title: A noval lightsheet scattering microscopy for long-term visualization of cell-ECM interaction

abstract: Visualizing interactions between cells and the extracellular matrix (ECM) mesh is important to understand cell behavior and regulatory mechanisms by the extracellular environment. However, long term visualization of three-dimensional (3D) matrix structures remains challenging mainly due to photobleaching or blind spots perpendicular to the imaging plane. Here, we combine label-free light-sheet scattering microcopy (LSSM) and fluorescence microscopy to solve these problems. We verified that LSSM can reliably visualize structures of collagen matrices from different origin including bovine, human and rat tail. The quality and intensity of collagen structure images acquired by LSSM did not decline with time. LSSM offers abundant wavelength choice to visualize matrix structures, maximizing combination possibilities with fluorescently-labelled cells, allowing visualizing of long-term ECM-cell interactions in 3D. Interestingly, we observed ultrathin thread-like structures between cells and matrix using LSSM, which were not observed by normal fluorescence microscopy. Transient local alignment of matrix by cell-applied forces can be observed. In summary, LSSM provides a powerful and robust approach to investigate the complex interplay between cells and ECM.

category: biophysics


title: The mechanosensitive TRPV2 calcium channel controls melanoma invasiveness and metastatic potential.

abstract:
Discovery of therapeutic targets against metastasis is of primary importance since being the main cause of cancer-related death. Melanoma is a highly aggressive cancer endowed with a unique capacity of rapidly metastasizing. Deregulation of calcium homeostasis has been involved in numerous cellular metastatic behaviors, although the molecular determinants supporting these processes often remain unclear. Here, we evidenced a prominent expression of the plasma membrane TRPV2 calcium channel as a distinctive feature of melanoma tumors, directly related to melanoma metastatic progression and dissemination. In vitro as well as in vivo, TRPV2 activity was sufficient to confer both migratory and invasive phenotypes to non-invasive melanoma cells, while conversely upon TRPV2 silencing, highly metastatic melanoma cells failed to retain their malignant behaviors. We established a model whereupon activation of the mechanosensitive TRPV2 channel, localized in highly dynamic nascent adhesion clusters, directly regulates calpain-dependent cleavage of the adhesive protein talin together with F-actin network. By operating at the crossroad of the tumor microenvironment and the intracellular machinery, mechanosensitive TRPV2 channel controls melanoma cells aggressiveness. Finally in human melanoma tumor samples, TRPV2 overexpression represents a molecular marker of advanced malignancy and bad prognosis, highlighting a new therapeutic option for migrastatics in the treatment of metastatic melanoma.

SignificanceOne essential feature of metastatic cells is enhanced motility and invasiveness. This study evidences TRPV2 channel control over metastatic melanoma invasiveness, highlights new migration regulatory mechanisms, and reveals this channel as a biomarker and migrastatic target for the treatment of advanced melanoma.

category: cancer biology


title: Classical Mathematical Models for Prediction of Response to Chemotherapy and Immunotherapy

abstract:
Classical mathematical models of tumor growth have shaped our understanding of cancer and have broad practical implications for treatment scheduling and dosage. However, even the simplest textbook models have been barely validated in real world-data of human patients. In this study, we fitted a range of differential equation models to tumor volume measurements of patients undergoing chemotherapy or cancer immunotherapy for solid tumors. We used a large dataset of 1472 patients with three or more measurements per target lesion, of which 652 patients had six or more data points. We show that the early treatment response shows only moderate correlation with the final treatment response, demonstrating the need for nuanced models. We then perform a head-to-head comparison of six classical models which are widely used in the field: the Exponential, Logistic, Classic Bertalanffy, General Bertalanffy, Classic Gompertz and General Gompertz model. Several models provide a good fit to tumor volume measurements, with the Gompertz model providing the best balance between goodness of fit and number of parameters. Similarly, when fitting to early treatment data, the general Bertalanffy and Gompertz models yield the lowest mean absolute error to forecasted data, indicating that these models could potentially be effective at predicting treatment outcome. In summary, we provide a quantitative benchmark for classical textbook models and state-of-the art models of human tumor growth. We publicly release an anonymized version of our original data, providing the first benchmark set of human tumor growth data for evaluation of mathematical models.

Author SummaryMathematical oncology uses quantitative models for prediction of tumor growth and treatment response. The theoretical foundation of mathematical oncology is provided by six classical mathematical models: the Exponential, Logistic, Classic Bertalanffy, General Bertalanffy, Classic Gompertz and General Gompertz model. These models have been introduced decades ago, have been used in thousands of scientific articles and are part of textbooks and curricula in mathematical oncology. However, these models have not been systematically tested in clinical data from actual patients. In this study, we have collected quantitative tumor volume measurements from thousands of patients in five large clinical trials of cancer immunotherapy. We use this dataset to systematically investigate how accurately mathematical models can describe tumor growth, showing that there are pronounced differences between models. In addition, we show that two of these models can predict tumor response to immunotherapy and chemotherapy at later time points when trained on early tumor growth dynamics. Thus, our article closes a conceptual gap in the literature and at the same time provides a simple tool to predict response to chemotherapy and immunotherapy on the level of individual patients.


title: A Developmentally Prometastatic Niche to Hepatoblastoma in Neonatal Liver mediated by the Cxcl1/Cxcr2 Axis

abstract:
Background and RationaleHepatoblastoma (HB) is the most common pediatric liver cancer. Its predominant occurrence in very young children led us to investigating whether the neonatal liver provides a protumorigenic niche to HB development.

MethodsHB development was compared between orthotopic transplantation models established in postnatal day 5 and 60 mice (P5Tx and P60Tx models). Single-cell RNA-sequencing was performed using tumor and liver tissues from both models and the top candidate cell types and genes identified are investigated for their roles in HB cell growth, migration, and survival.

ResultsWe found that various HB cell lines including HepG2 cells were consistently and considerably more tumorigenic and metastatic in the P5Tx model than in the P60Tx models. Sc-RNAseq of the P5Tx and P60Tx HepG2 models revealed that the P5Tx tumor was more hypoxic and had a larger number of activated hepatic stellate cells (aHSCs) in the tumor-surrounding liver which express significantly higher levels of Cxcl1 than those from the P60Tx model. We found these differences were developmentally present in normal P5 and P60 liver. We showed that the Cxcl1/Cxcr2 axis mediated HB cell migration and was critical to HB cell survival under hypoxia. Treating HepG2 P60Tx model with recombinant CXCL1 protein induced intrahepatic and pulmonary metastasis and CXCR2 knockout in HepG2 cells abolished their metastatic potential in the P5Tx model. Lastly, we showed that in metastatic HB patient tumors there was a similar larger population of aHSCs in the tumor-surrounding liver than in localized tumors, and tumor hypoxia was uniquely associated with HB patient prognosis among pediatric cancers.

ConclusionWe demonstrated that the neonatal liver provides a prometastatic niche to HB development via the Cxcl1/Cxcr2 axis.


title: Shared hotspot mutations in spontaneously arising cancers position dog as an unparalleled comparative model for precision therapeutics

abstract: Naturally occurring canine cancers have remarkable similarities to their human counterparts. In order to determine whether these similarities occur at the molecular level, we investigated hotspot mutations in a variety of spontaneously arising canine cancers and found high concordance in oncogenic drivers between cancers in both species. These findings suggest that canines may present a powerful and complementary model for preclinical investigations for targeted cancer therapeutics. Through analysis of 708 client-owned dogs from 96 breeds (plus mixed breeds) with 23 common tumor types, we discovered mutations in 50 well-established oncogenes and tumor suppressors, and compared them to those reported in human cancers. TP53 is the most commonly mutated gene, detected in 30.81% of canine tumors overall and >40% in hemangiosarcoma and osteosarcoma. Canine tumors share mutational hotspots with human tumors in oncogenes including PIK3CA, KRAS, NRAS, BRAF, KIT and EGFR. Hotspot mutations with significant (P<0.0001) association to tumor type include NRAS G61R and PIK3CA H1047R in hemangiosarcoma, ERBB2 V659E in pulmonary carcinoma, and BRAF V588E in urothelial carcinoma. This work positions canines as excellent spontaneous models of human cancers that can help to investigate a wide spectrum of targeted therapies.


title: Wiring multiple microenvironment proteomes uncovers the biology in head and neck cancer

abstract: The poor prognosis of head and neck cancer (HNC) is associated with the presence of metastasis within the lymph nodes (LNs). Herein, the proteome of 140 multisite samples from a 59-HNC patient cohort, including primary and matched LN-negative or -positive tissues, saliva, and blood cells, reveals insights into the biology and potential metastasis biomarkers that may assist in clinical decision making. Protein profiles are strictly associated with immune modulation across datasets, and this provides the basis for investigating immune markers associated with metastasis. The proteome of LN metastatic cells recapitulates the proteome of the primary tumor sites. Conversely, the LN microenvironment proteome highlights the candidate prognostic markers. By integrating prioritized peptide, protein, and transcript levels with machine learning models, we identified a nodal metastasis signature in the blood and saliva. In summary, we present the deepest proteome characterization wiring multiple sampling sites in HNC, thus providing a promising basis for understanding tumoral biology and identifying metastasis-associated signatures.


title: Sexual identity of enterocytes regulates rapamycin-mediated intestinal homeostasis and lifespan extension

abstract: Pharmacological attenuation of mTOR by rapamycin and other compounds presents a promising route for delay of ageing-related pathologies, including intestinal cancers. Here, we show that rapamycin treatment in Drosophila extends lifespan in females but not in males. Female-specific, age-related gut pathology and impaired intestinal barrier function are both markedly slowed by rapamycin treatment, mediated by increased autophagy. Upon rapamycin treatment, female intestinal enterocytes increase autophagy, via the H3/H4 histone-Bchs axis, while male enterocytes show high basal levels of autophagy that do not increase further upon rapamycin treatment. Sexual identity of enterocytes alone, determined by the expression of transformerFemale, dictates sexually dimorphic cell size, H3/H4-Bchs expression, basal rates of autophagy, fecundity, intestinal homeostasis and extension of lifespan in response to rapamycin. This study highlights that tissue sex determines regulation of metabolic processes by mTOR and the efficacy of mTOR-targeted, anti-ageing drug treatments.

category: physiology


title: RAGE antagonist peptide mitigates AGE-mediated endothelial hyperpermeability and accumulation of glycoxidation products in human ascending aortas and in a murine model of aortic aneurysm

abstract:
BackgroundAortic dissection and aneurysm are the result of altered biomechanical forces associated with structural weakening of the aortic wall caused by genetic or acquired factors. Current guidelines recommend replacement of the ascending aorta when the diameter is >5.5 cm in tricuspid aortic valve patients. Aortopathies are associated with altered wall stress and stiffness as well as endothelial cell dysfunction and synthetic vascular smooth muscle cell (VSMC) phenotype. We reported that these mechanisms are mediated by glycoxidation products [Reactive oxygen species (ROS) and Advance Glycation End products (AGE)]. This study addresses the role of glycoxidation on endothelial function and AGE-mediated aortic stiffness.

Hypothesis and aimsHere we investigate how circulating glycation products infiltrate the aortic wall via AGE-mediated endothelial hyperpermeability and contribute to both VSMC synthetic phenotype and extracellular matrix (ECM) remodeling in vivo and ex vivo. We also study how RAGE antagonist peptide (RAP) can rescue the effect of AGEs in vitro and in vivo in eNOS-/- vs WT mice.

Methods and resultsHuman ascending aortas (n=30) were analyzed for AGE, ROS, and ECM markers. In vitro glycation was obtained by treating VSMC or human and murine aortas with glyoxal. Endothelial permeability was measured under glycation treatment. Vascular stiffness was measured by a pressure myograph comparing wild-type mice {+/-} glyoxal. eNOS-/- mice, a model of increased endothelial permeability, were treated for 28 days with hyperlipidemic diet {+/-} Angiotensin II (1000ng/kg/min) with or without anti-glycation treatment (RAP 20mg/kg). Echo data of aortic diameter were collected. Murine vascular stiffness was measured by a pressure myograph (n=5/group). Glycoxidation products were detected in all human aortas independently of aortic diameter, with stronger accumulation on the lumen and the adventitia layer. AGEs increased endothelial permeability, induce synthetic phenotypic switch in human VSMCs, and inhibit cell migration. RAP pre-treatment rescue the effect of glyoxal on endothelial cells. Ex vivo glycation treatment of murine arteries impacted on ECM and increased stiffness. Aortic stiffness was higher in eNOS-/- vs WT mice. Ang II-mediated aortopathies results in aortic dilation, and AGE/ROS accumulation, which is rescued by RAGE antagonist peptide treatment of eNOS-/- mice.

ConclusionsGlycoxidation reaction mediate EC permeability, VSMCs phenotype, and ECM remodeling leading to dysfunctional microstructure of the ascending aorta, altered vascular stiffness and increasing aortic susceptibility to dilation and rupture. Moreover, we show that RAP can mitigate AGE-mediated endothelial hyper-permeability in vitro and impact on ascending aneurysm in vivo


title: Two structurally different oomycete MAMPs induce distinctive plant immune responses.

abstract: Plants recognize a variety of external signals and induce appropriate mechanisms to increase their tolerance to biotic and abiotic stresses. Precise recognition of attacking pathogens and induction of effective resistance mechanisms are critical functions for plant survival. Some molecular patterns unique to a certain group of microbes (MAMPs, microbe-associated molecular patterns) are sensed by plant cells as non-self molecules via pattern recognition receptors. While a variety of MAMPs of bacterial and fungal origin have been identified, reports on MAMPs of oomycete pathogens are relatively limited. This study aimed to identify unique MAMP elicitors from the oomycete pathogen Phytophthora infestans, the causal agent of potato late blight. Using reactive oxygen species (ROS) production and phytoalexin production in potato as markers for the purification of oomycete elicitors, we identified two structurally different groups of elicitors, namely ceramides and diacylglycerols. P. infestans ceramide (Pi-Cer) elicitors induced ROS production, while diacylglycerol (Pi-DAG) elicitors, containing eicosapentaenoic acid (EPA) as a substructure, induced the formation of phytoalexins in potato. Pi-Cer and Pi-DAG are also contained in the mycelia of another oomycete pathogen Pythium aphanidermatum, indicating that they are MAMPs of oomycetes. When Arabidopsis was treated with Pi-Cer and EPA, partially overlapping but different sets of genes were induced. Furthermore, simultaneous treatment with Pi-Cer and EPA did not have a cumulative effect on induced genes, but rather the expression of some genes induced by EPA was attenuated by the co-treatment with Pi-Cer. These results indicate that plants may combine the signals from simultaneously recognized MAMP elicitors to specifically adapt the defense response to a particular pathogen.

category: plant biology


title: Optimal confidence for unaware visuomotor deviations

abstract: Numerous studies have shown that humans can successfully correct deviations to ongoing movements without being aware of them, suggesting limited conscious monitoring of visuomotor performance. Here, we ask whether such limited monitoring impairs the capacity to judiciously place confidence ratings to reflect decision accuracy (metacognitive sensitivity). To this end, we recorded functional magnetic resonance imaging data while thirty-one participants reported visuomotor cursor deviations and rated their confidence retrospectively. We show that participants use a summary statistic of the unfolding visual feedback (the maximum cursor error) to detect deviations but that this information alone is insufficient to explain detection performance. The same summary statistics is used by participants to optimally adjust their confidence ratings, even for unaware deviations. At the neural level, activity in the ventral striatum tracked high confidence, whereas a broad network including the anterior prefrontal cortex encoded cursor error but not confidence, shedding new light on a role of the anterior prefrontal cortex for action monitoring rather than confidence. Together, our results challenge the notion of limited action monitoring and uncover a new mechanism by which humans optimally monitor their movements as they unfold, even when unaware of ongoing deviations.

category: neuroscience


title: Flexible neural coding in sensory, parietal, and frontal cortices during goal-directed virtual navigation

abstract: We do not understand how neural nodes operate within the recurrent action-perception loops that characterize naturalistic self-environment interactions, nor how brain networks reconfigure during changing computational demands. Here, we record local field potentials (LFPs) and spiking activity simultaneously from the dorsomedial superior temporal area (MSTd), parietal area 7a, and dorsolateral prefrontal cortex (dlPFC) as monkeys navigate in virtual reality to "catch fireflies". This task requires animals to actively sample from a closed-loop visual environment while concurrently computing latent variables: the evolving distance and angle to a memorized firefly. We observed mixed selectivity in all areas, with even a traditionally sensory area (MSTd) tracking latent variables. Strikingly, global encoding profiles and unit-to-unit coupling suggested a functional subnetwork between MSTd and dlPFC, and not between these are 7a, as anatomy would suggest. When sensory evidence was rendered scarce, lateral connectivity through neuron-to-neuron coupling within MSTd strengthened but its pattern remains fixed, while neuronal coupling adaptively remapped within 7a and dlPFC. The larger the remapping in 7a/dlPFC and the greater the stability within MSTd, the less was behavior impacted by loss of sensory evidence. These results highlight the distributed nature of neural coding during closed-loop action- perception naturalistic behaviors and suggest internal models may be housed in the pattern of fine-grain lateral connectivity within parietal and frontal cortices.


title: Molecular basis of ocean acidification sensitivity and adaptation in Mytilus galloprovincialis

abstract: One challenge in global change biology is to identify the mechanisms underpinning physiological sensitivities to environmental change and to predict their potential to adapt to future conditions. Using ocean acidification as the representative stressor, molecular pathways associated with abnormal larval development of a globally distributed marine mussel are identified. The targeted developmental stage was the trochophore stage, which is, for a few hours, pH sensitive and is the main driver of developmental success. RNA sequencing and in situ RNA hybridization were used to identify processes associated with abnormal development, and DNA sequencing was used to identify which processes evolve when larvae are exposed to low pH for the full duration of their larval stage. Trochophores exposed to low pH exhibited 43 differentially expressed genes. Thirteen genes, none of which have previously been identified in mussel trochophores, including three unknown genes, were expressed in the shell field. Gene annotation and in situ hybridization point to two core processes associated with the response to low pH: development of the trochophore shell field and the cellular stress response. Encompassing both of these processes, five genes demonstrated changes in allele frequency that are indicative of rapid adaptation. Thus, genes underpinning the most pH-sensitive developmental processes also exhibit scope to adapt via genetic variation currently maintained in the mussel population. These results provide evidence that protecting species existing genetic diversity is a critical management action to maximize the potential for rapid adaptation under a changing environment.

category: ecology


title: Genomic insights into variation in thermotolerance between hybridizing swordtail fishes

abstract: Understanding how organisms adapt to changing environments is a core focus of research in evolutionary biology. One common mechanism is adaptive introgression, which has received increasing attention as a potential route to rapid adaptation in populations struggling in the face of ecological change, particularly global climate change. However, hybridization can also result in deleterious genetic interactions that may limit the benefits of adaptive introgression. Here, we used a combination of genome-wide quantitative trait locus mapping and differential gene expression analyses between the swordtail fish species Xiphophorus malinche and X. birchmanni to study the consequences of hybridization on thermotolerance. While these two species are adapted to different thermal environments, we document a complicated architecture of thermotolerance in hybrids. We identify a region of the genome that contributes to reduced thermotolerance in individuals heterozygous for X. malinche and X. birchmanni ancestry, as well as widespread misexpression in hybrids of genes that respond to thermal stress in the parental species, particularly in the circadian clock pathway. We also show that a previously mapped hybrid incompatibility between X. malinche and X. birchmanni contributes to reduced thermotolerance in hybrids. Together, our results highlight the challenges of understanding the impact of hybridization on complex ecological traits and its potential impact on adaptive introgression.

category: evolutionary biology


title: ZELDA: a 3D Image Segmentation and Parent-Child relation plugin for microscopy image analysis in napari

abstract:
Bioimage analysis workflows allow the measurement of sample properties such as fluorescence intensity and polarization, cell number, and vesicles distribution, but often require the integration of multiple software tools. Furthermore, it is increasingly appreciated that to overcome the limitations of the 2D-view-based image analysis approaches and to correctly understand and interpret biological processes, a 3D segmentation of microscopy data sets becomes imperative.

Despite the availability of numerous algorithms for the 2D and 3D segmentation, the latter still offers some challenges for the end-users, who often do not have either an extensive knowledge of the existing software or coding skills to link the output of multiple tools. While several commercial packages are available on the market, fewer are the open-source solutions able to execute a complete 3D analysis workflow.

Here we present ZELDA, a new napari plugin that easily integrates the cutting-edge solutions offered by python ecosystem, such as scikit-image for image segmentation, matplotlib for data visualization, and napari multi-dimensional image viewer for 3D rendering. This plugin aims to provide interactive and zero-scripting customizable workflows for cell segmentation, vesicles counting, parent-child relation between objects, signal quantification, and results presentation; all included in the same open-source napari viewer, and "few clicks away".

category: bioinformatics


title: Transcriptional regulation of photoprotection in dark-to-light transition - more than just a matter of excess light energy

abstract: In nature, photosynthetic organisms are exposed to different light spectra and intensities depending on the time of day and atmospheric and environmental conditions. When photosynthetic cells absorb excess light, they induce non-photochemical quenching to avoid photo-damage and trigger expression of  photoprotective genes. In this work, we used the green alga Chlamydomonas reinhardtii to assess the impact of light intensity, light quality, wavelength, photosynthetic electron transport and CO2 on induction of the  photoprotective genes (LHCSR1, LHCSR3 and PSBS) during dark-to-light transitions. Induction (mRNA accumulation) occurred at very low light intensity, was independently modulated by blue and UV-B radiation through specific photoreceptors, and only LHCSR3 was strongly controlled by CO2 levels through a putative enhancer function of CIA5, a transcription factor that controls genes of the carbon concentrating mechanism. We propose a model that integrates inputs of independent signaling pathways and how they may help the cells anticipate diel conditions and survive in a dynamic light environment.


title: Photoprotection is regulated by light-independent CO2 availability

abstract:
Photosynthetic algae cope with suboptimal levels of light and CO2. In low CO2 and excess light, the green alga Chlamydomonas reinhardtii activates a CO2 Concentrating Mechanism (CCM) and photoprotection; the latter is mediated by LHCSR1/3 and PSBS. How light and CO2 signals converge to regulate photoprotective responses remains unclear. Here we show that excess light activates expression of photoprotection- and CCM-related genes and that depletion of CO2 drives these responses, even in total darkness. High CO2 levels, derived from respiration or impaired photosynthetic fixation, repress LHCSR3 and CCM genes while stabilizing the LHCSR1 protein. We also show that CIA5, which controls CCM genes, is a major regulator of photoprotection, elevating LHCSR3 and PSBS transcript accumulation while inhibiting LHCSR1 accumulation. Our work emphasizes the importance of CO2 in regulating photoprotection and the CCM, demonstrating that the impact of light on photoprotection is often indirect and reflects intracellular CO2 levels.

TeaserPhotoprotection- and CCM-related genes can be activated by changes in CO2 availability even in the absence of light.


title: Previously uncharacterized rectangular bacteria in the dolphin mouth

abstract: Much remains to be explored regarding the diversity of host-associated microbes. Here, we report the discovery of microbial structures in the mouths of bottlenose dolphins that we refer to as rectangular cell-like units (RCUs). DNA staining revealed multiple paired bands that suggested cells in the act of dividing along the longitudinal axis. Deep sequencing of samples enriched in RCUs through micromanipulation indicated that the RCUs are bacterial and distinct from Simonsiella, a genus with somewhat similar morphology and division patterning found in oral cavities of animals. Cryogenic transmission electron microscopy and tomography showed that RCUs are composed of parallel membrane-bound segments, likely individual cells, encapsulated by an S-layer-like periodic surface covering. RCUs displayed pilus-like appendages protruding as bundles of multiple threads that extend parallel to each other, and splay out at the tips and/or intertwine, in stark contrast to all known types of bacterial pili that consist of single, hair-like structures. These observations highlight the diversity of novel microbial forms and lifestyles that await discovery and characterization using tools complementary to genomics such as microscopy.

category: microbiology


title: MRGM: a mouse reference gut microbiome reveals a large functional discrepancy for gut bacteria of the same genus between mice and humans

abstract:
The gut microbiome is associated with human diseases and interacts with dietary components and drugs. In vivo mouse models may be effective for studying diet and drug effects on the gut microbiome. We constructed a mouse reference gut microbiome (MRGM, https://www.mbiomenet.org/MRGM/) that includes newly-assembled genomes from 878 metagenomes. Leveraging samples with ultra-deep metagenomic sequencing (>130 million read pairs), we demonstrated quality improvement in assembled genomes for mouse gut microbes as sequencing depth increased. MRGM provides a catalog of 46,267 non-redundant genomes with [≥]70% completeness and [≤]5% contamination comprising 1,689 representative bacterial species and 15.2 million non-redundant proteins. Importantly, MRGM significantly improved the taxonomic classification rate of sequencing reads from mouse fecal samples compared to previous databases. Using MRGM, we determined that reliable low-abundance taxa profiles of the mouse gut microbiome require sequencing >10 million reads. Despite the high overall functional similarity of the mouse and human gut microbiomes, only ~10% of MRGM species are shared with the human gut microbiome. Although ~80% of MRGM genera are present in the human gut microbiome, ~70% of the shared genera have <40% of core gene content for the respective genus with human counterparts. These suggest that although metabolic processes of the human gut microbiome largely occur in the mouse gut microbiome, functional translations between them according to genus-level taxonomic commonality require caution.

Key PointsO_LIMRGM provides 46,267 genomes comprising 1,689 bacterial species of mouse gut microbiome.
C_LIO_LIDespite high overlap of genera, functional discrepancy between mouse and human gut microbiota is large.
C_LIO_LILineage-specific markers underestimate the completeness of assembled genomes for uncharacterized taxa.
C_LI


title: Alternative Cdc20 translational isoforms bypass the spindle assembly checkpoint to control mitotic arrest duration

abstract: Mitotic chromosome segregation defects activate the Spindle Assembly Checkpoint (SAC), which inhibits the APC/C co-activator Cdc20 to induce a prolonged cell cycle arrest. Once errors are corrected, the SAC is silenced thereby allowing anaphase onset and mitotic exit to proceed. However, in the presence of persistent, unresolvable errors, cells can undergo "mitotic slippage", exiting mitosis into a tetraploid G1 state and escaping the cell death that results from a prolonged arrest. The molecular logic that allows cells to balance these dueling mitotic arrest and slippage behaviors remains unclear. Here we demonstrate that human cells modulate their mitotic arrest duration through the presence of conserved, alternative Cdc20 translational isoforms. Translation initiation at downstream start sites results in truncated Cdc20 isoforms that are resistant to SAC-mediated inhibition and promote mitotic exit even in the presence of mitotic perturbations. Targeted molecular changes or naturally-occurring mutations in cancer cells that alter the relative Cdc20 isoform levels or its translational regulatory control modulate both mitotic arrest duration and anti-mitotic drug sensitivity. Our work reveals a critical role for the differential translational regulation of Cdc20 in mitotic arrest timing, with important implications for the diagnosis and treatment of human cancers.

category: cell biology


title: The role of mitochondrial energetics in the origin and diversification of eukaryotes

abstract:
The origin of eukaryotic cell size and complexity is thought by some to have required an energy excess provided by mitochondria, whereas others claim that mitochondria provide no energetic boost to eukaryotes. Recent observations show that energy demand scales continuously and linearly with cell volume across both prokaryotes and eukaryotes, and thus suggest that eukaryotes do not have an increased energetic capacity over prokaryotes. However, amounts of respiratory membranes and ATP synthases scale super-linearly with cell surface area. Furthermore, the energetic consequences of the contrasting genomic architectures between prokaryotes and eukaryotes have yet to be precisely quantified. Here, we investigated (1) potential factors that affect the cell volumes at which prokaryotes become surface area-constrained, (2) the amount of energy that is divested to increasing amounts of DNA due to the contrasting genomic architectures of prokaryotes and eukaryotes, and (3) the costs and benefits of respiring symbionts for a host cell. Our analyses suggest that prokaryotes are not necessarily constrained by their cell surfaces at cell volumes of 100-103 m3, and that the genomic architecture of eukaryotes is only slightly advantageous at genomes sizes of 106-107 bp. This suggests that eukaryotes may have first evolved without the need for mitochondria as these ranges hypothetically encompass the Last Eukaryote Common Ancestor and its proto-eukaryotic ancestors. At these slightly larger cell volumes, a host cell may also have derived a greater advantage (lower cost) from harboring a population of ATP-producing symbionts. However, our analyses also show that increasingly larger and fast-dividing prokaryotes would have a shortage of surface area devoted to respiration and would disproportionally divest more energy to DNA synthesis at larger genome sizes. We thus argue that, even though mitochondria may not have been required by the first eukaryotes, the successful diversification of eukaryotes into larger and faster-growing cells was ultimately contingent upon the origin of mitochondria.

SignificanceThere has been a lot of theorizing about the evolution of eukaryotes from prokaryotes, but no consensus seems to be on the horizon. Our quantitative analyses on the required amount of respiratory membrane, and the amount of energy diverted to DNA synthesis, by both prokaryotes and eukaryotes, suggest that mitochondria provided rather small advantages to the first eukaryotes, but were advantageous for the macro-evolutionary diversification of eukaryotes. This conclusion provides a middle road in the debate between those that claim that the origin of eukaryotes required a massive energy boost provided by mitochondria, and those that argue that the origin of mitochondria did not represent a quantum leap in energetic advantages to eukaryotes.


title: Habitat determines convergent evolution of cephalic horns in vipers

abstract: Phenotypic convergence of traits in similar environments can provide insights into the evolutionary processes shaping trait evolution. Among squamate reptiles, horn-like cephalic appendages have evolved under various selective pressures, including selection for defence, crypsis or sexual selection. Yet, among snakes, particularly vipers, the functional and evolutionary significance of horns are unknown. We used a comparative phylogenetic approach with habitat and diet data on 263 viper taxa to shed light on the selective pressures underlying horn evolution in vipers. We detected significant correlations with habitat but not diet. The relative positions of horns are ecologically divergent in that supranasal horns are positively correlated with terrestrial forest habitats while supraocular horns are negatively correlated with terrestrial forest habitats and associated with arboreal or sparsely vegetated habitats. Multiple independent origins of supranasal or supraocular horns in similar habitats provide evidence of adaptive convergence. Comparisons with other snake lineages suggest that cephalic appendages may have evolved under selection for crypsis in ambush foraging snakes.


title: Excessive fetal growth affects HSC quiescence maintenance through epigenetic programming of EGR1 transcriptional network

abstract: Excessive fetal growth is associated with DNA methylation alterations in human hematopoietic stem and progenitor cells (HSPC), but their functional impact remains elusive. We implemented an integrative analysis combining single-cell epigenomics, single-cell transcriptomics, and in vitro analyses to functionally link DNA methylation changes to putative alterations of HSPC functions. We showed in hematopoietic stem cells (HSC) from large for gestational age neonates that both DNA hypermethylation and chromatin rearrangement target a specific network of transcription factors known to sustain stem cell quiescence. In parallel, we found a decrease expression of key genes regulating HSC differentiation including EGR1, KLF2, SOCS3, and JUNB. Our functional analyses showed that this epigenetic programming was associated with a decreased ability for HSCs to stay quiescent. Taken together, our multimodal approach using single-cell (epi)genomics showed that human fetal overgrowth affects hematopoietic stem cells quiescence maintenance via epigenetic programming.

category: genomics


title: Early epigenetic influence on EGR1, KLF2 and KLF4 transcriptional network associates with alteration of HSCs quiescence


title: Oral administration of bacterial Beta cell expansion factor A (BefA) alleviates diabetes in mice with Type 1 and Type 2 diabetes

abstract: Diabetes mellitus (DM) is a group of metabolic diseases, which is of urgent need to develop new therapeutic DM oral drugs with less side effects and sound therapeutic efficacy. In this study, a Beta cell expansion factor A (BefA) production strain of Escherichia Coli BL21-pet 28C-BefA was constructed, and the anti-diabetes effect of BefA was evaluated using type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) mice models. The T1DM mice results indicated that BefA significantly reduced the blood glucose level, exerted protective function of islet {beta} cell morphology, down-regulated the TLR-4, p-NF{kappa}B/NF{kappa}B, Bax/Bcl-2 expressions and the secretion level of IL-1{beta}, TNF-, increased the expression of PDX-1 protein and insulin secretion in a concentration-dependent manner, and restored the disturbed microbial diversity to normal level. Similar with the T1DM mice, BefA obviously increased islet {beta} cells, reduced inflammatory reaction and apoptosis in T2DM mice, and also improved liver lipid metabolism by down-regulating the expression of CEBP-, ACC, Fasn and inhibiting the synthesis of triglyceride and induce Cpt-1, Hmgcs2, Ppar in a concentration-dependent manner. In the present study, we verified therapeutic effect and potential mechanisms of BefA in mammal for the first time, providing basic data for its clinical application.

category: biochemistry


title: Benchmarking AlphaFold for protein complex modeling reveals accuracy determinants

abstract: High resolution experimental structural determination of protein-protein interactions has led to valuable mechanistic insights, yet due to the massive number of interactions and experimental limitations there is a need for computational methods that can accurately model their structures. Here we explore the use of the recently developed deep learning method, AlphaFold, to predict structures of protein complexes from sequence. With a benchmark of 152 diverse heterodimeric protein complexes, multiple implementations and parameters of AlphaFold were tested for accuracy. Remarkably, many cases had highly accurate models generated as top-ranked predictions, greatly surpassing the performance of unbound protein-protein docking, whereas antibody-antigen docking was largely unsuccessful. While AlphaFold-generated accuracy predictions were able to discriminate near-native models, previously developed scoring protocols improved performance. Our study demonstrates that end-to-end deep learning can accurately model transient protein complexes, and identifies areas for improvement to guide future developments to reliably model any protein-protein interaction of interest.


title: NanoSplicer: Accurate identification of splice junctions using Oxford Nanopore sequencing

abstract:
MotivationLong read sequencing methods have considerable advantages for characterising RNA isoforms. Oxford nanopore sequencing records changes in electrical current when nucleic acid traverses through a pore. However, basecalling of this raw signal (known as a squiggle) is error prone, making it challenging to accurately identify splice junctions. Existing strategies include utilising matched short-read data and/or annotated splice junctions to correct nanopore reads but add expense or limit junctions to known (incomplete) annotations. Therefore, a method that could accurately identify splice junctions solely from nanopore data would have numerous advantages.

ResultsWe developed "NanoSplicer" to identify splice junctions using raw nanopore signal (squiggles). For each splice junction the observed squiggle is compared to candidate squiggles representing potential junctions to identify the correct candidate. Measuring squiggle similarity enables us to compute the probability of each candidate junction and find the most likely one. We tested our method using 1. synthetic mRNAs with known splice junctions 2. biological mRNAs from a lung-cancer cell-line. The results from both datasets demonstrate NanoSplicer improves splice junction identification, especially when the basecalling error rate near the splice junction is elevated. Our method is implemented in the software package NanoSplicer, available at https://github.com/shimlab/NanoSplicer.


title: Multiple paths lead to salt tolerance - pre-adaptation vs dynamic responses from two closely related extremophytes

abstract: Salt tolerance is a complex trait with much of the underlying genetic variation and integrated response strategies yet to be discovered from stress adapted plants. Schrenkiella parvula and Eutrema salsugineum are salt-tolerant extremophytes related to Arabidopsis thaliana in Brassicaceae. We investigated their response strategies contrasted against the salt-sensitive model, A. thaliana to cope with salt stresses via transcriptomic, metabolomic, and ionomic adjustments. The extremophytes exemplified divergent routes to achieve nutrient balance, build osmotolerance, boost antioxidant capacity, and extend transcriptomic support for modified ion transport and stress signaling. Those led to similar molecular phenotypes adapted to salt stress in the extremophytes, absent in A. thaliana. The predominant transcriptomic signals in all three species were associated with salt stress. However, root architecture modulation mediated by negative regulation of auxin and ABA signaling supported minimally-affected root growth unique to each extremophyte during salt treatments. Overall, E. salsugineum exhibited pre-adapted responses at the metabolome level, whereas S. parvula showed dynamic metabolomic responses coupled to a pre-adapted transcriptome to survive salt stress. Our work shows that the two extremophytes share common salt tolerance features, but differ substantially in pathways leading to the convergent, adaptive traits.


title: Inactivation of SARS Coronavirus 2 and COVID-19 patient samples for contemporary immunology and metabolomics studies

abstract: In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from Wuhan, China spurring the Coronavirus Disease-19 (COVID-19) pandemic that has resulted in over 219 million confirmed cases and nearly 4.6 million deaths worldwide. Intensive research efforts ensued to constrain SARS-CoV-2 and reduce COVID-19 disease burden. Due to the severity of this disease, the US Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level 3 (BSL3) containment laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred and manipulated at lower containment levels (i.e., BSL2), and maintain the fidelity of downstream assays to expedite the development of medical countermeasures (MCMs). We demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with paraformaldehyde solution or other commonly-used branded reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and antibody detection assays, heat inactivation following cDNA amplification of single-cell emulsions for droplet-based single-cell mRNA sequencing applications, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of protocols for viral inactivation of SARS-CoV-2 and COVID-19 patient samples for downstream contemporary immunology assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.


title: ICA treatment diabets induced bone loss via primary cilia/Gli2/Osteocalcin signaling pathway

abstract: Diabetes mellitus, as a metabolic system disorder disease, aggravates the disease burden of patients and affects the quality of human life. Diabetes-associated bone complications lead to decreased bone mechanical strength and osteoporosis. Evidences show that chronic hyperglycemia and metabolic intermediates, such as inflammatory factor, reactive oxygen species (ROS) and advanced glycation end products (AGEs), are regarded as dominant hazardous factors of primary cilia/Gli2 signal disorders. Case studies have demonstrated abnormal bone metabolism in diabetics, however, how diabetes damages primary cilia/Gli2 signal is largely unknown. Therefore, we studied the effects of diabetes on femoral primary cilia by establishing a Streptozocin (STZ)-induced diabetic (Sprague Dawley) SD rat model and diabetic bone loss cell model in vitro. Our results confirmed that diabetes impaired femur primary cilia, osteoblast differentiation and mineralization by inhibiting primary cilia/Gli2 signaling pathway, additionally, Icariin(ICA) treatment could rescue the impairment of osteoblast differentiation caused by high glucose medium in vitro. ICA activated primary cilia/Gli2/osteocalcin signaling pathway of osteoblasts by protecting primary cilia from glucotoxicity imposed by diabetes, intact primary cilia could be as anchoring sites, in which Gli2 was processed and modified, and matured Gli2 entered the nucleus to initiate downstream osteocalcin gene transcription. Additionally, ICA inhibited ROS production of mitochondria, thus balanced mitochondrial energy metabolism and oxidative phosphorylation. All results suggest that ICA can protect the primary cilia and mitochondria of osteoblast by reducing intracellular ROS, thereby recover primary cilia/Gli2 signaling pathway to facilitate osteoblast differentiation and mineralization, suggesting that ICA has potential as a novel type of drug treating bone loss induced by diabetes.


title: Computational correction of cell-specific gene-independent effects in CRISPR-Cas9 essentiality screens: REStricted CUbic SplinEs with Mixed Models (RESCUE-MM)

abstract: Increased gene copy number has been associated with a greater antiproliferative response upon genome editing, regardless of the true essentiality profile of the targeted gene. Many methods have been developed to adjust for genomic copy number technical artifacts. Existing methods use a two-step correction by pre-processing the data prior to the final analysis. It has been shown that two-step corrections can produce unreliable results, due to the creation of a correlation structure in the corrected data. If this structure is unaccounted for, gene-essentiality levels can be inflated or underestimated, affecting the False Discovery Rate (FDR). We propose a one-step correction using restricted cubic splines (RCS) to be a simpler alternative which reduces the bias in downstream analyses. Moreover, most existing methods combine guide-level results to yield gene-level estimates which can misrepresent the true gene essentiality profile depending on the guide-averaging method. Our model-based approach (RESCUE-MM) for copy number correction provides a more flexible framework that allows for guide-level essentiality estimation while accommodating more complex designs with grouped data. We provide comparisons to existing copy number correction methods and suggest how to include copy number adjustment in a one-step correction fashion in multiple experimental designs.


title: Single-cell transcriptional analysis of the immune tumour microenvironment during myeloma disease evolution

abstract: Multiple myeloma is universally preceded by a premalignant disease state. However, efforts to develop preventative therapeutic strategies are hindered by an incomplete understanding of the immune mechanisms associated with progression. Using single-cell RNA-sequencing, we profiled 104,880 cells derived from the bone marrow of V{kappa}*MYC mice across the myeloma progression spectrum, of which 97,720 were identified as non-malignant cells of the tumour microenvironment. Analysis of the non-malignant cells comprising the immune microenvironment identified mechanisms associated with disease progression in innate and adaptive immune cell populations. This included activation of IL-17 signaling in myeloid cells from precursor mice, accompanied by upregulation of Il6 gene expression in basophils. In the T/Natural killer cell compartment, we identified Tox-expressing CD8+ T cells enriched in the tumour microenvironment of mice with overt disease, with co-expression of LAG3 and PD-1, as well as elevated T cell exhaustion signatures in mice with early disease. We subsequently showed that early intervention with combinatorial blockade of LAG3 and PD-1 using neutralizing monoclonal antibodies delayed tumor progression and improved survival of V{kappa}*MYC mice. Together, this work provides insight into the biology of myeloma evolution and nominates a treatment strategy for early disease.


title: A focal adhesion kinase-YAP signaling axis drives drug tolerant persister cells and residual disease in lung cancer

abstract: Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant tumor cells which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.


title: The proteasome regulator PSME4 drives immune evasion and abrogates anti-tumor immunity in NSCLC

abstract:
Protein degradation by proteasomes is important for the immune response against tumors. Antigens generated by the proteasome promote immune cell infiltration into tumors and improve tumors responses to immunotherapy. For example, immunoproteasomes - a subset of proteasomes induced by inflammatory signals - may improve the response of melanomas to immune checkpoint inhibitors (ICI) by eliciting tumor inflammation. Yet, it is unclear whether and how protein degradation by proteasomes impacts cancer progression and contributes to immune evasion and resistance. Here, we profile the proteasome-cleaved peptides in lung cancers and find that PSME4 serves as a novel inhibitory regulator of the immunoproteasome, playing an anti-inflammatory role in cancer. Biochemical assays combined with scRNA-seq, immunopeptidomics and in vivo analyses demonstrate that PSME4 promotes an immunosuppressive environment around the tumor and abrogates anti-tumor immunity by inhibiting antigen presentation and attenuating tumor inflammation. Furthermore, we find that PSME4 expression is correlated with responsiveness to ICI across several cancer types. Our findings suggest that PSME4-mediated regulation of proteasome activity is a novel mechanism of immune evasion in non-small-cell lung carcinoma and may be targeted therapeutically for restoring anti-tumor immunity.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/464690v1_ufig1.gif" ALT="Figure 1">
View larger version (39K):
org.highwire.dtl.DTLVardef@e87910org.highwire.dtl.DTLVardef@641843org.highwire.dtl.DTLVardef@175092corg.highwire.dtl.DTLVardef@ad34c2_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIMapping the degradation landscape in Non-Small Cell Lung Cancer (NSCLC) uncovers altered proteasome activity and composition
C_LIO_LIProteasome regulator PSME4 plays an anti-inflammatory role in NSCLC by attenuating immunoproteasome activity
C_LIO_LIPSME4 restricts tumor antigen presentation and cytokine secretion, defining a  cold tumor environment
C_LIO_LIPSME4 drives tumor immune evasion and is associated with resistance to immunotherapy
C_LI


title: Characterization of raloxifene as potential pharmacological agent against SARS-CoV-2 and its variants

abstract:
The new coronavirus that emerged, called SARS-CoV-2, is the causative agent of the COVID-19 pandemic. The identification of potential drug candidates that can rapidly enter clinical trials for the prevention and treatment of COVID-19 is an urgent need, despite the recent introduction of several new vaccines for the prevention and protection of this infectious disease, which in many cases becomes severe. Drug repurposing (DR), a process for studying existing pharmaceutical products for new therapeutic indications, represents one of the most effective potential strategies employed to increase the success rate in the development of new drug therapies. We identified raloxifene, a known Selective Estrogen Receptor Modulator (SERM), as a potential pharmacological agent for the treatment of COVID-19 patients. Following a virtual screening campaign on the most relevant viral protein targets, in this work we report the results of the first pharmacological characterization of raloxifene in relevant cellular models of COVID-19 infection. The results obtained on all the most common viral variants originating in Europe, United Kingdom, Brazil, South Africa and India, currently in circulation, are also reported, confirming the efficacy of raloxifene and, consequently, the relevance of the proposed approach.

Taken together, all the information gathered supports the clinical development of raloxifene and confirms that the drug can be proposed as a viable new option to fight the pandemic in at least some patient populations. The results obtained so far have paved the way for a first clinical study to test the safety and efficacy of raloxifene, just concluded in patients with mild to moderate COVID-19.

category: pharmacology and toxicology


title: NLR immune receptor-nanobody fusions confer plant disease resistance

abstract:
Plant pathogens cause recurrent epidemics that threaten crop yield and global food security. Efforts to retool the plant immune system have been limited to modifying natural components and can be nullified by the emergence of new pathogen races. Therefore, there is a need to develop made-to-order synthetic plant immune receptors with resistance tailored to the pathogen genotypes present in the field. Here we show that plant immune receptors can be used as scaffolds for VHH nanobody fusions that bind fluorescent proteins (FPs). The receptor-nanobody fusions signal in the presence of the corresponding FP and confer resistance against plant viruses expressing FPs. Given that nanobodies can be raised against virtually any molecule, immune receptor-nanobody fusions have the potential to generate resistance against all major plant pathogens and pests.

One-Sentence SummaryPlant immune receptor-nanobody fusions enable made-to-order disease resistance genes.


title: The humanized nanobody RBD-1-2G tolerates the spike N501Y mutation to neutralize SARS-CoV-2

abstract: Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

category: synthetic biology


title: A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike


title: Tsetse RNA Virome: Novel Iflavirus Genomes in Glossina morsitans and Other Tsetse Species

abstract: Tsetse flies (Glossina spp.) are vectors of Human and Animal African trypanosomiasis. The tsetse microbiome has been extensively studied in the context of bacterial endosymbiont-host interactions, however, remarkably little is known about the tsetse virome with only one well-characterized DNA virus, the salivary gland hypertrophy virus (SGHV). Here we report the genomes of four iflaviruses identified in tsetse flies and their distribution in public RNA-seq libraries, mainly from laboratory colonies. Strikingly, the iflavirus identified in G. morsitans, provisionally named Glossina iflavirus 1 (GliflaV1), is present in all 136 RNA-seq libraries of G. morsitans maintained at different institutions, and displays a broad tissue tropism and high abundance, reaching up to 15% of library content. Its ubiquitous distribution and presence in the reproductive tissues, intrauterine larvae, and teneral flies suggest it is part of the initial core microbiota maternally transmitted to the progeny. None of the G. morsitans samples harbor iflaviruses identified in the other three, more closely related, tsetse species which, conversely, do not harbor the iflavirus from G. morsitans. Though apparently asymptomatic, these infections may influence tsetse host fitness, developmental or biological processes which might be relevant in the context of tsetse population control strategies, mass rearing, and paratransgenesis, and open up new opportunities to study the quadripartite system of interactions among the invertebrate host, the parasitic protozoan, and both viral and bacterial symbionts.

category: zoology


title: A transcriptomic axis predicts state modulation of cortical interneurons

abstract: Transcriptomics has revealed the exquisite diversity of cortical inhibitory neurons1-7, but it is not known whether these fine molecular subtypes have correspondingly diverse activity patterns in the living brain. Here, we show that inhibitory subtypes in primary visual cortex (V1) have diverse correlates with brain state, but that this diversity is organized by a single factor: position along their main axis of transcriptomic variation. We combined in vivo 2-photon calcium imaging of mouse V1 with a novel transcriptomic method to identify mRNAs for 72 selected genes in ex vivo slices. We used transcriptomic clusters (t-types)4 to classify inhibitory neurons imaged in layers 1-3 using a three-level hierarchy of 5 Families, 11 Classes, and 35 t-types. Visual responses differed significantly only across Families, but modulation by brain state differed at all three hierarchical levels. Nevertheless, this diversity could be predicted from the first transcriptomic principal component, which predicted a cell types brain state modulation and correlations with simultaneously recorded cells. Inhibitory t-types with narrower spikes, lower input resistance, weaker adaptation, and less axon in layer 1 as determined in vitro8 fired more in resting, oscillatory brain states. Transcriptomic types with the opposite properties fired more during arousal. The former cells had more inhibitory cholinergic receptors, and the latter more excitatory receptors. Thus, despite the diversity of V1 inhibitory neurons, a simple principle determines how their joint activity shapes state-dependent cortical processing.


title: Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in nonhuman primates is coincident with an anamnestic antibody response in the lower airway

abstract: mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. We immunized rhesus macaques at weeks 0 and 4 and assessed immune responses over one year in blood, upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody binding titers also decreased in bronchoalveolar lavage (BAL). Four days after challenge, virus was unculturable in BAL and subgenomic RNA declined [~]3-log10 compared to control animals. In nasal swabs, sgRNA declined 1-log10 and virus remained culturable. Anamnestic antibody responses (590-fold increase) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

category: immunology


title: SARS-CoV-2 Infection Impacts Carbon Metabolism and Depends on Glutamine for Replication in Syrian Hamster Astrocytes

abstract: Coronaviruses belong to a well-known family of enveloped RNA viruses and are the causative agent of the common cold. Although the seasonal coronaviruses do not pose a threat to human life, three members of this family, i.e., SARS-CoV, MERS-CoV and recently, SARS-CoV2, may cause severe acute respiratory syndrome and lead to death. Unfortunately, COVID-19 has already caused more than 4.4 million deaths worldwide. Although much is better understood about the immunopathogenesis of the lung disease, important information about systemic disease is still missing, mainly concerning neurological parameters. In this context, we sought to evaluate immunometabolic changes using in vitro and in vivo models of hamsters infected with SARS-CoV-2. Here we show that, besides infecting hamsters astrocytes, SARS-CoV-2 induces changes in protein expression and metabolic pathways involved in carbon metabolism, glycolysis, mitochondrial respiration, and synaptic transmission. Interestingly, many of the differentially expressed proteins are concurrent with proteins that correlate with neurological diseases, such as Parkinsonss disease, multiple sclerosis, amyotrophic lateral sclerosis, and Huntingtons disease. Metabolic analysis by high resolution real-time respirometry evidenced hyperactivation of glycolysis and mitochondrial respiration. Further metabolomics analysis confirmed the consumption of many metabolites, including glucose, pyruvate, glutamine, and alpha ketoglutarate. Interestingly, we observed that glutamine was significantly reduced in infected cultures, and the blockade of mitochondrial glutaminolysis significantly reduced viral replication and pro-inflammatory response. SARS-CoV-2 was confirmed in vivo as hippocampus, cortex, and olfactory bulb of intranasally infected hamsters were positive for viral genome several days post-infection. Altogether, our data reveals important changes in overall protein expression, mostly of those related to carbon metabolism and energy generation, causing an imbalance in important metabolic molecules and neurotransmitters. This may suggest that some of the neurological features observed during COVID-19, as memory and cognitive impairment, may rely on altered energetic profile of brain cells, as well as an unbalanced glutamine/glutamate levels, whose importance for adequate brain function is unquestionable.


title: Discovery and Biosynthesis of Persiathiacins: Unusual Polyglycosylated Thiopeptides Active against Multi-drug resistant Tuberculosis

abstract: Thiopeptides are ribosomally biosynthesized and post-translationally modified peptides (RiPPs) that potently inhibit the growth of Gram-positive bacteria by targeting multiple steps in protein biosynthesis. The poor pharmacological properties of thiopeptides, in particular their low aqueous solubility, has hindered their development into clinically useful antibiotics. Antimicrobial activity screens of a library of Actinobacterial extracts led to discovery of the novel polyglycosylated thiopeptides persiathiacins A and B from Actinokineospora sp. UTMC 2475 and Actinokineospora sp. UTMC 2448. Persiathiacin A is active against methicillin-resistant Staphylococcus aureus (MRSA) and several Mycobacterium tuberculosis strains, including drug-resistant and multidrug-resistant clinical isolates, and does not significantly affect the growth of ovarian cancer cells at concentrations up to 400 M. In vitro translation assays showed that, like other thiopeptide antibiotics, persiathiacin A targets protein biosynthesis. Polyglycosylated thiopeptides are extremely rare and nothing is known about their biosynthesis. Sequencing and analysis of the Actinokineospora sp. UTMC 2448 genome enabled identification of the putative persiathiacin biosynthetic gene cluster. A cytochrome P450 encoded by this gene cluster catalyses the hydroxylation of nosiheptide in vitro and in vivo, consistent with the proposal that the cluster directs persiathiacin biosynthesis. Several genes in the cluster encode homologues of enzymes known to catalyse the assembly and attachment of deoxysugars during the biosynthesis of other classes of glycosylated natural products. The discovery of the persiathiacins and their biosynthetic gene cluster thus provides the basis for the development of biosynthetic engineering approaches to the creation of novel (poly)glycosylated thiopeptide derivatives with enhanced pharmacological properties.


title: Experimental microevolution of Trypanosoma cruzi reveals hybridization and clonal mechanisms driving rapid diversification of genome sequence and structure.

abstract: Protozoa and fungi are known to have extraordinarily diverse mechanisms of genetic exchange. However, the presence and epidemiological relevance of genetic exchange in Trypanosoma cruzi, the agent of Chagas disease, has been controversial and debated for many years. Field studies have identified both predominantly clonal and sexually recombining natural populations. Two of six natural T. cruzi lineages (TcV and TcVI) show hybrid mosaicism, using analysis of single-gene locus markers. The formation of hybrid strains in vitro has been achieved and this provides a framework to study the mechanisms and adaptive significance of genetic exchange. Using whole genome sequencing of a set of experimental hybrids strains, we have confirmed that hybrid formation initially results in tetraploid parasites. The hybrid progeny showed novel mutations that were not attributable to either (diploid) parent showing an increase in amino acid changes. In long-term culture, up to 800 generations, there was progressive, gradual erosion of progeny genomes towards triploidy, yet retention of elevated copy number was observed at several core housekeeping loci. Our findings indicate hybrid formation by fusion of diploid T. cruzi, followed by sporadic genome erosion, but with substantial potential for adaptive evolution, as has been described as a genetic feature of other organisms, such as some fungi.


title: ARP2/3- and resection-coupled genome reorganization facilitates translocations

abstract: DNA end-resection and nuclear actin-based movements orchestrate clustering of double-strand breaks (DSBs) into homology-directed repair (HDR) domains. Here, we analyze how actin nucleation by ARP2/3 affects damage-dependent and -independent 3D genome reorganization and facilitates pathologic repair. We observe that DNA damage, followed by ARP2/3-dependent establishment of repair domains enhances local chromatin insulation at a set of damage-proximal boundaries and affects compartment organization genome-wide. Nuclear actin polymerization also promotes interactions between DSBs, which in turn facilitates aberrant intra- and inter-chromosomal rearrangements. Notably, BRCA1 deficiency, which decreases end-resection, DSB mobility, and subsequent HDR, nearly abrogates recurrent translocations between AsiSI DSBs. In contrast, loss of functional BRCA1 yields unique translocations genome-wide, reflecting a critical role in preventing spontaneous genome instability and subsequent rearrangements. Our work establishes that the assembly of DSB repair domains is coordinated with multiscale alterations in genome architecture that enable HDR despite increased risk of translocations with pathologic potential.

category: molecular biology


title: Rapid decline in Western monarch butterflies leads to phenological and geographic Allee effects

abstract: Allee effects are processes that become disrupted at low population sizes, causing further declines to eventual extinction. Identifying Allee effects in natural populations is important for both ecological theory and conservation efforts but challenging because populations typically only experience them briefly before collapsing. In 2018, the Western monarch butterfly population fell below the threshold that scientists predicted would trigger Allee effects. Here we show that since 2018, the Western monarch population has expanded more slowly during spring and summer breeding and filled a smaller breeding range. Other aspects of the monarch life cycle (winter survival, fecundity, morphology) have not declined after the crash. However, winter survival is much lower and wing sizes of overwintering monarchs are larger now than in the 20th century, suggesting higher mortality and selection for increased dispersal in recent decades. Delayed arrival to parts of the Western monarch breeding range is a novel Allee effect, which could lead to temporal mismatches between monarchs and their and host plants.


title: Quantification of breeding sites parameters in shaping bacterial communities in Aedes aegypti (Diptera: Culicidae).

abstract: Studies have demonstrated the importance of breeding site, few had disentangled the role of microbiome, physico-chemical and biological factors of water as well as landuse on larval microbial communities and their recruitment in mosquito. A quantitative exploration of the interplay of multiple factors on mosquito microbiome was performed using a dataset obtained through a field survey undertaken in French Guiana. Two complementary hypotheses were tested (i) the most dissimilar larval microbiome structures in breeding sites displayed the most contrasting water properties and land-use, (ii) a higher specificity level of environmental parameters have an incidence on larval microbiome. Variance partitioning approach validated the two hypothesis by providing evidence that water bacterial community is a most significant driver shaping the structure of the bacteriome in mosquito than other environmental parameters from the breeding sites. However, land-use does not play such important role to explain variance. Our results consolidate and complement the knowledge shaping mosquito microbiota but also highlighted the large unknown in understanding the ecology of the recruitment into host.


title: Assessment of Agaricus bisporus Mushroom as Protective Agent Against Ultraviolet Exposure

abstract: Mushrooms are versatile materials with applications including but not limited to food, cosmetics, and pharmaceutical industries. In this work, the potential of the common button mushroom, Agaricus bisporus, as a protective agent against ultraviolet exposure was assessed. The assessment was done by investigating the radical scavenging activity, sun protecting capability, and tyrosinase inhibiting properties of Agaricus bisporus ethanolic extract. The extraction was carried out using absolute ethanol as its solvent at low to room temperatures. The bioactive components of the ethanolic extract were analysed for its phenolic and flavonoid contents quantitatively, while other phytochemical agents were analysed qualitatively. The Agaricus bisporus ethanolic extract was found exhibit varying degree of activity in all of the assessment. We found low radical scavenging ability with %RSA IC50 of [~]5456 g/mL, low to moderate sun protecting factor of [~]5.355 at 5000 ppm concentration, and high tyrosinase inhibition property with IC50 of [~]2 g/mL. The high tyrosinase inhibition property was found to correlate with relatively high total phenolic content of [~]1143 mg GAE/100g for Agaricus bisporus and the presence of terpenoid in the ethanolic extract.


title: SINBAD: a flexible tool for single cell DNA methylation data

abstract: DNA methylation is an epigenetic mark that has vital importance in both development and disease. Single cell bisulfite sequencing technologies enable profiling of the methylome at high resolution, providing the basis for dissecting the heterogeneity and dynamics of DNA methylation in complex tissues and over time. Despite the rapid increase in the number of experimental protocols for methylome sequencing, analytical tools designed specifically for single-cell data are lacking. We developed a computational tool, SINBAD, for efficient and standardized pre-processing, quality assessment and analysis of single cell methylation data. Starting from multiplexed sequencing reads, major analysis modules of SINBAD include preprocessing, read mapping, methylation quantification, multivariate analysis, and gene signature profiling. SINBAD provides a flexible platform to implement interoperable and robust processing of single-cell methylome data.


title: Dynamics of lipids in the yeast lipid droplets

abstract: The physical properties and chemical composition of lipid droplets inside cells of the yeast Cryptococcus curvatus were investigated using quasi-elastic neutron scattering and mass spectrometry with complementary surface modeling using 3D microscopy. With temperature decrease from 310 to 280 K, their phase remained fluid, i.e., the droplets remained in the physiological state, unlike synthetic lipid membranes that transition to a gel phase. The lipid dynamics in the droplet was described by a model implying diffusion of the lipid and its hydrocarbon chains. The diffusion coefficient of the lipid chains (274x10-3 [A]2/ps at 310 K) was much higher than that observed in a synthetic lipid membrane because of the larger volume (up to 12 [A]) for the local dynamics in the droplet. These physical properties were correlated with the types of lipids composing the droplet. Based on that, the lipid packing and resulting energetic value of the yeast droplets are discussed in relation to their usefulness as biofuels.

category: biophysics


title: Building tools for machine learning and artificial intelligence in cancer research: best practices and a case study with the PathML toolkit for computational pathology

abstract: Imaging datasets in cancer research are growing exponentially in both quantity and information density. These massive datasets may enable derivation of insights for cancer research and clinical care, but only if researchers are equipped with the tools to leverage advanced computational analysis approaches such as machine learning and artificial intelligence. In this work, we highlight three themes to guide development of such computational tools: scalability, standardization, and ease of use. We then apply these principles to develop PathML, a general-purpose research toolkit for computational pathology. We describe the design of the PathML framework and demonstrate applications in diverse use-cases. PathML is publicly available at www.pathml.com.

category: cancer biology


title: Cytidine deaminase protects pancreatic cancer cells from replicative stress and drives resistance to DNA-targeting drugs

abstract: Chronic DNA replication stress and genome instability are two hallmarks of cancer that fuel oncogenesis and tumor diversity. Therapeutic approaches aimed to leverage tumor-specific replication stress to intolerable levels or to expose vulnerabilities for synthetic lethality purposes have recently gained momentum, especially for pancreatic cancer, a disease with no cure. However, the current knowledge regarding the molecular mechanisms involved in the replication stress response in pancreatic tumors is limited. Cytidine deaminase (CDA) is involved in the pyrimidine salvage pathway for DNA and RNA synthesis. Loss of CDA induces genomic instability in Bloom Syndrome, and CDA protects tumor cells from chemotherapy with pyrimidine analogs. Here, we show that CDA is overexpressed in genetically unstable pancreatic tumors, associates with a DNA replication signature, and is instrumental for experimental tumor growth. In cancer cells, CDA promotes DNA replication, increases replication fork speed, and controls replication stress and genomic stability levels. CDA expression is predictive of DNA-damaging drug efficacy and targeting CDA relieves resistance to chemotherapy in patients models, both in vitro and in vivo. Our findings shed new light on the mechanisms by which pancreatic cancer cells control replication stress, and highlight targeting of CDA as a potential therapeutic strategy to defeat tumor resistance to treatment.


title: Spatial coding in the hippocampus of flying owls

abstract: The elucidation of spatial coding in the hippocampus requires exploring diverse animal species. While robust place-cells are found in the mammalian hippocampus, much less is known about spatial coding in the hippocampus of birds - and nothing is known about avian spatial representation during flight. Here we used a wireless-electrophysiology system to record single neurons in the hippocampus and related pallial structures from freely flying barn owls (Tyto alba) - a central-place nocturnal predator species with excellent navigational abilities. The owls 3D position was monitored while it flew back and forth between two perches. We found place cells - neurons that robustly represented the owls location during flight, and its flight-direction - as well as neurons that coded the owls perching position between flights. Spatial coding was invariant to changes in lighting conditions and to the position of a salient object in the room. Place cells were found in the anterior hippocampus and in the adjacent posterior hyperpallium apicale, and to a much lesser extent in the visual Wulst (visual-cortex homologue). The finding of place-cells in flying owls suggests commonalities in spatial coding across a variety of species - including rodents, bats and owls.


title: Quercetin combined with Concanavalin A as a safe vaccine adjuvant that stimulates PBMCs proliferation, alleviates lipid peroxidation and boosts iNOS activity in the elderly.

abstract:
Immunosenescence, oxidative stress, and low vaccine efficacy are important symptoms of aging. The goal of our study was to see if quercetin had anti-aging and stimulating effects on PBMC immune cells in vitro. In the presence of concanavalin a, PBMCs were isolated from healthy elderly and young people and cultured in a complete RPMI-1640 medium supplemented with quercetin. Cell proliferation was assessed using the MTT colorimetric assay after a 48-hour incubation period. Spectrophophotometric assays were used to assess oxidative biomarkers (PC, MDA, and GSH). The ELISA method was used to determine the amount of IL-2 released. A Griess reagent was used to investigate iNOS activity. When compared to young control cells, aged PBMCs had lower proliferation potency, lower IL-2 and NO release, and higher MDA and PC levels. Importantly, quercetin-treated aged PBMCs have a high proliferative response comparable to young cells, restored iNOS activity, and increased levels of GSH antioxidant defences. In comparison to untreated aged PBMCs, treated PBMCs have lower lipo-oxidative damage but higher PC levels. Quercetin may be used as a promising dietary vaccinal adjuvant in the elderly, it has significant effects in reducing immunosenescence hallmarks, as well as mitigating the lipo-oxidative stress in PBMCs cells.

Practical abstractQuercetin is a common dietary polyphenol found in fruits and vegetables that is well known for its antioxidant activity in radical scavenging and anticancer properties characterized by immune system stimulation. In vitro, quercetin boosts immune responses in the elderly and reduces immunosenescence symptoms, according to our findings. As a result, this molecule could be a safe and promising vaccine adjuvant for boosting immunity and reducing aging complications in immune cells as well as reducing oxidative stress in PBMC cells.


title: Quercetin handles cellular oxidant / antioxidant systems and mitigates immunosenescence hallmarks in human PBMCs: an in vitro study


title: Interleukin 7 receptor drives Early T lineage Progenitor expansion

abstract:
Interleukin 7 (IL-7) and IL-7 receptor (IL-7r) are essential for T lymphocyte differentiation, by driving proliferation and survival of specific developmental stages. While early T lineage progenitors (ETP), the most immature thymocyte population known, have a history of IL-7r expression, it is unclear whether IL-7r is required at this stage. Here, we show that mice lacking IL-7 or IL-7r have a marked loss of ETPs that results mostly from a cell-autonomous defect in proliferation and survival, although no changes were detected in Bcl2 protein levels. Further, a fraction of ETPs responded to IL-7 stimulation ex vivo by phosphorylating Stat5, and IL-7r was enriched in the most immature Flt3+Ccr9+ ETPs. Consistently, IL-7 promoted the expansion of Flt3+ but not Flt3-ETPs on OP9-DLL4 cocultures, without affecting differentiation at either stage. Taken together, our data show that IL-7/IL-7r is necessary following thymus seeding, by promoting proliferation and survival of the most immature thymocytes.

SummaryPaiva et al. show that IL-7/IL-7r signaling upon thymus seeding is essential for proliferation and survival of the most immature early T lineage progenitors (ETP), thereby determining the physiological ETP cellularity.


title: A Prion that regulates genome diversification

abstract: Epigenetic mechanisms mediate diverse gene expression programs in growth and development. Here we report a protein-based epigenetic element, a prion, formed by the conserved DNA helicase Mph1/FANCM. [MIX+] is a cytoplasmically inherited state with propagation driven by the non-amyloid prion templating of Mph1. [MIX+] provides resistance to DNA damage, a gain-of-function trait that requires helicase activity. [MIX+] reduces mitotic mutation rates, but promotes meiotic crossovers, driving measurable phenotypic diversification in wild outcrosses. Remarkably, [MIX+] can be induced by DNA-damaging stresses in which it is beneficial. Thus, [MIX+] fuels a quasi-Lamarckian form of inheritance that promotes survival of the current generation and diversification of the next.


title: Stemness activity underlying whole brain regeneration in a basal chordate

abstract: Central nervous system (CNS) regeneration extent is highly diverse across the metazoans, with adult mammals demonstrating limited ability1,2. Understanding how neurons regenerate following injury remains a central challenge in regenerative medicine. Although conserved pathways associated with neural regeneration have been identified3,4, a study describing the stepwise morphogenetic changes that take place throughout a complete CNS regeneration is lacking. Utilizing the highly regenerative tunicate model Polycarpa mytiligera5, we characterized the morphological, cell proliferation, and transcriptomic dynamics that lead to entire CNS regeneration. The regenerated CNS of adult P. mytiligera expressed key neurodevelopmental markers that are not otherwise present in the adult CNS. Removal of the entire CNS resulted in high cell proliferation in the regenerated area. Transcriptome analysis revealed enhanced stem-cell related gene activity, with high expression of P53 and piRNA pathways preceding the activation of Notch, Wnt, and Nanos pathways. The CNS regeneration atlas created here depicts the transcriptomic landscape of the entire CNS regeneration process, revealing the core pathways that regulate neuronal response to injury, and the regeneration stage at which they are most pronounced. The molecular and cellular mechanisms controlling regenerative capacity that this atlas reveals could be used to develop approaches to enhancing neurogenesis in closely-related chordate species, including humans.

category: developmental biology


title: Density-dependent selection in Drosophila: evolution of egg size and hatching time

abstract: Many different laboratory studies of adaptation to larval crowding in Drosophila spp. have all yielded the evolution of pre-adult competitive ability, even though the ecological context in which crowding was experienced varied across studies. However, the evolution of competitive ability was achieved through different suites of traits in studies wherein crowding was imposed in slightly different ways. Earlier studies showed the evolution of increased competitive ability via increased larval feeding rate and tolerance to nitrogenous waste, at the cost of food to biomass conversion efficiency. However, more recent studies, with crowding imposed at relatively low food levels, showed the evolution of competitive ability via decreased larval development time and body size, and an increase in the time efficiency of conversion of food to biomass, with no change in larval feeding rate or waste tolerance. Taken together, these studies have led to a more nuanced understanding of how the specific details of larval numbers, food amounts etc. can affect which traits evolve to confer increased competitive ability. Here, we report results from a study in which egg size and hatching time were assayed on three sets of populations adapted to larval crowding experienced in slightly different ways, as well as their low density ancestral control populations. Egg size and hatching time are traits that may provide larvae with initial advantages under crowding through increased starting larval size and a temporal head-start, respectively. In each set of populations adapted to some form of larval crowding, the evolution of longer and wider eggs was seen, compared to controls, thus making egg size the first consistent correlate of the evolution of increased larval competitive ability across Drosophila populations experiencing crowding in slightly different ways. Among the crowding-adapted populations, those crowded at the lowest overall eggs/food density, but the highest density of larvae in the feeding band, showed the largest eggs, on an average. All three sets of crowding-adapted populations showed shorter average egg hatching time than controls, but the difference was significant only in the case of populations experiencing the highest feeding band density. Our results underscore the importance of considering factors other than just eggs/food density when studying the evolution of competitive ability, as also the advantages of having multiple selection regimes within one experimental set up, allowing for a more nuanced understanding of the subtlety with which adaptive evolutionary trajectories can vary across even fairly similar selection regimes.


title: Compatibility logic of human enhancer and promoter sequences

abstract: Gene regulation in the human genome is controlled by distal enhancers that activate specific nearby promoters. One model for the specificity of enhancer-promoter regulation is that different promoters might have sequence-encoded preferences for distinct classes of enhancers, for example mediated by interacting sets of transcription factors or cofactors. This "biochemical compatibility" model has been supported by observations at individual human promoters and by genome-wide measurements in Drosophila. However, the degree to which human enhancers and promoters are intrinsically compatible or specific has not been systematically measured, and how their activities combine to control RNA expression remains unclear. To address these questions, we designed a high-throughput reporter assay called enhancer x promoter (ExP) STARR-seq and applied it to examine the combinatorial compatibilities of 1,000 enhancer and 1,000 promoter sequences in human K562 cells. We identify a simple logic for enhancer-promoter compatibility - virtually all enhancers activated all promoters by similar amounts, and intrinsic enhancer and promoter activities combine multiplicatively to determine RNA output (R2=0.82). In addition, two classes of enhancers and promoters showed subtle preferential effects. Promoters of housekeeping genes contained built-in activating sequences, corresponding to motifs for factors such as GABPA and YY1, that correlated with both stronger autonomous promoter activity and enhancer activity, and weaker responsiveness to distal enhancers. Promoters of context-specific genes lacked these motifs and showed stronger responsiveness to enhancers. Together, this systematic assessment of enhancer-promoter compatibility suggests a multiplicative model tuned by enhancer and promoter class to control gene transcription in the human genome.

category: genetics


title: Bicolor angelfish (Centropyge bicolor) provides the first chromosome-level genome of the Pomacanthidae family

abstract: The Bicolor Angelfish, Centropyge bicolor, is a tropical coral reef fish. It is named for its striking two-color body. However, a lack of high-quality genomic data means little is known about the genome of this species. Here, we present a chromosome-level C. bicolor genome constructed using Hi-C data. The assembled genome is 650 Mbp in size, with a scaffold N50 value of 4.4 Mbp, and a contig N50 value of 114 Kbp. Protein-coding genes numbering 21,774 were annotated. Our analysis will help others to choose the most appropriate de novo genome sequencing strategy based on resources and target applications. To the best of our knowledge, this is the first chromosome-level genome for the Pomacanthidae family, which might contribute to further studies exploring coral reef fish evolution, diversity and conservation.


title: Fast and parallel nanoscale 3D tracking of heterogeneous mammalian chromatin dynamics

abstract: Chromatin organization and dynamics are critical for gene regulation. In this work we present a methodology for fast and parallel 3D tracking of multiple chromosomal loci of choice over many thousands of frames on various time scales. We achieved this by developing and combining fluorogenic and replenishable nanobody arrays, engineered point spread functions, and light sheet illumination. The result is gentle live-cell 3D tracking with excellent spatiotemporal resolution throughout the mammalian cell nucleus. Correction for both sample drift and nuclear translation facilitated accurate long-term tracking of the chromatin dynamics. We demonstrate tracking of both fast dynamics (50 Hz) and over time scales extending to several hours, and we find both large heterogeneity between cells and apparent anisotropy in the dynamics in the axial direction. We further quantify the effect of inhibiting actin polymerization on the dynamics and find an overall increase in both the apparent diffusion coefficient D* and anomalous diffusion exponent , and a transition to more isotropic dynamics in 3D after such treatment. We think that our methodology in the future will allow researchers to obtain a better fundamental understanding of chromatin dynamics and how it is altered during disease progression and after perturbations of cellular function.


title: A suspension technique for efficient large-scale cancer organoid culturing and perturbation screens

abstract: Organoid cell culture methodologies are enabling the generation of cell models from healthy and diseased tissue. Patient-derived cancer organoids that recapitulate the genetic and histopathological diversity of patient tumours are being systematically generated, providing an opportunity to investigate novel cancer biology and therapeutic approaches. The use of organoid cultures for many applications, including genetic and chemical perturbation screens, is limited due to the technical demands and cost associated with their handling and propagation. Here we report and benchmark a suspension culture technique for cancer organoids which allows for the expansion of models to tens of millions of cells with increased efficiency in comparison to standard organoid culturing protocols. Using whole-genome DNA and RNA sequencing analyses, as well as medium-throughput drug sensitivity testing and genome-wide CRISPR-Cas9 screening, we demonstrate that cancer organoids grown as a suspension culture are genetically and phenotypically similar to their counterparts grown in standard conditions. This culture technique simplifies organoid cell culture and extends the range of organoid applications, including for routine use in large-scale perturbation screens.


title: Kinetic and data-driven modeling of pancreatic 	β-cell central carbon metabolism and insulin secretion

abstract:
Pancreatic {beta}-cells respond to increased extracellular glucose levels by initiating a metabolic shift. That change in metabolism is part of the process of glucose-stimulated insulin secretion and is of particular interest in the context of diabetes. However, we do not fully understand how the coordinated changes in metabolic pathways and metabolite products influence insulin secretion. In this work, we apply systems biology approaches to develop a detailed kinetic model of the intracellular central carbon metabolic pathways in pancreatic {beta}-cells upon stimulation with high levels of glucose. The model is calibrated to published metabolomics datasets for the INS1 823/13 cell line, accurately capturing the measured metabolite fold-changes. We first employed the calibrated mechanistic model to estimate the stimulated cells fluxome. We then used the predicted network fluxes in a data-driven approach to build a partial least squares regression model. By developing the combined kinetic and data-driven modeling framework, we gain insights into the link between {beta}-cell metabolism and glucose-stimulated insulin secretion. The combined modeling framework was used to predict the effects of common anti-diabetic pharmacological interventions on metabolite levels, flux through the metabolic network, and insulin secretion. Our simulations reveal targets that can be modulated to enhance insulin secretion. The model is a promising tool to contextualize and extend the usefulness of metabolomics data and to predict dynamics and metabolite levels that are difficult to measure in vitro. In addition, the modeling framework can be applied to identify, explain, and assess novel and clinically-relevant interventions that may be particularly valuable in diabetes treatment.

Author SummaryDiabetes is among the most common chronic illnesses, occurring when the {beta}-cells in the pancreas are unable to produce enough insulin to properly manage the bodys blood sugar levels. {beta}-cells metabolize nutrients to produce energy needed for insulin secretion in response to high glucose, and there is a potential to harness {beta}-cell metabolism for treating diabetes. However, {beta}-cell metabolism is not fully characterized. We have developed a computational modeling framework to better understand the relationship between cellular metabolism and insulin production in the pancreatic {beta}-cell. With this modeling framework, we are able to simulate metabolic perturbations, such as the knockdown of the activity of a metabolic enzyme, and predict the effect on the metabolic network and on insulin production. This work can therefore be applied to investigate, in a time- and cost-efficient manner, {beta}-cell metabolism and predict effective therapies that target the cells metabolic network.

category: systems biology