title: ProtView: A versatile tool for in silico protease evaluation and selection in a proteomic and proteogenomic context

abstract: Tools have been created to generate in silico proteome digests with different protease enzymes and provide useful information for selecting optimal digest schemes for specific needs. This can save on time and resources and generate insights on the observable proteome. However, there remains a need for a tool that evaluates digest schemes beyond protein and amino acid coverages in the proteomic domain. Here, we present ProtView, a versatile in-silico protease/protease combination and digest evaluation workflow that maps in silico digested peptides to both protein and genome references, so that the potential observable sections of the proteome, transcriptome and genome can be identified. This supports the identification and quantification of the proteomic evidence of transcriptional, co-transcriptional, post-transcriptional and translational regulations. Benchmarking against biological data comparing multiple proteases shows that ProtView can correctly estimate the relative performances among the digest schemes. ProtView provides this information in a way that is easy to interpret, allowing for digest schemes to be evaluated before carrying out an analysis, in a broader context to optimize proteomic and proteogenomic experiments. ProtView is available at https://github.com/SSPuliasis/ProtView.


title: A MYB transcription factor, PlMYB308, plays an essential role in flower senescence of herbaceous peony

abstract: Herbaceous peony is an important cut-flower plant cultivated across the world, but its short vase life substantially restricts the economic value of this crop. It is well established that endogenous hormones regulate the senescing process, but the molecular mechanism of them in flower senescence is still unclear. Here, we isolated a MYB transcription factor gene PlMYB308 from herbaceous peony flowers. Transcript abundance of PlMYB308 was strongly up-regulated in senescing petals. Silencing of PlMYB308 resulted in delayed peony flower senescence, and dramatically increased gibberellin (GA) but reduced ethylene and abscisic acid (ABA) levels in petals. Ectopic overexpression of PlMYB308 in tobacco accelerated flower senescence, and reduced GA but increased ethylene and ABA accumulation. Correspondingly, biosynthetic genes of ethylene, ABA, and GA showed variable expression levels in petals after silencing or overexpression of PlMYB308. A dual-luciferase assay showed that PlMYB308 specifically bound to the PlACO1 promoter. High expression levels of PlMYB308 were accompanied by low petal anthocyanin accumulation in senescing petals. A further bimolecular fluorescence complementation assay revealed an interaction between PlMYB308 and PlbHLH33, which was supposed to inhibit the anthocyanin biosynthesis. Taken together, our results suggest that the PlMYB308-PlACO1 and PlMYB308-PlbHLH33 regulatory checkpoints perhaps positively and negatively operate the production of ethylene and anthocyanin, respectively, and thus contribute to the senescence with impaired pigmentation in herbaceous peony flowers.

category: biophysics


title: How To Extend The Capabilities Of A Commercial Two-Photon Microscope To Perform Super-Resolution Imaging, Wavelength Mixing And Label-Free Microscopy.

abstract: Multimodal microscopy combines multiple non-linear techniques that take advantage of different optical processes to generate contrast and increase the amount of information that can be obtained from biological samples. However, the most advanced optical architectures are typically custom-made and require complex alignment procedures, as well as daily maintenance by properly trained personnel for optimal performance. Here, we describe a hybrid system we constructed to overcome these disadvantages by modifying a commercial upright microscope. We show that our multimodal imaging platform can be used to seamlessly perform two-photon STED, wavelength mixing and label-free microscopy in both ex vivo and in vivo samples. The system is highly stable and endowed with remote alignment hardware that ensures simplified operability for non-expert users. This optical architecture is an important step forward towards a wider practical applicability of non-linear optics to bioimaging.


title: Discovery of highly potent pancoronavirus fusion inhibitors that also effectively inhibit COVID-19 variants from the UK and South Africa

abstract: We report here the discovery of several highly potent small molecules that showed low nM potency against SARS-CoV (IC50: as low as 13 nM), SARS-CoV-2 (IC50: as low as 23 nM), and MERS-CoV (IC50: as low as 76 nM) in pseudovirus based assays with excellent selectivity indices (SI: as high as > 5000) demonstrating their pancoronavirus inhibition. Some compounds also show 100% inhibition of CPE (IC100) at 1.25 {micro}M against an authentic SARS-CoV-2 (US_WA-1/2020). Furthermore, the most active inhibitors also potently inhibited variants of concerns (VOCs), such as the UK (B.1.1.7), South Africa (B.1.351), and Delta variant (B.1.617.2), originated in India. We confirmed that one of the potent inhibitors binds to the prefusion spike protein trimer of SARS-CoV-2 by SPR. Besides, we showed that they inhibit virus-mediated cell-cell fusion. The ADME data of one of the most active inhibitors, NBCoV1, show drug-like properties. In vivo PK of NBCoV1 in rats demonstrated excellent half-life (t1/2) of 11.3 h, mean resident time (MRT) of 14.2 h, and oral bioavailability. We expect the lead inhibitors to pave the way for further development to preclinical and clinical candidates.

category: pharmacology and toxicology


title: Discovery of highly potent pancoronavirus fusion inhibitors that also effectively inhibit COVID-19 variants from the UK (Alpha), South Africa (Beta), and India (Delta)


title: Integrated Omics Analysis Reveals Sirtuin Signaling is Central to Hepatic Response to a High Fructose Diet

abstract:
BackgroundDietary high fructose (HFr) is a known metabolic disruptor contributing to development of obesity and diabetes in Western societies. Initial molecular changes from exposure to HFr on liver metabolism may be essential to understand the perturbations leading to insulin resistance and abnormalities in lipid and carbohydrate metabolism. We studied vervet monkeys (Clorocebus aethiops sabaeus) fed a HFr (n=5) or chow diet (n=5) for 6 weeks, and obtained clinical measures of liver function, blood insulin, cholesterol and triglycerides. In addition, we performed untargeted global transcriptomics, proteomics, and metabolomics analyses on liver biopsies to determine the molecular impact of a HFr diet on coordinated pathways and networks that differed by diet.

ResultsWe show that integration of omics data sets improved statistical significance for some pathways and networks, and decreased significance for others, suggesting that multiple omics datasets enhance confidence in relevant pathway and network identification. Specifically, we found that sirtuin signaling and a peroxisome proliferator activated receptor alpha (PPARA) regulatory network were significantly altered in hepatic response to HFr. Integration of metabolomics and miRNAs data further strengthened our findings.

ConclusionsOur integrated analysis of three types of omics data with pathway and regulatory network analysis demonstrates the usefulness of this approach for discovery of molecular networks central to a biological response. In addition, metabolites aspartic acid and docosahexaenoic acid (DHA), protein ATG3, and genes ATG7, HMGCS2 link sirtuin signaling and the PPARA network suggesting molecular mechanisms for altered hepatic gluconeogenesis from consumption of a HFr diet.

category: systems biology


title: A Compressive Sensing Approach to Inferring Cognitive Representations with Reverse Correlation

abstract:
Uncovering high-level cognitive representations of categories such as faces is an elusive goal that has been frequently sought using reverse correlation, a technique employed in fields ranging from neurophysiology to cognitive psychology. In reverse correlation, subjects are asked to make perceptual judgments (e.g., "do you see a face?") about richly varying stimuli (e.g., white noise), and observed responses are then regressed against stimuli to yield reconstructions of the underlying cognitive representation. However, many thousands of stimulus-response pairs are frequently required, which severely limits the breadth of studies that are feasible using this powerful method. Techniques that are currently employed to improve efficiency, such as filtering the reconstruction, nevertheless bias the outcome. Here, we show that an advanced signal processing technique for improving sampling efficiency - compressive sensing - is directly compatible with reverse correlation. A trio of simulations are performed to demonstrate that compressive sensing can reduce the required stimulus-response pairs by up to 90% without biasing the reconstruction or can retrospectively improve the accuracy of the reconstructions on existing data. This work concludes by outlining the potential of compressive sensing to improve representation reconstruction throughout the field of neuroscience and beyond.

Significance StatementUncovering cognitive representations is an elusive goal that is increasingly pursued using the reverse correlation method, wherein human subjects make judgments about vague stimuli. Employing reverse correlation often entails collecting thousands of stimulus-response pairs, which severely limits the breadth of studies that are feasible using the method. Here we show that this methodological barrier can be overcome using compressive sensing, an advanced signal processing technique designed to improve sampling efficiency. Three sets of simulations are performed to demonstrate that compressive sensing can improve the accuracy of reconstructed cognitive representations and dramatically reduce the required number of stimulus-response pairs. This work concludes by outlining the potential of compressive sensing to improve representation reconstruction throughout the field of neuroscience and beyond.

category: neuroscience


title: Human spindle variability

abstract:
In humans, sleep spindles are 10-16 Hz oscillations lasting approximately 0.5-2 s. Spindles, along with cortical slow oscillations, facilitate memory consolidation by enabling synaptic plasticity. Early recordings of spindles at the scalp found anterior channels had overall slower frequency than central-posterior channels. This robust, topographical finding led to dichotomizing spindles as  slow versus  fast, modelled as two distinct spindle generators in frontal versus posterior cortex. Using a large dataset of intracranial sEEG recordings (n=20, 365 bipolar recordings), we show that the difference in spindle frequency between frontal and parietal channels is comparable to the variability in spindle frequency within the course of individual spindles, across different spindles recorded by a given site, and across sites within a given region. Thus, fast and slow spindles only capture average differences that obscure a much larger underlying overlap in frequency. Furthermore, differences in mean frequency are only one of several ways that spindles differ. For example, compared to parietal, frontal spindles are smaller, tend to occur after parietal when both are engaged, and show a larger decrease in frequency within-spindles. However, frontal and parietal spindles are similar in being longer, less variable, and more widespread than occipital, temporal, and Rolandic spindles. These characteristics are accentuated in spindles which are highly phase-locked to posterior hippocampal spindles. We propose that rather than a strict parietal-fast/frontal-slow dichotomy, spindles differ continuously and quasi-independently in multiple dimensions, with variability due about equally to within-spindle, within-region and between-region factors.

Significance StatementSleep spindles are 10-16 Hz neural oscillations generated by cortico-thalamic circuits that promote memory consolidation. Spindles are often dichotomized into slow-anterior and fast-posterior categories for cognitive and clinical studies. Here, we show that the anterior-posterior difference in spindle frequency is comparable to that observed between different cycles of individual spindles, between spindles from a given site, or from different sites within a region. Further, we show that spindles vary on other dimensions such as duration, amplitude, spread, primacy and consistency, and that these multiple dimensions vary continuously and largely independently across cortical regions. These findings suggest that multiple continuous variables rather than a strict frequency dichotomy may be more useful biomarkers for memory consolidation or psychiatric disorders.


title: SYNAPTIC DYSFUNCTION UNDERLIES ALTERATIONS IN THE INITIATION OF GOAL-DIRECTED BEHAVIORS: IMPLICATIONS FOR HIV-1 ASSOCIATED APATHY

abstract: Individuals living with human immunodeficiency virus type 1 (HIV-1) exhibit an increased prevalence of neuropsychiatric comorbities (e.g., apathy) relative to their seronegative counterparts. Given the profound functional consequences associated with apathy, conceptualizing the multidimensional neuropsychiatric syndrome, and associated neural mechanisms, following chronic HIV-1 viral protein exposure remains a critical need. HIV-1 associated apathy was examined by quantifying goal-directed behaviors, indexed using voluntary wheel running, during the diurnal and nocturnal cycle. Apathetic behaviors in the HIV-1 Tg rat were characterized by a profound decrease in the number of running bouts during both the diurnal and nocturnal cycle, supporting a prominent deficit in the self-initiation of spontaneous behaviors. Additionally, HIV-1 Tg animals exhibited a decreased reinforcing efficacy of voluntary wheel running during the nocturnal cycle. Following the completion of voluntary wheel running, synaptic dysfunction in medium spiny neurons (MSNs) of the nucleus accumbens (NAc) was examined as a potential neural mechanism underlying HIV-1 associated apathy. HIV-1 Tg animals displayed prominent synaptic dysfunction in MSNs of the NAc, characterized by decreased synaptic connectivity and a population shift towards an immature dendritic spine phenotype relative to control animals. Synaptic dysfunction accounted for 42.0% to 68.5% of the variance in the number of running bouts affording a key neural mechanism underlying the self-initiation of spontaneous behaviors. The establishment of a fundamental neural mechanism underlying apathy affords a key target for the development of novel therapeutics and cure strategies for affective alterations associated with HIV-1.


title: Patch-Clamp Proteomics of Single Neuronal Somas in Tissue Using Electrophysiology and Subcellular Capillary Electrophoresis Mass Spectrometry

abstract: Understanding of the relationship between cellular function and molecular composition holds a key to next-generational therapeutics but requires measurement of all types of molecules in cells. Developments in sequencing enabled semi-routine measurement of single-cell genomes and transcriptomes, but analytical tools are scarce for detecting diverse proteins in tissue-embedded cells. To bridge this gap for neuroscience research, we report the integration of patch-clamp electrophysiology with subcellular shot-gun proteomics by high-resolution mass spectrometry (HRMS). Recording of electrical activity permitted identification of dopaminergic neurons in the substantia nigra pars compacta. Ca. 20-50% of the neuronal soma content, containing an estimated 100 pg of total protein, was aspirated into the patch pipette filled with ammonium bicarbonate. About ~1 pg of somal protein, or ~0.25% of the total cellular proteome, was analyzed on a custom-built capillary electrophoresis (CE) high-resolution mass spectrometer (HRMS). A series of experiments were conducted to systematically enhance detection sensitivity through refinements in sample processing and detection, allowing us to quantify ~275 different proteins from somal aspirate-equivalent protein digests. From single neurons, patch-clamp proteomics of the soma quantified 91, 80, and 95 different proteins from 3 different dopaminergic neurons, or 157 proteins in total. Quantification revealed detectable proteomic differences between the somal protein samples. Analysis of canonical knowledge predicted rich interaction networks between the proteins. The integration of patch-clamp electrophysiology with subcellular CE-HRMS proteomics expands the analytical toolbox of neuroscience.


title: Activation of the Zinc-sensing receptor GPR39 promotes T cell reconstitution after hematopoietic stem cell transplant

abstract:
Prolonged lymphopenia represents a major clinical problem after cytoreductive therapies such as chemotherapy and the conditioning required for hematopoietic stem cell transplant (HCT), contributing toward the risk of infections and malignant relapse. Restoration of T cell immunity is dependent on tissue regeneration in the thymus, the primary site of T cell development; although the capacity of the thymus to repair itself diminishes over lifespan. However, although boosting thymic function and T cell reconstitution is of considerable clinical importance, there are currently no approved therapies for treating lymphopenia. Here we found that Zinc (Zn), is critically important for both normal T cell development as well as repair after acute damage. Accumulated Zn in thymocytes during development was released into the extracellular milieu after HCT conditioning, where it triggered regeneration by stimulating endothelial cell-production of BMP4 via the cell surface receptor GPR39. Dietary supplementation of Zn was sufficient to promote thymic function in a mouse model of allogeneic HCT, including enhancing the number of recent thymic emigrants in circulation; although direct targeting of GPR39 with a small molecule agonist enhanced thymic function without the need for prior Zn accumulation in thymocytes. Together, these findings not only define an important pathway underlying tissue regeneration, but also offer an innovative preclinical approach to treat lymphopenia in HCT recipients.

KEY POINTSO_LIThymocytes release zinc after HCT conditioning is sensed by GPR39 and promotes epithelial repair
C_LIO_LIPharmacologic stimulation of GPR39 promotes T cell reconstitution after HCT
C_LI

category: immunology


title: Macrophages in the uterus are functionally specialised and continually replenished from the circulation

abstract:
Macrophages are innate immune cells that fight infection but also regulate tissue regeneration and remodelling. In the uterus, although tissue remodelling is essential for establishment and maintenance of pregnancy, the specialisation of macrophages is not well characterised compared to other mucosal tissues. Here we show that uterine macrophages are functionally specialised, expressing multiple markers of alternative activation associated with tissue remodelling and repair, and responding more highly to the type 2 cytokine IL-4 than other mucosal tissue macrophages. Uterine macrophages were continuously replenished from circulating bone marrow-derived CCR2+ monocytes that fluctuated dramatically in number throughout the reproductive cycle, and had properties distinct from the macrophages that they became, including differential responses to microbial stimulation. Importantly, many of these properties of uterine monocytes and macrophages were conserved between mice and humans. These findings further our understanding of immune regulation of uterine tissue integrity and have important implications for differences in immune responses to infections at different phases of the reproductive cycle.

SUMMARYUterine macrophages are specialised, alternatively activated cells that are replenished from circulating bone marrow-derived monocytes. Monocyte and macrophage properties fluctuate markedly throughout the reproductive cycle, with many features conserved between mice and humans, and exhibiting differential responses to microbial stimulation.


title: Modelling rheumatoid arthritis: A hybrid modelling framework to describe pannus formation in a small joint

abstract: Rheumatoid arthritis (RA) is a chronic inflammatory disorder that causes pain, swelling and stiffness in the joints, and negatively impacts the life of affected patients. The disease does not have a cure yet, as there are still many aspects of this complex disorder that are not fully understood. While mathematical models can shed light on some of these aspects, to date there are few such models that can be used to better understand the disease. As a first step in the mechanistic understanding of RA, in this study we introduce a new hybrid mathematical modelling framework that describes pannus formation in a small proximal interphalangeal (PIP) joint. We perform numerical simulations with this new model, to investigate the impact of different levels of immune cells (macrophages and fibroblasts) on the degradation of bone and cartilage. Since many model parameters are unknown and cannot be estimated due to a lack of experiments, we also perform a sensitivity analysis of model outputs to various model parameters (single parameters or combinations of parameters). Finally, we discuss how our model could be applied to investigate current treatments for RA, for example, methotrexate, TNF-inhibitors or tocilizumab, which can impact different model parameters.


title: KappaBle fluorescent reporter mice enable dynamic and low-background single-cell detection of NF-kB activity in vivo

abstract: Nuclear factor-{kappa}B (NF-{kappa}B) is a transcription factor with a key role in a great variety of cellular processes from embryonic development to immunity, the outcome of which depends on the fine-tuning of NF-{kappa}B activity. The development of sensitive and faithful reporter systems to accurately monitor the activation status of this transcription factor is therefore desirable. To address this need, over the years a number of different approaches have been used to generate NF-{kappa}B reporter mice, which can be broadly subdivided into bioluminescence- and fluorescence-based systems. While the former enables whole-body visualization of the activation status of NF-{kappa}B, the latter have the potential to allow the analysis of NF-{kappa}B activity at single cell level. However, fluorescence-based reporters frequently show poor sensitivity and excessive background or are incompatible with high-throughput flow cytometric analysis. In this work we describe the generation and analysis of ROSA26 knockin NF-{kappa}B reporter (KappaBle) mice containing a destabilized EGFP, which showed sensitive, dynamic, and faithful monitoring of NF-{kappa}B activity at the single-cell level of various cell types during inflammatory and infectious diseases.


title: Optimization of single dose VSV-based COVID-19 vaccination in hamsters

abstract: The ongoing COVID-19 pandemic has resulted in global effects on human health, economic stability, and social norms. The emergence of viral variants raises concerns about the efficacy of existing vaccines and highlights the continued need the for the development of efficient, fast-acting, and cost-effective vaccines. Here, we demonstrate the immunogenicity and protective efficacy of two vesicular stomatitis virus (VSV)-based vaccines encoding the SARS-CoV-2 spike protein either alone (VSV-SARS2) or in combination with the Ebola virus glycoprotein (VSV-SARS2-EBOV). Intranasally vaccinated hamsters showed an early CD8+ T cell response in the lungs and a greater antigen-specific IgG response, while intramuscularly vaccinated hamsters had an early CD4+ T cell and NK cell response. Intranasal vaccination resulted in protection within 10 days with hamsters not showing clinical signs of pneumonia when challenged with three different SARS-CoV-2 variants. This data demonstrates that VSV-based vaccines are viable single-dose, fast-acting vaccine candidates that are protective from COVID-19.

category: microbiology


title: Time to stop; agent-based modelling of chemoaffinity with competition

abstract: In the classic Chemoaffinity theory, the retinotectal axon projection is thought to use pairs of orthogonal signalling gradients in the retina to specify the eventual location of synapses made on the surface of the tectum/superior colliculus. Similar orthogonal gradients in the tectum provide a coordinate system which allows the axons to match their prespecified destination with the correct location. Although the Ephrins have been shown to guide axons toward their destination, there has yet to emerge a complete account of the local interactions which halt the axonal growth cones in the correct locations to recreate the topography of the retinal cells. The model of Simpson and Goodhill (2011) provides an account of the basic topographic arrangement of cells on the tectum, as well as reproducing well known surgical and genetic manipulation experiments. However, it suffers from the absence of a local chemotactic guidance mechanism. Instead, each agent in their model is given instantaneous knowledge of the vector that would move it toward its pre specified destination. In addition to the globally supervised chemoaffinity term, Simpson and Goodhill (2011) introduced a competitive interaction for space between growth cone agents and a receptor-ligand axon-axon interaction in order to account for the full set of experimental manipulations. Here, we propose the replacement of the chemoaffinity term with a gradient following model consisting of axonal growth cone agents which carry receptor molecule expression determined by their somas location of origin on the retina. Growth cones move on the simulated tectum guided by two pairs of opposing, orthogonal signalling molecules representing the Ephrin ligands. We show that with only the chemoaffinity term and a receptor-ligand based axon-axon interaction term (meaning that all growth cone interactions are by receptor-ligand signalling), a full range of experimental manipulations to the retinotectal system can be reproduced. Furthermore, we show that the observation that competition is not and essential requirement for axons to find their way (Gosse et al., 2008) is also accounted for by the model, due to the opposing influences of signalling gradient pairs. Finally, we demonstrate that, assuming exponentially varying receptor expression in the retina, ligand expression should either be exponential if the receptor-ligand signal induces repulsion (i.e. gradient descent) or logarithmic if the signal induces attraction (gradient ascent). Thus, we find that a model analogous to the one we presented in James et al. (2020) that accounts for murine barrel patterning is also a candidate mechanism for the arrangement of the more continuous retinotectal system.

category: developmental biology


title: Implications of sample treatment on characterization of the riverine environmental metabolome

abstract:
High-resolution mass spectrometry techniques are widely used in the environmental sciences to characterize natural organic matter and, when utilizing these instruments, researchers must make multiple decisions regarding sample pre-treatment and the instrument ionization mode. To identify how these choices alter organic matter characterization and resulting conclusions, we analyzed a collection of 17 riverine samples from East River, CO (USA) under four PPL-based Solid Phase Extraction (SPE) treatment and electrospray ionization polarity (e.g., positive and negative) combinations: SPE (+), SPE (-), non-SPE (-), and non-SPE (+). The greatest number of formula assignments were achieved with SPE-treated samples due to the removal of compounds that could interfere with ionization. Furthermore, the SPE (-) treatment captured the most formulas across the widest chemical compound diversity. In addition to a reduced number of assigned formulas, the non-SPE datasets resulted in altered thermodynamic interpretations that could cascade into incomplete assumptions about the availability of organic matter pools for microbial heterotrophic respiration. Thus, we infer that the SPE (-) treatment is the best single method for characterizing environmental organic matter pools unless the focus is on lipid-like compounds, in which case we recommend a combination of SPE (-) and SPE (+) to adequately characterize these molecules.

SynopsisWe provide data-driven sample treatment and ionization mode recommendations to researchers who aim to use high-resolution mass spectrometry to characterize environmental organic matter.

Abstract Art O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

category: ecology


title: Consequences of coupled barriers to gene flow for the build-up of genomic differentiation

abstract: Theory predicts that when different barriers to gene flow become coincident, their joint effects enhance reproductive isolation and genomic divergence beyond their individual effects, but empirical tests of this  coupling hypothesis are rare. Here, we analyze patterns of gene exchange among populations of European corn borer moths that vary in the number of acting barriers, allowing for comparisons of genomic variation when barrier traits or loci are in coincident or independent states. We find that divergence is mainly restricted to barrier loci when populations differ by a single barrier, whereas the coincidence of temporal and behavioral barriers is associated with divergence of two chromosomes harboring barrier loci. Furthermore, differentiation at temporal barrier loci increases in the presence of behavioral divergence and differentiation at behavioral barrier loci increases in the presence of temporal divergence. Our results demonstrate how the joint action of coincident barrier effects leads to levels of genomic differentiation that far exceed those of single barriers acting alone, consistent with theory arguing that coupling allows indirect selection to combine with direct selection and thereby lead to a stronger overall barrier to gene flow. Thus, the state of barriers - independent or coupled - strongly influences the accumulation of genomic differentiation.

category: evolutionary biology


title: Single-cell transcriptome analysis reveals mesenchymal stem cells in cavernous hemangioma

abstract:
A cavernous hemangioma, well-known as vascular malformation, is present at birth, grows proportionately with the child, and does not undergo regression. Although a cavernous hemangioma has well-defined histopathological characteristics, its origin and formation remain unknown. In the present study, we characterized the cellular heterogeneity of cavernous hemangioma using single-cell RNA sequencing (scRNA-seq). The main contribution of this study is the discovery of mesenchymal stem cells (MSCs) that cause tumour formation in cavernous hemangioma and we propose that these MSCs may be abnormally differentiated or incompletely differentiated from epiblast stem cells.

Other new findings include the responsive ACKR1 positive endothelial cell (ACKR1+EC) and BTNL9 positive endothelial cell (BTNL9+EC) and the BTNL9-caused checkpoint blockade enhanced by the CXCL12-CXCR4 signalling. The activated CD8+T and NK cells may highly express CCL5 for their infiltration in cavernous hemangiomas, independent on the tumor cell-derived CCL5-IFNG-CXCL9 pathway. The highly co-expression of CXCR4 and GZMB suggested that plasmacytoid dendritic cells (pDCs) function for anti-tumour as CD8+T cells in cavernous hemangiomas. The oxidised low-density lipoprotein (oxLDL) in the TME of cavernous hemangiomas may play an important role as a signalling molecular in the immune responses. Notably, we propose that oxLDL induces the oxLDL-OLR1-NLRP3 pathway by over-expression of OLR1 in M1-like macrophages, whereas oxLDL induces the oxLDL-SRs-C1q (SRs are genes encoding scavenger receptors of oxLDL except OLR1) pathway by over-expression of other scavenger receptors in M2-like macrophages.

The present study revealed the origin of cavernous hemangiomas and discovered marker genes, cell types and molecular mechanisms associated with the origin, formation, progression, diagnosis or therapy of cavernous hemangiomas. The information from the present study makes important contributions to the understanding of cavernous hemangioma formation and progression and facilitates the development of gold standard for molecular diagnosis and effective drugs for treatment.

category: cancer biology


title: Neurodevelopmental processes in the prefrontal cortex derailed by chronic HIV-1 viral protein exposure

abstract: Due to the widespread access to, and implementation of, combination antiretroviral therapy, individuals perinatally infected with human immunodeficiency virus type 1 (HIV-1) are living into adolescence and adulthood. Perinatally infected adolescents living with HIV-1 (pALHIV) are plagued by progressive, chronic neurocognitive impairments; the pathophysiological mechanisms underlying these deficits, however, remains understudied. A longitudinal experimental design from postnatal day (PD) 30 to PD 180 was utilized to establish the development of pyramidal neurons, and associated dendritic spines, from layers II-III of the medial prefrontal cortex (mPFC). Three putative neuroinflammatory markers (i.e., IL-1{beta}, IL-6, and TNF-) were evaluated early in development (i.e., PD 30) as a potential mechanism underlying synaptic dysfunction in the mPFC. Constitutive expression of HIV-1 viral proteins induced prominent neurodevelopmental alterations, independent of biological sex, in pyramidal neurons from layers II-III of the mPFC. Specifically, HIV-1 transgenic rats exhibited prominent deficits in dendritic and synaptic pruning, a developmental decrease in synaptic connectivity, and an age-related decline in synaptic efficacy. Examination of dendritic spine morphology revealed an age-related population shift towards a more immature dendritic spine phenotype in HIV-1 transgenic animals. There was no compelling evidence for neuroinflammation in the mPFC during early development. Understanding the neural mechanisms underlying chronic neurocognitive impairments in pALHIV may afford a key target for innovative therapeutics and cure strategies; an urgent need given the growing population of pALHIV.


title: Metaproteomics as a tool for studying the protein landscape of human-gut bacterial species

abstract:
Host-microbiome interactions and the microbial community have broad impact in human health and diseases. Most microbiome based studies are performed at the genome level based on next-generation sequencing techniques, but metaproteomics is emerging as a powerful technique to study microbiome functional activity by characterizing the complex and dynamic composition of microbial proteins. We conducted a large-scale survey of human gut microbiome metaproteomic data to identify generalist species that are ubiquitously expressed across all samples and specialists that are highly expressed in a small subset of samples associated with a certain phenotype. We were able to utilize the metaproteomic mass spectrometry data to reveal the protein landscapes of these species, which enables the characterization of the expression levels of proteins of different functions and underlying regulatory mechanisms, such as operons. Finally, we were able to recover a large number of open reading frames (ORFs) with spectral support, which were missed by de novo protein-coding gene predictors. We showed that a majority of the rescued ORFs overlapped with de novo predicted proteincoding genes, but on opposite strands or on different frames. Together, these demonstrate applications of metaproteomics for the characterization of important gut bacterial species. Results are available for public access at https://omics.informatics.indiana.edu/GutBac.

Author summaryMany reference genomes for studying human gut microbiome are available, but knowledge about how microbial organisms work is limited. Identification of proteins at individual species or community level provides direct insight into the functionality of microbial organisms. By analyzing more than a thousand metaproteomics datasets, we examined protein landscapes of more than two thousands of microbial species that may be important to human health and diseases. This work demonstrated new applications of metaproteomic datasets for studying individual genomes. We made the analysis results available through the GutBac website, which we believe will become a resource for studying microbial species important for human health and diseases.

category: bioinformatics


title: Sex differences in central and peripheral fatigue induced by sustained isometric ankle plantar flexion

abstract: The main aim of this study was to determine sex differences in central and peripheral fatigue produced by a sustained isometric exercise of ankle plantar flexors in healthy young adults. Ten males and fourteen females performed a sustained isometric ankle exercise until task failure. Maximal voluntary isometric contraction torque (plantarflexion), voluntary activation level (using the twitch interpolation technique), and twitch contractile properties (twitch peak torque, twitch half relaxation time, and low frequency fatigue index) were measured before, immediately after, and throughout a recovery period (1, 2, 5, and 10 min) following the exercise protocol in order to characterize neuromuscular fatigue. Fatigue had a significant effect (p [≤] 0.05) on all dependent variables. Other than for the maximal voluntary contraction torque, where males showed a greater fatigue-related decrease than females, males and females showed generally similar changes with fatigue. Altogether, our findings indicate no major differences in central or peripheral fatigue mechanisms between males and females to explain a somewhat greater fatigability in males.


title: The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes

abstract: Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50{micro}M) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0-100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3 and the 5 splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 {micro}M). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.

category: molecular biology


title: Automatic variable selection in ecological niche modeling: A case study using Cassins Sparrow (Peucaea cassinii)

abstract: MERRA/Max provides a feature selection approach to dimensionality reduction that enables direct use of global climate model outputs in ecological niche modeling. The system accomplishes this reduction through a Monte Carlo optimization in which many independent MaxEnt runs, operating on a species occurrence file and a small set of randomly selected variables in a large collection of variables, converge on an estimate of the top contributing predictors in the larger collection. These top predictors can be viewed as potential candidates in the variable selection step of the ecological niche modeling process. MERRA/Maxs Monte Carlo algorithm operates on files stored in the underlying filesystem, making it scalable to large data sets. Its software components can run as parallel processes in a high-performance cloud computing environment to yield near real-time performance. In tests using Cassins Sparrow (Peucaea cassinii) as the target species, MERRA/Max selected a set of predictors from Worldclims Bioclim collection of 19 environmental variables that have been shown to be important determinants of the species bioclimatic niche. It also selected biologically and ecologically meaningful predictors from a more diverse set of 86 environmental variables derived from NASAs Modern-Era Retrospective Analysis for Research and Applications Version 2 (MERRA-2) reanalysis, an output product of the Goddard Earth Observing System Version 5 (GEOS-5) modeling system. We believe these results point to a technological approach that could expand the use global climate model outputs in ecological niche modeling, streamline the modeling process, and, eventually, enable automated bioclimatic modeling as a practical, readily accessible, low-cost, commercial cloud service.


title: A critical role of brain network architecture in a continuum model of autism spectrum disorders spanning from healthy individuals with genetic liability to individuals with ASD

abstract: Studies have shown cortical alterations in individuals with autism spectrum disorders (ASD) as well as in individuals with high polygenic risk for ASD. An important addition to the study of altered cortical anatomy is the investigation of the underlying brain network architecture that may reveal brain-wide mechanisms in ASD and in polygenic risk for ASD. Such an approach has been proven useful in other psychiatric disorders by revealing that brain network architecture shapes (to an extent) the disorder-related cortical alterations. This study uses data from a clinical dataset - 560 male subjects (266 individuals with ASD and 294 healthy individuals, CTL, mean age at 17.2 years) from the Autism Brain Imaging Data Exchange database, and data of 391 healthy individuals (207 males, mean age at 12.1 years) from the Pediatric Imaging, Neurocognition and Genetics database. ASD-related cortical alterations (group difference, ASD-CTL, in cortical thickness) and cortical correlates of polygenic risk for ASD were assessed, and then statistically compared with structural connectome-based network measures (such as hubs) using spin permutation tests. Next, we investigated whether polygenic risk for ASD could be predicted by network architecture by building machine-learning based prediction models, and whether the top predictors of the model were identified as disease epicenters of ASD. We observed that ASD-related cortical alterations as well as cortical correlates of polygenic risk for ASD implicated cortical hubs more strongly than non-hub regions. We also observed that age progression of ASD-related cortical alterations and cortical correlates of polygenic risk for ASD implicated cortical hubs more strongly than non-hub regions. Further investigation revealed that structural connectomes predicted polygenic risk for ASD (r=0.30, p<0.0001), and two brain regions (the left inferior parietal and left suparmarginal) with top predictive connections were identified as disease epicenters of ASD. Our study highlights a critical role of network architecture in a continuum model of ASD spanning from healthy individuals with genetic risk to individuals with ASD. Our study also highlights the strength of investigating polygenic risk scores in addition to multi-modal neuroimaging measures to better understand the interplay between genetic risk and brain alterations associated with ASD.


title: Dopamine-dependent cAMP dynamics in basal amygdala glutamatergic neurons

abstract: Dopaminergic inputs to basal amygdala (BA) instruct learning of motivational salience. Here, we investigated the dynamics of dopamine release and downstream signaling during multiple salient events occurring within tens of seconds. We established in vitro and in vivo real-time tracking and manipulation of cAMP - a key intracellular plasticity signal downstream of dopamine receptor activation. Optogenetically-evoked release of dopamine drove proportional increases in cAMP in almost all BA glutamatergic neurons, suggesting widespread actions of dopamine across neurons preferring positive or negative valence. These cAMP responses decayed more slowly than dopamine release, potentially extending the window of plasticity. cAMP levels accumulated following direct photostimulation of cAMP but not repeated stimulation of dopamine axons, due to potent depression of dopamine release. cAMP and protein kinase A (PKA) responses to repeated appetitive or aversive stimuli also exhibited pronounced depression. Thus, history-dependent dynamics of dopamine and cAMP may regulate learning of temporally clustered, salient stimuli.


title: Multifaceted N-degron recognition and ubiquitylation by GID/CTLH E3 ligases

abstract: N-degron E3 ubiquitin ligases recognize specific residues at the N-termini of substrates. Although molecular details of N-degron recognition are known for several E3 ligases, the range of N-terminal motifs that can bind a given E3 substrate binding domain remains unclear. Here, studying the Gid4 and Gid10 substrate receptor subunits of yeast "GID"/human "CTLH" multiprotein E3 ligases, whose known substrates bear N-terminal prolines, we discovered capacity for high-affinity binding to diverse N-terminal sequences determined in part by context. Screening of phage displaying peptide libraries with exposed N-termini identified novel consensus motifs with non-Pro N-terminal residues distinctly binding Gid4 or Gid10 with high affinity. Structural data reveal that flexible loops in Gid4 and Gid10 conform to complementary folds of diverse interacting peptide sequences. Together with analysis of endogenous substrate degrons, the data show that degron identity, substrate domains harboring targeted lysines, and varying E3 ligase higher-order assemblies combinatorially determine efficiency of ubiquitylation and degradation.

category: biochemistry


title: A novel widespread bacterial structure related to the flagellar type III secretion system

abstract: The flagellar type III secretion system (fT3SS) is a suite of membrane-embedded and cytoplasmic proteins responsible for building the bacterial flagellar motility machinery. Homologous proteins form the injectisome machinery bacteria use to deliver effector proteins into eukaryotic cells, and other family members have recently been reported to be involved in the formation of membrane nanotubes. Here we describe a novel, ubiquitous and evolutionarily widespread hat-shaped structure embedded in the inner membrane of bacteria, of yet-unidentified function, that is related to the fT3SS, adding to the already rich repertoire of this family of nanomachines.


title: CRISPRs are associated with bacterial expansion in the human gut during the first year of life

abstract: The dynamics of the gut microbiome in infancy have a profound impact on health in adulthood, but the ecological mechanism underlying the dynamics between bacteria and bacteriophages remains poorly understood. CRISPR is a bacterial adaptive immune system to resist bacteriophages; however, the role they play in the dynamics in infants gut microbiota is unknown. In this work, using large-scale metagenomic sequencing data from 82 Sweden infants gut microbiomes, 1882 candidate CRISPRs were identified and their dynamics were analyzed. The results showed CRISPRs were distributed in dominant bacteria and could target distinct bacteriophages at different time points with largely alternated spacers. In the putative identical CRISPRs, we found the CRISPRs could acquire new spacers and loss old spacers during the first year. In addition, it is the first time to report that gender was a major factor to determine the bacterial richness and the number of CRISPRs and host range of bacteriophages was narrow in silico. Therefore, we concluded that CRISPRs were associated with bacterial expansion. This work improves the understanding of the ecological mechanism behind dynamics in the early life of the human gut and substantially expands the repertoire of predicted CRISPRs providing a resource to study the function of bacterial unknown genes and to enhance the performance of these beneficial bacteria by CRISPR gene-editing technology.


title: The m6A reader YTHDC2 is essential for escape from KSHV SOX-induced RNA decay

abstract: The role m6A modifications have increasingly been associated with diverse set of roles in modulating viruses and influencing the outcomes of viral infection. Here we report that the landscape of m6A deposition is drastically shifted during KSHV (Kaposi Sarcoma Associated herpesvirus) lytic infection for both viral and host transcripts. In line with previous reports, we also saw an overall decrease in host methylation in favor of viral mRNA along with 5 hypomethylation and 3 hypermethylation. During KSHV lytic infection, a major shift in overall mRNA abundance is driven by the viral endoribonuclease SOX, which induces the decay of greater than 70% of transcripts. Here, we reveal that Interlukin-6 (IL-6) mRNA, a well-characterized SOX-resistant transcript, is m6A modified during lytic infection. Furthermore, we show that this modification falls within the IL-6 SOX Resistance Element (SRE), an RNA element in IL-6 3 UTR that was previously shown to be sufficient for protection from SOX cleavage. We show that the presence of this m6A modification is essential to confer SOX resistance to the IL-6 mRNA. We next show that this modification recruits the m6A reader YTHDC2 and found that YTHDC2 is necessary for the escape of the IL-6 transcript. These results shed light on how the host cell has evolved to use RNA modifications to circumvent viral manipulation of RNA fate during KSHV infection.


title: Mutations at the Alphavirus E2/E1 inter-dimer interface have host-specific phenotypes

abstract:
Alphaviruses are enveloped viruses transmitted by arthropod vectors to vertebrate hosts. The surface of the virion contains 80 glycoprotein spikes embedded in the membrane and these spikes mediate attachment to the host cell and initiate viral fusion. Each spike consists of a trimer of E2-E1 heterodimers. These heterodimers interact at two interfaces: (1) the intra-dimer interactions between E2 and E1 of the same heterodimer, and (2) the inter-dimer interactions between E2 of one heterodimer and E1 of the adjacent heterodimer (E1). We hypothesized that the inter-dimer interactions are essential for trimerization of the E2-E1 heterodimers into a functional spike. In this work, we made a mutant virus (CPB) where we replaced six inter-dimeric residues in the E2 protein of Sindbis virus (WT SINV) with those from the E2 protein from chikungunya virus, and studied its effect in both mammalian and mosquito cell lines. CPB produced fewer infectious particles in mammalian cells than in mosquito cells, relative to WT SINV. When CPB virus was purified from mammalian cells, particles showed reduced amounts of glycoproteins relative to capsid protein, and contained defects in particle morphology compared to virus derived from mosquito cells. Using cryo-EM, we determined that the spikes of CPB had a different conformation than WT SINV. Last, we identified two revertants, E2-H333N and E1-S247L, that restored particle growth and assembly to different degrees. We conclude the inter-dimer interface is critical for spike trimerization and is a novel target for potential antiviral drug design.

IMPORTANCEAlphaviruses, which can cause disease when spread to humans by mosquitoes, have been classified as an emerging pathogen, with infections occurring worldwide. The spikes on the surface of the alphavirus particle are absolutely required for the virus to enter a new host cell and initiate an infection. Using a structure-guided approach, we made a mutant virus that alters spike assembly in mammalian cells but not mosquito cells. This is important because it identifies a region in the spike that could be a target for antiviral drug design.


title: Alterations in gut microbiome composition and function in irritable bowel syndrome and increased probiotic abundance with daily supplementation

abstract:
BackgroundIrritable bowel syndrome (IBS) is characterized by abdominal discomfort and irregular bowel movements and stool consistency. Because there are different symptoms associated with IBS, it is difficult to diagnose the role of the microbiome in IBS.

ObjectiveHere, we present a study that includes metagenomic sequencing of stool samples from subjects with the predominant subtypes of IBS and a healthy cohort. We collected longitudinal samples from individuals with IBS who took daily made-to-order precision probiotic and prebiotic supplementation throughout the study.

Materials and MethodsThis study includes a population of 489 individuals with IBS and 122 healthy controls. All stool samples were subjected to shotgun metagenomic sequencing. Precision probiotics and prebiotics were formulated for all subjects with longitudinal timepoints.

ResultsThere was significant variation explained in the microbiome between the healthy and IBS cohorts. Individuals with IBS had a lower gut microbiome diversity and reduced anti-inflammatory microbes compared to the healthy controls. Eubacterium rectale and Faecalibacterium prausnitzii were associated with healthy microbiomes while Shigella species were associated with IBS. Pathway analysis indicated a functional imbalance of short chain fatty acids, vitamins, and a microbial component of Gram-negative bacteria in IBS compared to healthy controls. In the longitudinal dataset, there was a significant difference in microbiome composition between timepoints 1 and 3. There was also a significant increase in the overall microbiome score and relative abundances of probiotic species used to target the symptoms associated with IBS.

ConclusionsWe identified microbes and pathways that differentiate healthy and IBS microbiomes. In response to precision probiotic supplementation, we identified a significant improvement in the overall microbiome score in individuals with IBS. These results suggest an important role for probiotics in managing IBS symptoms and modulation of the microbiome as a potential management strategy.

ImportanceAn estimated 35 million people in the United States and 11.5% of the population globally are affected by IBS. Immunity, genetics, environment, diet, small intestinal bacterial overgrowth (SIBO), and the gut microbiome are all factors that contribute to the onset or triggers of IBS. With strong supporting evidence that the gut microbiome may influence symptoms associated with IBS, elucidating the important microbes that contribute to the symptoms and severity is important to make decisions for targeted treatment. As probiotics have become more common in treating IBS symptoms, identifying effective probiotics may help inform future studies and treatment.


title: Adaptation and phenotypic diversification of Bacillus thuringiensis 407 biofilm are accompanied by a fuzzy spreader morphotype

abstract: Bacillus cereus group (Bacillus cereus sensu lato) has a diverse ecology, including various species that produce biofilms on abiotic and biotic surfaces. While genetic and morphological diversification enable the adaptation of multicellular communities, this area remains largely unknown in the Bacillus cereus group. In this work, we dissected the experimental evolution of Bacillus thuringiensis 407 Cry-during continuous recolonization of plastic beads. We observed the evolution of a distinct colony morphotype that we named fuzzy spreader (FS) variant. Most multicellular traits of the FS variant displayed higher competitive ability versus the ancestral strain, suggesting an important role for diversification in the adaptation of B. thuringiensis to the biofilm lifestyle. Further genetic characterization of FS variant revealed the disruption of a guanylyltransferase gene by an insertion sequence (IS) element, which could be similarly observed in the genome of a natural isolate. The evolved FS and the deletion mutant in the guanylyltransferase gene (Bt407{Delta}rfbM) displayed similarly altered aggregation and hydrophobicity compared to the ancestor strain, suggesting that adaptation process highly depends on the physical adhesive forces.


title: Interrogation of the integrated mobile genetic elements in gut-associated Bacteroidaceae with a consensus prediction approach

abstract:
Exploration of mobile genetic element (MGE) diversity and relatedness is vital to understanding microbial communities, especially the gut microbiome, where the mobilization of antibiotic resistance and pathogenicity genes has important clinical consequences. Current MGE prediction tools are biased toward elements similar to previously-identified MGEs, especially tailed phages of proteobacterial hosts. Further, there is a need for methods to examine relatedness and gene sharing among MGEs. We present VICSIN, a consensus approach for MGE prediction and clustering of predictions to provide classification. Testing of VICSIN on datasets of Pseudomonas aeruginosa and Bacteroides fragilis genomes suggests VICSIN is the optimal approach to predict integrated MGEs from poorly-explored host taxa, because of its increased sensitivity and accuracy. We applied VICSIN to a dataset of gut-associated Bacteroidaceae genomes, identifying 816 integrated MGEs falling into 95 clusters, most of which are novel. VICSINs fast and simple network-building scheme revealed a high degree of gene sharing within and between related MGE clusters. Shared gene functions across MGEs include core mobilization functions and accessory gene content, such as type VI secretion systems and antibiotic resistance genes. The MGEs identified here encode a large portion of unknown gene content, emphasizing the fact that the full diversity of MGEs and the factors they encode remain very poorly understood. Together, this work motivates more exploration of the gut mobilome, which is likely one of the most potent drivers of microbial evolution in the human microbiome.

IMPORTANCEMobile genetic elements (MGEs), including phages and integrative and conjugative elements (ICEs), drive the diversity and function of microbial communities through horizontal gene transfer. Current tools to predict MGEs in genomic sequence data are highly focused on phages, and are biased against the discovery of novel MGEs. We present VICSIN, a consensus approach to MGE prediction that is able to find a diversity of MGEs, particularly in poorly-understood bacterial taxa. By applying VICSIN to a large database of diverse Bacteroidaceae genomes, we have been able to get a distinct view of the gut mobilome, extending beyond the phageome. These novel MGEs belong to related groups, sharing a significant amount of functional gene content within and between groups, supporting a mosaic model of evolution for ICEs. Understanding how phages evolve in Bacteroidaceae hosts, however, remains elusive and highlights the need for more experimental research.


title: Robust coordination of collective oscillatory signaling requires single-cell excitability and fold-change detection

abstract: Complex multicellular behaviors are coordinated at the level of biochemical signaling networks, yet how this decentralized mechanism enables robust control in variable environments and over many orders of magnitude of spatiotemporal scales remains an open question. A stunning example of these behaviors is found in the microbe Dictyostelium discoideum, which uses the small molecule cyclic AMP (cAMP) to drive the propagation of collective signaling oscillations leading to multicellular development. The critical design features of the Dictyostelium signaling network remain unclear despite decades of mathematical modeling and experimental interrogation because each model makes different assumptions about the network architecture and in general, normalizing models for direct comparison presents a major challenge. We overcome this challenge by using recent experimental data to normalize the time and response scales of five major signal relay network models to one another and assess their ability to recapitulate experimentally-observed population and single-cell dynamics. We find that to successfully reproduce the full range of observed dynamical behaviors, single cells must be excitable and respond to the relative fold-change of environmental signals. This suggests these features represent robust principles for coordinating cellular populations through oscillatory signaling and that single-cell excitable dynamics are a generalizable route for controlling population behaviors.


title: Transposon Screen of Surface Accessibility in S. aureus

abstract:
Bacterial cell walls represent one of the most prominent targets of antibacterial agents. These agents include natural products (e.g., vancomycin) and proteins stemming from the innate immune system (e.g., peptidoglycan-recognition proteins and lysostaphin). Among bacterial pathogens that infect humans, Staphylococcus aureus (S. aureus) continues to impose a tremendous healthcare burden across the globe. S. aureus has evolved countermeasures that can directly restrict the accessibility of innate immune proteins, effectively protecting itself from threats that target key cell well components. We recently described a novel assay that directly reports on the accessibility of molecules to the peptidoglycan layer within the bacterial cell wall of S. aureus. The assay relies on site-specific chemical remodeling of the peptidoglycan with a biorthogonal handle. Here, we disclose the application of our assay to a screen of a nonredundant transposon mutant library for susceptibility of the peptidoglycan layer with the goal of identifying genes that contribute to the control of cell surface accessibility. We discovered several genes that resulted in higher accessibility levels to the peptidoglycan layer and showed that these genes modulate sensitivity to lysostaphin. These results indicate that this assay platform can be leveraged to gain further insight into the biology of bacterial cell surfaces.

Table of Contents Figure O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici.

abstract:
Acquired resistance is a threat for antifungal efficacy in medicine and agriculture. The diversity of possible resistance mechanisms, as well as the highly adaptive traits of pathogens make it difficult to predict evolutionary outcomes of treatments. We used directed evolution as an approach to assess the risk of resistance to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. Fenpicoxamid inhibits complexIII of the respiratory chain at the ubiquinone reduction site (Qi site) of the mitochondrially encoded cytochrome b, a different site than the widely-used strobilurins which the respiratory complex by binding to the ubiquinol oxidation site (Qo site). We identified the G37V change, within the cytochrome b Qi site, as the most likely resistance mechanism to be selected in Z. tritici. This change triggered high fenpicoxamid resistance and halved the enzymatic activity of cytochrome b, despite no significant penalty for in vitro growth. In addition, we identified a negative cross-resistance between isolates harboring G37V or G143A, a Qo site change previously selected by strobilurins. Moreover, double mutants were less resistant to both QiIs and QoIs compared to single mutants. This work is a proof of concept that experimental evolution can be used to predict adaptation to fungicides, and provides new perspectives for the management of QiIs.

Originality-Significance StatementO_LIThe highly adaptive traits of pathogens render evolutionary outcomes of antifungal treatments difficult to predict.
C_LIO_LIWe used directed evolution to assess the risk of resistance to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici.
C_LIO_LIWe identified a target modification as the most likely resistance mechanism to be selected.
C_LIO_LIThis change triggered high fenpicoxamid resistance and halved the activity of the target enzyme despite no significant penalty for in vitro growth.
C_LIO_LIThis work supports the use of experimental evolution as a method to predict adaptation to fungicides and provides important information for the management of QiIs.
C_LI


title: Tight association between microbial eukaryote and Imitervirales communities in the Pacific Arctic Ocean

abstract: Viruses are important regulatory factors of marine microbial community including microeukaryotes. However, little is known about their role in the northern Chukchi Sea of the Arctic basin, which remains oligotrophic conditions in summer. To elucidate linkages of microbial eukaryotic community with viruses as well as environmental variables, we investigated the community structures of microeukaryotes (3-144 {micro}m and 0.2-3 {micro}m size fractions) and Imitervirales (0.2-3 {micro}m size fraction), a major group of viruses infecting marine microeukaryotes. Surface water samples were collected at 21 ocean stations located in the northeastern Chukchi Sea (NECS), an adjacent area outside the Beaufort Gyre (Adjacent Sea; AS), and two melt ponds on sea ice in the summer of 2018. At the ocean stations, nutrient concentrations were low in most of the locations expect at the shelf in the AS. The community variations were significantly correlated between eukaryotes and Imitervirales, even within the NECS characterized by relatively homogeneous environmental conditions. The association of the eukaryotic community with the viral community was stronger than that with geographical and physicochemical environmental factors. These results suggest that Imitervirales are actively infecting their hosts even in cold and oligotrophic sea water in the Arctic Ocean.


title: The human disease gene CLEC16A encodes an intrinsically disordered protein region required for mitochondrial quality control

abstract: CLEC16A regulates mitochondrial health through mitophagy and is associated with over 20 human diseases. While CLEC16A has ubiquitin ligase activity, the key structural and functional regions of CLEC16A, and their relevance for human disease, remain unknown. Here, we report that a disease-associated CLEC16A variant lacks a C-terminal intrinsically disordered protein region (IDPR) that is critical for mitochondrial quality control. Using carbon detect NMR, we find that the CLEC16A C terminus lacks secondary structure, validating the presence of an IDPR. Loss of the CLEC16A C-terminal IDPR in vivo impairs pancreatic {beta}-cell mitophagy, mitochondrial function, and glucose-stimulated insulin secretion, ultimately causing glucose intolerance. Deletion of the CLEC16A C-terminal IDPR increases its self-ubiquitination and destabilizes CLEC16A, thus impairing formation of a critical CLEC16A-dependent mitophagy complex. Importantly, CLEC16A stability is dependent on proline bias within the C-terminal IDPR, but not amino acid sequence order or charge. Together, we clarify how an IDPR in CLEC16A prevents diabetes, thus implicating the disruption of IDPRs as novel pathological contributors to diabetes and other CLEC16A-associated diseases.


title: FOXO1 represses Sprouty2 and Sprouty4 expressionin endothelial cells to promote arterial specification and vascular remodeling in the mouse yolk sac

abstract: The establishment of a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss of function mouse models have shown that FOXO1, a Forkhead family transcription factor, is required for yolk sac vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that loss of FoxO1 in E8.25 endothelial cells results in increased Sprouty2 and Sprouty4 transcripts, reduced expression of arterial genes, and decreased Flk1/Vegfr2 mRNA levels without affecting overall endothelial cell identity, survival, or proliferation. Using a Dll4-BAC-nlacZ reporter line, we found that one of the earliest expressed arterial genes, Delta like 4 (Dll4), is significantly reduced in the yolk sac of FoxO1 mutants without being substantially affected in the embryo proper. We show that in the yolk sac, FOXO1 not only binds directly to a subset of previously identified Sprouty2 gene regulatory elements (GREs), as well as newly identified, evolutionarily conserved Sprouty4 GREs, but can also repress their expression. Additionally, over expression of Sprouty4 in transient transgenic embryos largely recapitulates reduced expression of arterial genes seen in endothelial FoxO1 mutant mouse embryos. Together, these data reveal a novel role for FOXO1 as a key early transcriptional repressor controlling both pre-flow arterial specification and subsequent vessel remodeling within the murine yolk sac.


title: Prey identity affects fitness of a generalist consumer in a brown food web

abstract: The use of ever-advancing sequencing technologies has revealed incredible biodiversity at the microbial scale, and yet we know little about the ecological interactions in these communities. For example, in the phytotelmic community found in the purple pitcher plant, Sarrecenia purpurea, ecologists typically consider the bacteria as a functionally homogenous group. In this food web, bacteria decompose detritus and are consumed by protozoa that are considered generalist consumers. Here we tested whether a generalist consumer benefits from all bacteria equally. We isolated and identified 22 strains of bacteria, belonging to six genera, from S. purpurea plants. We grew the protozoa, Tetrahymena sp. with single isolates and strain mixtures of bacteria and measured Tetrahymena fitness. We found that different bacterial strains had different effects on protozoan fitness, both in isolation and in mixture. Our results demonstrate that not accounting for composition of prey communities may affect the predicted outcome of predator-prey interactions.


title: Using hypertemporal Sentinel-1 data to predict forest growing stock volume

abstract:
In this study, we assess the potential of long time series of Sentinel-1 SAR data to predict forest growing stock volume and evaluate the temporal dynamics of the predictions. The boreal coniferous forests study site is located near the Hyytiala forest station in central Finland and covers an area of 2,500 km2 with nearly 17,000 stands. We considered several prediction approaches (linear, support vector and random forests regression) and fine-tuned them to predict growing stock volume in several evaluation scenarios. The analyses used 96 Sentinel-1 images acquired over three years. Different approaches for aggregating SAR images and choosing feature (predictor) variables were evaluated. Our results demonstrate considerable decrease in RMSEs of growing stock volume as the number of images increases. While prediction accuracy using individual Sentinel-1 images varied from 85 to 91 m3/ha RMSE (relative RMSE 50-53%), RMSE with combined images decreased to 75.6 m3/ha (relative RMSE 44%). Feature extraction and dimension reduction techniques facilitated achieving the near-optimal prediction accuracy using only 8-10 images. When using assemblages of eight consecutive images, the GSV was predicted with the greatest accuracy when initial acquisitions started between September and January.

HighlightsO_LITime series of 96 Sentinel-1 images is analysed over study area with 17,762 forest stands.
C_LIO_LIRigorous evaluation of tools for SAR feature selection and GSV prediction.
C_LIO_LIImproved periodic seasonality using assemblages of consecutive Sentinel-1 images.
C_LIO_LIAnalysis of combining images acquired in "frozen" and "dry summer" conditions.
C_LIO_LICompetitive estimates using calculation of prediction errors with stand-area weighting.
C_LI


title: Complex genetic patterns and distribution limits mediated by native congeners of the worldwide invasive red-eared slider turtle

abstract: Non-native (invasive) species offer a unique opportunity to study the geographic distribution and range limits of species, wherein the evolutionary change driven by interspecific interactions between native and non-native closely related species is a key component. The red-eared slider turtle, Trachemys scripta elegans (TSE), has been introduced and successfully established worldwide. It can coexist with its native congeners T. cataspila, T. venusta and T. taylori in Mexico. We performed comprehensive fieldwork, executed a battery of genetic analyses and applied a novel species distribution modeling approach to evaluate their historical lineage relationships and contemporary population genetic patterns. Our findings support the historical common ancestry between native TSE and non-native (TSEalien), while also highlighting the genetic differentiation of the exotic lineage. Genetic patterns are associated with their range size/endemism gradient, the microendemic T. taylori showed significant reduced genetic diversity and high differentiation, whereas TSEalien showed the highest diversity and signals of population size expansion. Counter to our expectations, lower naturally occurring distribution overlap and little admixture patterns were found between and its congeners, exhibiting reduced gene flow and clear genetic separation across neighboring species despite having zones of contact. We demonstrate that these native Trachemys species have distinct climatic niche suitability, likely preventing establishment of and displacement by the non-native TSE. Additionally, we found major niche overlap between TSEalien and native species worldwide, supporting our prediction that sites with closer ecological optima to the invasive species have higher establishment risk than those that are closer to the niche-center of the native species.


title: Detection of F1 hybrids from single-genome data reveals frequent hybridization in Hymenoptera and particularly ants

abstract: Hybridization occupies a central role in many fundamental evolutionary processes, such as speciation or adaptation. Yet, despite its pivotal importance in evolution, little is known about the actual prevalence and distribution of hybridization across the tree of life. Here we develop and implement a new statistical method enabling the detection of F1 hybrids from single-individual genome sequencing data. Using simulations and sequencing data from known hybrid systems, we first demonstrate the specificity of the method, and identify its statistical limits. Next, we showcase the method by applying it to available sequencing data from more than 1500 species of Arthropods, including Hymenoptera, Hemiptera, Coleoptera, Diptera and Archnida. Among these taxa, we find Hymenoptera, and especially ants, to display the highest number of candidate F1 hybrids, suggesting higher rates of recent hybridization in these groups. The prevalence of F1 hybrids was heterogeneously distributed across ants, with taxa including many candidates tending to harbor specific ecological and life history traits. This work shows how large-scale genomic comparative studies of recent hybridization can be implemented, uncovering the determinants of hybridization frequency across whole taxa.


title: Powerful, efficient QTL mapping in Drosophila melanogaster using bulked phenotyping and pooled sequencing

abstract: Despite the value of Recombinant Inbred Lines (RILs) for the dissection of complex traits, large panels can be difficult to maintain, distribute, and phenotype. An attractive alternative to RILs for many traits leverages selecting phenotypically-extreme individuals from a segregating population, and subjecting pools of selected and control individuals to sequencing. Under a bulked or extreme segregant analysis paradigm, genomic regions contributing to trait variation are revealed as frequency differences between pools. Here we describe such an extreme quantitative trait locus, or X-QTL mapping strategy that builds on an existing multiparental population, the DSPR (Drosophila Synthetic Population Resource), and involves phenotyping and genotyping a population derived by mixing hundreds of DSPR RILs. Simulations demonstrate that challenging, yet experimentally tractable X-QTL designs (>=4 replicates, >=5000 individuals/replicate, and a selection intensity of 5-10%) yield at least the same power as traditional RIL-based QTL mapping, and can localize variants with sub-centimorgan resolution. We empirically demonstrate the effectiveness of the approach using a 4-fold replicated X-QTL experiment that identifies 7 QTL for caffeine resistance. Two mapped X-QTL factors replicate loci previously identified in RILs, 6/7 are associated with excellent candidate genes, and RNAi knock-downs support the involvement of 4 genes in the genetic control of trait variation. For many traits of interest to drosophilists a bulked phenotyping/genotyping X-QTL design has considerable advantages.

category: genetics


title: Genomic prediction of growth in a commercially, recreationally, and culturally important marine resource, the Australian snapper (Chrysophrys auratus)

abstract: Growth is one of the most important traits of an organism. For exploited species, this trait has ecological and evolutionary consequences as well as economical and conservation significance. Rapid changes in growth rate associated with anthropogenic stressors have been reported for several marine fishes, but little is known about the genetic basis of growth traits in teleosts. We used reduced genome representation data and genome-wide association approaches to identify growth-related genetic variation in the commercially, recreationally, and culturally important Australian snapper (Chrysophrys auratus, Sparidae). Based on 17,490 high-quality SNPs and 363 individuals representing extreme growth phenotypes from 15,000 fish of the same age and reared under identical conditions in a sea pen, we identified 100 unique candidates that were annotated to 51 proteins. We documented a complex polygenic nature of growth in the species that included several loci with small effects and a few loci with larger effects. Overall heritability was high (75.7%), reflected in the high accuracy of the genomic prediction for the phenotype (small vs large). Although the SNPs were distributed across the genome, most candidates (60%) clustered on chromosome 16, which also explains the largest proportion of heritability (16.4%). This study demonstrates that reduced genome representation SNPs and the right bioinformatic tools provide a cost-efficient approach to identify growth-related loci and to describe genomic architectures of complex quantitative traits. Our results help to inform captive aquaculture breeding programmes and are of relevance to monitor growth-related evolutionary shifts in wild populations in response to anthropogenic pressures.

category: genomics


title: Contiguously-hydrophobic sequences are functionally significant throughout the human exome

abstract:
Hydrophobic interactions have long been established as essential to stabilizing structured proteins as well as drivers of aggregation, but the impact of hydrophobicity on the functional significance of sequence variants has rarely been considered in a genome-wide context. Here we test the role of hydrophobicity on functional impact using a set of 70,000 disease and non-disease associated single nucleotide polymorphisms (SNPs), using enrichment of disease-association as an indicator of functionality. We find that functional impact is uncorrelated with hydrophobicity of the SNP itself, and only weakly correlated with the average local hydrophobicity, but is strongly correlated with both the size and minimum hydrophobicity of the contiguous hydrophobic domain that contains the SNP. Disease-association is found to vary by more than 6-fold as a function of contiguous hydrophobicity parameters, suggesting utility as a prior for identifying causal variation. We further find signatures of differential selective constraint on domain hydrophobicity, and that SNPs splitting a long hydrophobic region or joining two short regions of contiguous hydrophobicity are particularly likely to be disease-associated. Trends are preserved for both aggregating and non-aggregating proteins, indicating that the role of contiguous hydrophobicity extends well beyond aggregation risk.

Statement of SignificanceProteins rely on the hydrophobic effect to maintain structure and interactions with the environment. Surprisingly, no signs that amino acid hydrophobicity influences natural selection have been detected using modern genetic data. This may be because analyses that treat each amino acid separately do not reveal significant results, which we confirm here. However, because the hydrophobic effect becomes more powerful as more hydrophobic molecules are introduced, we tested whether unbroken stretches of hydrophobic amino acids are under selection. Using genetic variant data from across the human genome, we found evidence that selection pressure increases continually with the length of the unbroken hydrophobic sequence. These results could lead to improvements in a wide range of genomic tools as well as insights into disease and protein evolutionary history.


title: Quantitative accuracy and precision in multiplexed single-cell proteomics.

abstract: Single-cell proteomics workflows have considerably improved in sensitivity and reproducibility to characterize yet unknown biological phenomena. With the emergence of multiplexed single-cell proteomics, studies increasingly present single-cell measurements in conjunction with an abundant congruent carrier to improve precursor selection and enhance identifications. While these extreme carrier spikes are often >100-times more abundant than the investigated samples, undoubtedly the total ion current increases, but quantitative accuracy possibly is affected. We here focus on narrowly titrated carrier spikes (i.e., <20x) and assess their elimination for comparable sensitivity at superior accuracy. We find that subtle changes in the carrier ratio can severely impact measurement variability and describe alternative multiplexing strategies to evaluate data quality. Lastly, we demonstrate elevated replicate overlap while preserving acquisition throughput at improved quantitative accuracy with DIA-TMT and discuss optimized experimental designs for multiplexed proteomics of trace samples. This comprehensive benchmarking gives an overview of currently available techniques and guides conceptualizing the optimal single-cell proteomics experiment.


title: Highly Efficient Knockin in Human iPS Cells and Rat Embryos by CRISPR/Cas9 Molecular Optimization

abstract: The CRISPR/Cas9 system is now the gold standard for the generation of genetically modified cell and animal models but knockin is a bottleneck. One reason could be that there is no consensus regarding the concentrations of its components to be used. Here, we defined optimal Cas9 protein, guide RNA and short donor DNA concentrations on a GFP to BFP conversion model of human induced pluripotent stem cells and point mutations on rat transgenic embryos. With a molecular rational approach of the CRISPR/Cas9 system and study of ribonucleoprotein complex formation by nanodifferential scanning fluorimetry, we defined that Cas9/guide RNA 1/1 molar ratio with 0.2M and 0.4M of Cas9, coupled with 2M of ssODN are sufficient for optimal and high knockin frequencies in rat embryos and human induced pluripotent stem cells, respectively. These optimal conditions use lower concentrations of CRISPR reagents to form the RNP complex than most conditions published while achieving 50% of knockin. This study allowed us to reduce costs and toxicity while improving editing and knockin efficacy on two particularly key models to mimic human diseases.

category: bioengineering


title: Viral Phrenology

abstract: We introduce Viral Phrenology, a new scheme for understanding the genomic composition of spherical viruses base on the locations of their structural protrusions. We used icosahedral point arrays to classify 135 distinct viral capsids collected from over 600 capsids available in the VIPERdb. Using gauge points of point arrays, we found 149 unique structural protrusions. We then show how to use the locations of these protrusions to determine the genetic composition of the virus. We then show that ssDNA, dsDNA, dsRNA and ssRNA viruses use different arrangements for distributing their protrusions. We also found that Triangulation number is also partially dependent on the structural protrusions. This analysis begins to tie together Baltimore classification and Triangulation number using point arrays.


title: Reovirus sensitizes microsatellite stable colorectal cancer to anti-PD-1 treatment via cross-talk in innate and adaptive immune systems

abstract:
BackgroundMicrosatellite stable (MSS) colorectal cancer (CRC) represents ~85% of all CRCs. These tumors are poorly immunogenic and largely resistant to immunotherapy, necessitating a need to develop new immune enhancing strategies. Oncolytic reovirus has a high propensity to replicate in KRAS mutant tumors which account for ~50% of MSS CRCs. Current study explores the ability of reovirus to potentiate the effect of immune checkpoint inhibition in MSS CRC.

MethodsEffectiveness of reovirus infection was quantified through MTT assay for cell viability, and expression of immune-response genes by flow cytometry, RT-qPCR, and microarray. Computational analysis of differentially expressed genes was performed by TAC, DAVID and STRING. Combinatorial approach using anti-PD-1 monoclonal antibody was assessed in ex vivo and in vivo models. Live-cell imaging, tumor volume and survival were measured for quantification of anti-tumor activity. Expression of pattern recognition receptors (PRRs), cell surface and activation markers of immune cells, and PD-1/PD-L1 axis were studied using multi-color flow cytometry, immunoblotting, immunohistochemistry, and immunofluorescence.

ResultsReovirus infection exerted growth arrest and expression of immune-response genes in CRCs cell lines in a KRAS-dependent manner. However, microsatellite instability, rather than KRAS status determined immune-repose pathways, functionalities and biological processes post-reovirus infection. Furthermore, reovirus significantly enhanced the anti-tumor activity of anti-human PD-1 [nivolumab] treatment in MSS CRC cell lines ex vivo. Similarly, reovirus increased the activity of anti-mouse PD-1 treatment in the CT26 [MSS, KRASMut], but not the MC38 [MSI, KRASWt] syngeneic mouse model of CRC. Combinatorial treatment has reduced the proliferative index, increased apoptosis and differentially altered PD-L1/PD-1 signaling among CT26 and MC38 tumors. Activation of innate immune system and expression of PRRs and antigen presentation markers were observed under reovirus and anti-PD-1 treatment that additionally reduced immunosuppressive macrophages. This led to an increase in T cell subsets, increase in effector T cell activation, and decrease in exhaustion markers specifically within CT26 microenvironment.

ConclusionThe current study systematically evaluates immune characteristics and immune microenvironment of CRC under reovirus/anti-PD-1 combination treatment that proves increased effectiveness among MSS compared to MSI CRCs. This is a promising regimen warranting translation into clinical trials.

One Sentence SummaryOncolytic reovirus alters innate and adaptive immune system and potentiates MSS type colorectal cancer to checkpoint inhibition therapy.


title: Bone Structural, Biomechanical and Histomorphometric Characteristics of the Postcranial Skeleton of the Marsh Rice Rat (Oryzomys palustris)

abstract:
INTRODUCTIONThe rice rat (Oryzomys palustris) is a non-conventional laboratory rodent species used to model some human bone disorders. However, no studies have been conducted to characterize the postcranial skeleton. Therefore, we aimed to investigate age- and gender-related features of the appendicular skeleton of this species.

METHODSWe used femurs and tibiae from 94 rats of both genders aged 4-28 wks. Bone mineral content (BMC), bone mineral density (BMD), and biomechanical properties were determined in femurs. In addition, bone histomorphometry of tibiae was conducted to assess bone cells activities and bone turnover over time.

RESULTSBodyweight, bone length, total metaphysis BMC/BMD, cortical BMC/BMD, cortical thickness, and cortical area progressively augmented with age. Whereas the increase in these parameters plateaued at age 16-22 wks in female rats, they continued to rise to age 28 wks in male rats. Furthermore, bone strength parameters increased with age, with few differences between genders. We also observed a rapid decrease in longitudinal growth between ages 4-16 wks. Whereas young rats had a greater bone formation rate and bone turnover, older rice rats had greater bone volume and trabecular thickness, with no differences between genders.

CONCLUSIONS1) Sexual dimorphism in the rice rat becomes grossly evident at age 16 wks; 2) the age-related increases in bone mass, structural cortical parameters, and in some biomechanical property parameters plateau at an older age in male than in female rats; and 3) bone growth and remodeling significantly decreased with age indistinctive of the gender.

category: physiology


title: Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing

abstract: CRISPR-Cas9 offers unprecedented opportunities to modify genome sequences in primary human cells to study disease variants and reprogram cell functions for next-generation cellular therapies. CRISPR has several potential advantages over widely used retroviral vectors including: 1) site-specific transgene insertion via homology directed repair (HDR), and 2) reductions in the cost and complexity of genome modification. Despite rapid progress with ex vivo CRISPR genome engineering, many novel research and clinical applications would be enabled by methods to further improve knock-in efficiency and the absolute yield of live knock-in cells, especially with large HDR templates (HDRT). We recently reported that Cas9 target sequences (CTS) could be introduced into double-stranded DNA (dsDNA) HDRTs to improve knock-in, but yields and efficiencies were limited by toxicity at high HDRT concentrations. Here we developed a novel system that takes advantage of lower toxicity with single-stranded DNA (ssDNA). We designed hybrid ssDNA HDRTs that incorporate CTS sites and were able to boost knock-in percentages by >5-fold and live cell yields by >7-fold relative to dsDNA HDRTs with CTS. Knock-in efficiency and yield with ssCTS HDRTs were increased further with small molecule inhibitor combinations to improve HDR. We demonstrate application of these methods across a variety of target loci, knock-in constructs, and primary human cell types to reach ultra-high HDR efficiencies (>80-90%) which we use for pathogenic gene variant modeling and universal gene replacement strategies for IL2RA and CTLA4 mutations associated with mendelian immune disorders. Finally, we develop a GMP-compatible method for fully non-viral CAR-T cell manufacturing, demonstrating knock-in efficiencies of 46-62% and generating yields of >1.5 x 109 CAR+ T cells, well above current doses for adoptive cellular therapies. Taken together, we present a comprehensive non-viral approach to model disease associated mutations and re-write targeted genome sequences to program immune cell therapies at a scale compatible with future clinical application.

category: synthetic biology


title: Global anti-tumor immunity after localized, bioengineered Treg depletion

abstract: Over 90% of deaths from cancer occur due to solid tumors, occurring at a rate of [~]1,500 deaths per day in the US, highlighting a profound and unmet need for new therapies. Solid tumors evade clearance by T cells due to a variety of immunosuppressive properties of the tumor microenvironment. However, this immunosuppression cannot be easily blocked on a global level because systemic activation of the immune system elicits a host of complications. An ideal therapy for solid tumors would act locally to activate the immune response without evoking global adverse effects. Here we present a biodegradable, macroporous scaffold that is implanted adjacent to the tumor and suppresses the main obstacle to cancer immunosurveillance: intratumoral regulatory T cells. The scaffold also promotes the recruitment and activation of T cell effectors into the tumor, resulting in clearance of otherwise aggressive and fatal tumors in mice. Unexpectedly, the local depletion of Tregs results in an "immunological abscopal effect" acting on distant tumors. We demonstrate that this versatile platform can also deliver tumor-antigen-specific T cells directly to the peri-tumoral environment, bypassing difficulties in intravenous delivery including the environmental barriers imposed by the tumors vasculature. By orchestrating multiple local immunomodulatory treatments, this scaffold offers a general approach to engineer T-cell responses to solid tumors without systemic toxicities.


title: Acropetal and basipetal cardenolide transport in Erysimum cheiranthoides (wormseed wallflower)

abstract: Plant specialized metabolites are often subject to within-plant transport and have tissue-specific distribution patterns. Among plants in the Brassicaceae, the genus Erysimum is unique in producing not only glucosinolates but also cardenolides as defense against insect herbivory. Ten cardenolides were detected with varying abundance in different tissues of Erysimum cheiranthoides (wormseed wallflower). As is predicted by the optimal defense theory, cardenolides were most abundant in young leaves and reproductive tissues. The lowest concentrations were observed in senescing leaves and roots. Crosses between wildtype E. cheiranthoides and a mutant line with an altered cardenolide profile showed that the seed cardenolide phenotype is determined entirely by the maternal genotype. Prior to the development of the first true leaves, seedling cotyledons also had the maternal cardenolide profile. Hypocotyl grafting experiments showed that the root cardenolide profile is determined entirely by the aboveground plant genotype. In further grafting experiments, there was no evidence of cardenolide transport into the leaves, but a mixed cardenolide profile was observed in the stems and inflorescences of plants that had been grafted at vegetative and flowering growth stages, respectively. Together, these results indicate that E. cheiranthoides leaves are a site of cardenolide biosynthesis and therefore also the plant tissue that is most likely to be expressing the relevant biosynthetic genes.

category: plant biology


title: Recruitment of an ancient branching program to suppress carpel development in maize flowers

abstract: Floral morphology is immensely diverse. One developmental process acting to shape this diversity is growth suppression. For example, grass flowers exhibit extreme diversity in floral sexuality, arising through differential suppression of stamens or carpels. In maize, carpels undergo programmed cell death in half of the flowers initiated in ears and in all flowers in tassels. The HD-ZIP I transcription factor gene GRASSY TILLERS1 (GT1) is one of only a few genes known to regulate this process. To identify additional regulators of carpel suppression, we performed a gt1 enhancer screen, and found a genetic interaction between gt1 and ramosa3 (ra3). RA3 is a classic inflorescence meristem determinacy gene that encodes a trehalose-6-phosphate (T6P) phosphatase (TPP). Dissection of floral development revealed that ra3 single mutants have partially derepressed carpels, whereas gt1; ra3 double mutants have completely derepressed carpels. Surprisingly, gt1 suppresses ra3 inflorescence branching, revealing a role for gt1 in meristem determinacy. Supporting these genetic interactions, GT1 and RA3 proteins colocalize to carpel nuclei in developing flowers. Global expression profiling revealed common genes misregulated in single and double mutant flowers, as well as in derepressed gt1 axillary meristems. Indeed, we found that ra3 enhances gt1 vegetative branching, similar to the roles for the trehalose pathway and GT1 homologs in the eudicots. This functional conservation over ~160 million years of evolution reveals ancient roles for GT1-like genes and the trehalose pathway in regulating axillary meristem suppression, later recruited to mediate carpel suppression. Our findings expose hidden pleiotropy of classic maize genes, and show how an ancient developmental program was redeployed to sculpt floral form.