terminate at a specific time point, a process termed floral meristem termination (FMT). Variation in the timing of FMT can give rise to floral morphological diversity, but how this process is fine-tuned at a developmental and evolutionary level is poorly understood. Flowers from the genus Aquilegia share identical floral organ arrangement except for stamen whorl numbers (SWN), making Aquilegia a well-suited system for investigation of this process: differences in SWN between species represent differences in the timing of FMT. By crossing A. canadensis and A. brevistyla, quantitative trait locus (QTL) mapping has revealed a complex genetic architecture with seven QTL. We identified potential candidate genes under each QTL and characterized novel expression patterns of select candidate genes using in situ hybridization. To our knowledge, this is the first attempt to dissect the genetic basis of how natural variation in the timing of FMT is regulated and our results provide insight into how floral morphological diversity can be generated at the meristematic level.


title: Functional connectome of arousal and motor brainstem nuclei in living humans by 7 Tesla resting-state fMRI

abstract: Brainstem nuclei play a pivotal role in many functions, such as arousal and motor control. Nevertheless, the connectivity of arousal and motor brainstem nuclei is understudied in living humans due to the limited sensitivity and spatial resolution of conventional imaging, and to the lack of atlases of these deep tiny regions of the brain. For a holistic comprehension of sleep, arousal and associated motor processes, we investigated in 20 healthy subjects the resting-state functional connectivity of 18 arousal and motor brainstem nuclei in living humans. To do so, we used high spatial-resolution 7 Tesla resting-state fMRI, as well as a recently developed in-vivo probabilistic atlas of these nuclei in stereotactic space. Further, we verified the translatability of our brainstem connectome approach to conventional (e.g. 3 Tesla) fMRI. Arousal brainstem nuclei displayed high interconnectivity, as well as connectivity to the thalamus, hypothalamus, basal forebrain and frontal cortex, in line with animal studies and as expected for arousal regions. Motor brainstem nuclei showed expected connectivity to the cerebellum, basal ganglia and motor cortex, as well as high interconnectivity. Comparison of 3 Tesla to 7 Tesla connectivity results indicated good translatability of our brainstem connectome approach to conventional fMRI, especially for cortical and subcortical (non-brainstem) targets and to a lesser extent for brainstem targets. The functional connectome of 18 arousal and motor brainstem nuclei with the rest of the brain might provide a better understanding of arousal, sleep and accompanying motor function in living humans in health and disease.


title: High-throughput Evaluation of Epilepsy-associated KCNQ2 Variants Reveals Functional and Pharmacological Heterogeneity

abstract: Hundreds of KCNQ2 variants have been identified by genetic testing of children with early onset epilepsy and/or developmental disability. Voltage-clamp recording from heterologous cells has proved useful for establishing deleterious functional effects of KCNQ2 variants, but procedures adapting these assays for standardized, higher throughput data collection and reporting are lacking. In this study, we employed automated patch clamp recording to assess in parallel the functional and pharmacological properties of 79 missense and 2 in-frame deletion variants of KCNQ2. Among the variants we studied were a training set of 18 pathogenic variants previously studied by voltage-clamp recording, 24 mostly rare population variants, and 39 disease-associated variants with unclear functional effects. Variant KCNQ2 subunits were transiently expressed in a cell line stably expressing KCNQ3 to reconstitute the physiologically relevant channel complex. Variants with severe loss-of-function were also co-expressed 1:1 with WT KCNQ2 in the KCNQ3 cell line to mimic the heterozygous genotype and assess dominant-negative behavior. In total, we analyzed electrophysiological data recorded from 9,480 cells. The functional properties of WT KCNQ2/KCNQ3 channels and pharmacological responses to known blockers and activators determined by automated patch clamp recording were highly concordant with previous findings. Similarly, functional properties of 18 known pathogenic variants largely matched previously published results and the validated automated patch clamp assay. Many of the 39 previously unstudied disease-associated KCNQ2 variants exhibited prominent loss-of-function and dominant-negative effects, providing strong evidence in support of pathogenicity. All variants, exhibit response to retigabine (10 {micro}M), although there were differences in maximal responses. Variants within the ion selectivity filter exhibited the weakest responses whereas retigabine had the strongest effect on gain-of-function variants in the voltage-sensor domain. Our study established a high throughput method to detect deleterious functional consequences of KCNQ2 variants. We demonstrated that dominant-negative loss-of-function is a common mechanism associated with missense KCNQ2 variants but this does not occur with rare population variation in this gene. Importantly, we observed genotype-dependent differences in the response of KCNQ2 variants to retigabine.


title: Ultrasound-mediated delivery of novel tau-specific monoclonal antibody enhances brain uptake but not therapeutic efficacy

abstract: Tau-specific immunotherapy is an attractive therapeutic strategy for the treatment of Alzheimers disease and other tauopathies. However, targeting tau effectively remains a considerable challenge due to the restrictive nature of the blood-brain barrier (BBB), which excludes 99.9% of peripherally administered antibodies. We have previously shown that the delivery of tau-specific monoclonal antibody (mAb) with low-intensity scanning ultrasound in combination with intravenously injected microbubbles (SUS+MB) increases the passage of IgG antibodies into the brain. SUS+MB transiently opens tight junctions to allow paracellular transport, but also facilitates transcellular transport, particularly for larger cargoes. However, therapeutic efficacy after enhanced brain delivery has not been explored. To assess whether ultrasound-mediated delivery of tau-specific mAbs leads to an enhanced therapeutic response, K369I tau transgenic K3 mice were passively immunised once weekly for 12 weeks with a novel mAb, RNF5, in combination with SUS+MB. While none of the treatment arms improved behaviour or motor functions in these mice, we found that both RNF5 and SUS+MB treatments on their own reduced tau pathology, but, surprisingly, the combination of both (RNF5+SUS+MB) did not achieve an additive reduction in tau pathology. This was despite observing increased antibody penetration in the brain. Interestingly, a significant fraction of the antibody in the combination treatment was visualized in brain endothelial cells, suggesting that paracellular transport may not be the preferred uptake mechanism for RNF5. Taken altogether, more research is warranted to develop SUS+MB as a delivery modality for anti-tau antibodies.


title: Functional connectome of brainstem nuclei involved in autonomic, limbic, pain and sensory processing in living humans from 7 Tesla resting state fMRI

abstract: Despite remarkable advances in mapping the functional connectivity of the cortex, the functional connectivity of subcortical regions is understudied in living humans. This is the case for brainstem nuclei that control vital processes, such as autonomic, limbic, nociceptive and sensory functions. This is because of the lack of precise brainstem nuclei localization, of adequate sensitivity and resolution in the deepest brain regions, as well as of optimized processing for the brainstem. To close the gap between the cortex and the brainstem, on 20 healthy subjects, we computed a correlation-based functional connectome of 15 brainstem nuclei involved in autonomic, limbic, nociceptive, and sensory function (superior and inferior colliculi, ventral tegmental area-parabrachial pigmented nucleus complex, microcellular tegmental nucleus-prabigeminal nucleus complex, lateral and medial parabrachial nuclei, vestibular and superior olivary complex, superior and inferior medullary reticular formation, viscerosensory motor nucleus, raphe magnus, pallidus, and obscurus, and parvicellular reticular nucleus - alpha part) with the rest of the brain. Specifically, we exploited 1.1mm isotropic resolution 7 Tesla resting-state fMRI, ad-hoc coregistration and physiological noise correction strategies, and a recently developed probabilistic template of brainstem nuclei. Further, we used 2.5mm isotropic resolution resting-state fMRI data acquired on a 3 Tesla scanner to assess the translatability of our results to conventional datasets. We report highly consistent correlation coefficients across subjects, confirming available literature on autonomic, limbic, nociceptive and sensory pathways, as well as high interconnectivity within the central autonomic network and the vestibular network. Interestingly, our results showed evidence of vestibulo-autonomic interactions in line with previous work. Comparison of 7 Tesla and 3 Tesla findings showed high translatability of results to conventional settings for brainstem-cortical connectivity and good yet weaker translatability for brainstem-brainstem connectivity. The brainstem functional connectome might bring new insight in the understanding of autonomic, limbic, nociceptive and sensory function in health and disease.


title: Nandrolone Decanoate supplementation promotes AMPK activation and divergent brain connectivity rearrangements in adult and aged mice.

abstract: Decreased anabolic androgen levels are followed by impaired brain energy support and sensing with loss of neural connectivity during physiological aging, providing a neurobiological basis for hormone supplementation. Here, we investigated whether nandrolone decanoate (ND) administration mediates hypothalamic AMPK activation and glucose metabolism, thus affecting metabolic connectivity in brain areas of adult and aged mice. Metabolic interconnected brain areas of rodents can be detected by positron emission tomography using 18FDG-mPET. Albino CF1 mice at 3 and 18 months of age were separated into 4 groups that received daily subcutaneous injections of either ND (15 mg/kg) or vehicle for 15 days. At the in vivo baseline and on the 14th day, 18FDG-mPET analyses were performed. Hypothalamic pAMPKT172/AMPK protein levels were assessed, and basal mitochondrial respiratory states were evaluated in synaptosomes. A metabolic connectivity network between brain areas was estimated based on 18FDG uptake. We found that ND increased the pAMPKT172/AMPK ratio in both adult and aged mice but induced 18FDG uptake and mitochondrial basal respiration only in adult mice. Furthermore, ND triggered rearrangement in the metabolic connectivity of adult mice and aged mice compared to age-matched controls. Altogether, our findings suggest that ND promotes hypothalamic AMPK activation, and distinct glucose metabolism and metabolic connectivity rearrangements in the brains of adult and aged mice.


title: New adaptive peaks for crops - an example from improvement of drought-resilience of sorghum in Ethiopia

abstract:
O_LIAs the center of diversity for sorghum [Sorghum bicolor (L.) Moench], elite cultivars selected in Ethiopia are of central importance to sub-Saharan food security. Despite being presumably well adapted to their center of diversity, elite Ethiopian sorghums nonetheless experience constraints to productivity, for example associated with shifting rainfall patterns associated with climate change.
C_LIO_LIA sorghum backcross nested association mapping (BC-NAM) population developed by crossing thirteen diverse lines pre-identified to have various drought resilience mechanisms, with an Ethiopian elite cultivar, Teshale, was tested under three rain-fed environments in Ethiopia.
C_LIO_LI27, 15, and 15 QTLs with predominantly small additive effects were identified for days to flowering, days to maturity, and plant height, respectively. Many associations detected in this study corresponded closely to known or candidate genes or previously mapped QTLs, supporting their validity. Field tests show drought resilience to be improved by incorporation of adaptations from the diverse donor lines.
C_LIO_LIThe expectation that genotypes such as Teshale from near the center of diversity tend to have a history of strong balancing selection, with novel variations more likely to persist in small marginal populations, was strongly supported in that for these three traits, nearly equal numbers of alleles from the donor lines conferred increases and decreases in phenotype relative to the Teshale allele. Such rich variation provides a foundation for selection to traverse a  valley of reduced yield and arrive at a new  adaptive peak, exemplifying the nature of efforts that may be necessary to adapt many crops to new climate extremes.
C_LI

Societal Impact StatementIn Ethiopia, agriculture is the largest economic sector and contributes 48% of the nations GDP, and sorghum provides more than one third of the cereal diet and is widely grown for food, feed, brewing, and construction purposes. With a worldwide water crisis looming, developing drought tolerant sorghum is vital in rain-fed environments, particularly in sub-Saharan Africa. Addressing such issues often requires a far-reaching approach to identify and incorporate new traits into a gene pool, followed by a period of selection to re-establish an overall adaptive phenotype. This study revealed that with the enormous altitudinal variation of a country such as Ethiopia, somewhat different lines may be needed for different locales.


title: Genome-wide association study of open fieldbehavior in outbred heterogeneous stock ratsidentifies multiple loci implicated inpsychiatric disorders

abstract: Many personality traits are influenced by genetic factors. Rodents models provide an efficient system for analyzing genetic contribution to these traits. Using 1,246 adolescent heterogeneous stock (HS) male and female rats, we conducted a genome-wide association study (GWAS) of behaviors measured in an open field, including locomotion, novel object interaction, and social interaction. We identified 30 genome-wide significant quantitative trait loci (QTL). Using multiple criteria, including the presence of high impact genomic variants and co-localization of cis-eQTL, we identified 13 candidate genes (Adarb2, Ankrd26, Cacna1c, Clock, Crhr1, Ctu2, Cyp26b1, Eva1a, Fam114a1, Kcnj9, Mlf2, Rab27b, Sec11a) for these traits. Most of these genes have been implicated by human GWAS of various psychiatric traits. For example, Cacna1c, a gene known to be critical for social behavior in rodents and implicated in human schizophrenia and bipolar disorder, is a candidate gene for distance to the social zone. In addition, the QTL region for total distance to the novel object zone, on Chr1 at 144 Mb, is syntenic to a hotspot on human Chr15 (82.5-90.8 Mb) that contains 14 genes associated with psychiatric or substance abuse traits. Although some of the genes identified by this study appear to replicate findings from prior human GWAS, others likely represent novel findings that can be the catalyst for future molecular and genetic insights into human psychiatric diseases. Together, these findings provide strong support for the use of the HS population to study psychiatric disorders.


title: High-throughput imaging of Caenorhabditis elegans aging using collective activity monitoring

abstract: The genetic manipulability and short lifespan of C. elegans make it an important model for aging research. Widely applied methods for measurements of worm aging based on manual observation are labor intensive and low-throughput. Here, we describe the Worm Collective Activity Monitoring Platform (WormCamp), a system for assaying aging in C. elegans by monitoring activity of populations of worms in standard 24-well plates. We show that metrics based on the rate of decline in collective activity can be used to estimate the average lifespan and locomotor healthspan in the population. Using the WormCamp, we assay a panel of highly divergent natural isolates of C. elegans and show that both lifespan and locomotor healthspan display substantial heritability. To facilitate analysis of large numbers of worms, we developed a robotic imaging system capable of simultaneous automated monitoring of activity, lifespan, and locomotor healthspan in up to 2,304 populations containing a total of ~90,000 animals. We applied the automated system to conduct a large-scale RNA interference screen for genes that affect lifespan and locomotor healthspan. The WormCamp system is complementary to other current automated methods for assessing C. elegans aging and is well suited for efficiently screening large numbers of conditions.

category: bioengineering


title: Genomic identification, annotation, and comparative analysis of Vacuolar-type ATP synthase subunits in Diaphorina citri

abstract: Detailed annotation and comparative analysis were performed on the Asian citrus psyllid (ACP), Diaphorina citri, vacuolar-type ATP synthase (V-ATPase) to support the biological understanding and development of novel therapeutics to manage psyllid vectors. D. citri is a hemipteran insect that vectors the causative agent, the bacteria Candidatus Liberibacter asiaticus (CLas), of the citrus greening disease, Huanglongbing (HLB). Millions of citrus trees have been destroyed by citrus greening and every grove in Florida has been directly impacted. In eukaryotic organisms, V-ATPase is an abundant heterodimeric enzyme that serves the cell with essential compartment acidification through the active processes that transport protons across the membrane. Manual curation was completed on 15 putative V-ATPase genes in the D. citri genome. Comparative genomic analysis reveals that the D. citri V-ATPase subunits share domains and motifs with other insects, including the V-ATPase-A superfamily domain from the V-ATPase catalytic subunit A, which shares a 92% identity with Acyrthosiphon pisum. Phylogenetic analysis separates D. citri V-ATPase subunits into expected clades with orthologous sequences. Based on the results of annotation and comparative genomic analysis, RNAi therapies targeting D. citri V-ATPase genes, which have been successfully utilized in related hemipterans, are being pursued. Annotation of the D. citri genome is a critical step towards the development of directed-pest management that will lead to the reduced spread of the pathogens causing HLB throughout the citrus industry.


title: Inverse Potts model improves accuracy of phylogenetic profiling

abstract: Phylogenetic profiling is a powerful computational method for revealing the functions of function-unknown genes. Although conventional similarity evaluation measures in phylogenetic profiling showed high prediction accuracy, they have two estimation biases: an evolutionary bias and a spurious correlation bias. Existing studies have focused on the evolutionary bias, but the spurious correlation bias has not been analyzed. To eliminate the spurious correlation bias, we applied an evaluation measure based on the inverse Potts model (IPM) to phylogenetic profiling. We also proposed an evaluation measure to remove both the evolutionary and spurious correlation biases using the IPM. In an empirical dataset analysis, we demonstrated that these IPM-based evaluation measures improved the prediction performance of phylogenetic profiling. In addition, we found that the integration of several evaluation measures, including the IPM-based evaluation measures, had superior performance to a single evaluation measure. The source code is freely available at https://github.com/fukunagatsu/Ipm.


title: Protease-controlled secretion and display of intercellular signals

abstract: To program intercellular communication for biomedicine, it is crucial to regulate the secretion and surface display of signaling proteins. If such regulations are at the protein level, there are additional advantages, including compact delivery and direct interactions with endogenous signalling pathways. We created a modular, generalizable design called Retained Endoplasmic Cleavable Secretion (RELEASE), with engineered proteins retained in the endoplasmic reticulum and displayed/secreted in response to specific proteases. The design allows functional regulation of multiple synthetic and natural proteins by synthetic protease circuits to realize diverse signal processing capabilities, including logic operation and threshold tuning. By linking RELEASE to additional novel sensing and processing circuits, we were able to achieve elevated protein secretion in response to "undruggable" oncogene KRAS mutants. RELEASE should enable the local, programmable delivery of intercellular cues for a broad variety of fields such as neurobiology, cancer immunotherapy and cell transplantation.

category: synthetic biology


title: Explosive Synchronization-Based Brain Modulation Reduces Hypersensitivity in The Brain Network: A Computational Model Study

abstract:
Fibromyalgia (FM) is a chronic pain condition that is characterized by hypersensitivity to multi-modal sensory stimuli, widespread pain, and fatigue. We have previously proposed explosive synchronization (ES), a phenomenon wherein a small perturbation to a network can lead to an abrupt state transition, as a potential mechanism of the hypersensitive FM brain. Therefore, we hypothesized that converting a brain network from ES to general synchronization (GS) may reduce the hypersensitivity of FM brain. To find an effective brain network modulation to convert ES into GS, we constructed a large-scale brain network model near criticality (i.e., an optimally balanced state between order and disorders), which reflects brain dynamics in conscious wakefulness, and adjusted two parameters: local structural connectivity and signal randomness of target brain regions. The network sensitivity to global stimuli was compared between the brain networks before and after the modulation. We found that only increasing the local connectivity of hubs (nodes with intense connections) changes ES to GS, reducing the sensitivity, whereas other types of modulation such as decreasing local connectivity, increasing and decreasing signal randomness are not effective. This study would help to develop a network mechanism-based brain modulation method to reduce the hypersensitivity in FM.

Author summaryPhase transitions, the physical processes of transition between system states in nature, are divided into two broad categories: first and second-order phase transitions. For example, boiling water presents abrupt transition (a first-order) along with high sensitivity to temperature change, distinct from gradual magnetization near Curie temperature (a second-order). Recently, we found that chronic pain shows specific brain network configurations that can induce the first-order transition, so-called  explosive synchronization. In this modeling study, we tried to identify a modulation method that can convert a first-order transition into a second-order transition in the brain network, expecting that it may inhibit the hypersensitivity in chronic pain. We found that increasing structural connectivity of hubs changes the type of phase transition in the brain network, significantly reducing network sensitivity.


title: Preclinical Efficacy of IMM-BCP-01, a Highly Active Patient-Derived Anti-SARS-CoV-2 Antibody Cocktail

abstract:
Using an unbiased interrogation of the anti-viral memory B cell repertoire of convalescent COVID-19 patients, we identified three human antibodies that when combined demonstrated both robust viral suppressive properties against all tested SARS-CoV-2 variants of concern in vitro and profound anti-viral efficacy in vivo. In this report, we describe the pre-clinical characterization of an antibody cocktail, IMM-BCP-01, that consists of three unique, patient-derived recombinant antibodies directed at non-overlapping surfaces on the Spike protein, each with particularly effective antiviral activity. One antibody has a composite epitope blocking ACE2 binding, one antibody bridges two Spike proteins, and one antibody neutralizes virus by binding to a conserved epitope outside of ACE2 binding site. These antibodies, when administered after viral infection, potently decreased viral load in lungs of infected Syrian golden hamsters in a dose-dependent manner, elicited broad anti-viral neutralizing activity against multiple SARS-CoV-2 variants, and induced a robust anti-viral effector function response, including phagocytosis, and activation of classical complement pathway. Our pre-clinical data demonstrate that the unique three antibody cocktail IMM-BCP-01 is a potent and dose-efficient approach to treat early viral infection and prevent SARS-CoV-2 in susceptible individuals.

One sentence summaryWe describe three human antibodies that recognize unique non-overlapping epitopes on Spike protein and, when combined, exhibit highly potent in vitro activity against multiple SARS-CoV-2 variants of concern, including Delta, that translates into a striking dose-dependent efficacy against two SARS-CoV-2 isolates in vivo, augmented by a robust, rare epitope-driven Fc effector response.


title: Senataxin and RNase H2 act redundantly to suppress genome instability during class switch recombination

abstract: Class switch recombination generates antibody distinct isotypes critical to a robust adaptive immune system and defects are associated with auto-immune disorders and lymphomagenesis. Transcription is required during class switch to recruit the cytidine deaminase AID--an essential step for the formation of DNA double-strand breaks--and strongly induces the formation of R loops within the immunoglobulin heavy chain locus. However, the impact of R loops on double-strand break formation and repair during class switch recombination remains unclear. Here we report that cells lacking two enzymes involved in R loop removal-- Senataxin and RNase H2--exhibit increased R loop formation and genome instability at the immunoglobulin heavy chain locus without impacting class switch recombination efficiency, transcriptional activity, or AID recruitment. Senataxin and RNase H2-deficient cells also exhibit increased insertion mutations at switch junctions, a hallmark of alternative end joining. Importantly, these phenotypes were not observed in cells lacking Senataxin or RNase H2B alone. We propose that Senataxin acts redundantly with RNase H2 to mediate timely R loop removal, promoting efficient repair while suppressing AID-dependent genome instability and insertional mutagenesis.


title: Post-transcriptionally regulated genes are essential for pluripotent stem cell survival

abstract: The effects of transcription factors on the maintenance and differentiation of pluripotent stem cells (PSCs) have been well studied. However, the importance of post-transcriptional regulatory mechanisms, which cause the quantitative dissociation of mRNA and protein expression, has not been explored in detail. Here, by combining transcriptome and proteome profiling, we identified 228 post-transcriptionally regulated genes with strict upregulation of the protein level in PSCs. Among them, we found that 84 genes were vital for the survival of PSCs and HDFs, including 20 genes that were specifically necessary for the survival of PSCs. These 20 proteins were upregulated only in PSCs and not in differentiated cells derived from the three germ layers. Subcellular fractionation of the mRNA showed that the expression of most of the 20 proteins was regulated at the mRNA localization stage from the nucleus to the cytoplasm, but their translation efficiency was constant. Together, these results revealed that post-transcriptionally regulated genes have a crucial role in PSC survival.


title: Profiling of Nascent Lariat Intermediates Reveals Key Genetic Determinants of the Timing of Human Co-transcriptional Splicing

abstract:
As splicing is intimately coupled with transcription, understanding splicing mechanisms requires an understanding of splicing timing, which is currently limited. Here, we developed CoLa-seq (co-transcriptional lariat sequencing), a genomic assay that reports splicing timing relative to transcription through analysis of nascent lariat intermediates. In human cells, we mapped 165,282 branch points and characterized splicing timing for over 70,000 introns. Splicing timing varies dramatically across introns, with regulated introns splicing later than constitutive introns. Machine learning-based modeling revealed genetic elements predictive of splicing timing, notably the polypyrimidine tract, intron length, and regional GC content, which illustrate the significance of the broader genomic context of an intron and the impact of co-transcriptional splicing. The importance of the splicing factor U2AF in early splicing rationalizes surprising observations that most introns can splice independent of exon definition. Together, these findings establish a critical framework for investigating the mechanisms and regulation of co-transcriptional splicing.

HighlightsO_LICoLa-seq enables cell-type specific, genome-wide branch point annotation with unprecedented efficiency.
C_LIO_LICoLa-seq captures co-transcriptional splicing for tens of thousands of introns and reveals splicing timing varies dramatically across introns.
C_LIO_LIModeling uncovers key genetic determinants of splicing timing, most notably regional GC content, intron length, and the polypyrimidine tract, the binding site for U2AF2.
C_LIO_LIEarly splicing precedes transcription of a downstream 5 SS and in some cases accessibility of the upstream 3 SS, precluding exon definition.
C_LI


title: SARS-COV-2 γ variant acquires spike P681H or P681R for improved viral fitness

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) evolves and generates different variants through a continuously branching model. Four variants of concern have been the major pandemic drivers around the world. One important question is how they may evolve and generate subvariants, some of which may be even more virulent and drive the pandemic further. While investigating how {gamma} (or P.1) variant has been evolving, I noticed the spike substitution P681H in a group of genomes encoding a new subvariant, which has been designated P.1.7. This subvariant has become the dominant P.1 sublineage in Brazil, Italy, Spain and Peru, supporting that P681H confers evolutionary advantage to P.1.7. In Brazil and Peru, P.1.7 was still responsible for ~30% and ~40% cases, respectively, in August 2021. However, it has been competed out by {delta}1 (a {delta} subvariant) in both countries, Italy and Spain, suggesting that P.1.7 is not as virulent as {delta}1. In addition, 160 P.1 genomes possess a related substitution, P681R, and 120 of them encode a new subvariant, designated P.1.8. This P.1 subvariant carries two additional spike substitutions, T470N and C1235F, located at the receptor-binding pocket and cytoplasmic tail of spike protein, respectively. More P.1.8 genomes have been identified than P.1 genomes that encode P681R but not T470N and C1235F, suggesting that these two substitutions improve virulence of P.1.8 subvariant. Some P.1 genomes carry other substitutions (such as N679K, V687L and C1250F) that affect the furin cleavage site or cytoplasmic tail of spike protein. Thus, to improve viral fitness and expand its evolutionary cage, {gamma} variant acquires mutations to finetune the furin cleavage site and cytoplasmic tail of spike protein.

category: evolutionary biology


title: Comparative linkage mapping uncovers massive chromosomal inversions that suppress recombination between locally adapted fish populations

abstract: The role of recombination in genome evolution has long been studied in theory, but until recently empirical investigations had been limited to a small number of model species. Here we compare the recombination landscape and genome collinearity between two populations of the Atlantic silverside (Menidia menidia), a small fish distributed across the steep latitudinal climate gradient of the North American Atlantic coast. Using ddRADseq, we constructed separate linkage maps for locally adapted populations from New York and Georgia and their inter-population lab cross. First, we used one of the linkage maps to improve the current silverside genome assembly by anchoring three large unplaced scaffolds to two chromosomes. Second, we estimated sex-specific recombination rates, finding 2.75-fold higher recombination rates in females than males--one of the most extreme examples of heterochiasmy in a fish. While recombination occurs relatively evenly across female chromosomes, it is restricted to only the terminal ends of male chromosomes. Furthermore, comparisons of female linkage maps revealed suppressed recombination along several massive chromosomal inversions spanning nearly 16% of the genome and segregating between locally adapted populations. Finally, we discerned significantly higher recombination rates across chromosomes in the northern population. In addition to providing valuable resources for ongoing evolutionary and comparative genomic studies, our findings represent a striking example of structural variation that impacts recombination between adaptively divergent populations, providing empirical support for theorized genomic mechanisms facilitating adaptation despite gene flow.