title: Binomial models uncover biological variation during feature selection of droplet-based single-cell RNA sequencing

abstract: Single-cell RNA sequencing (scRNA-seq) aims to characterize how variation in gene expression is distributed across cells in tissues and organisms. Yet, effective comprehension of these extremely high-dimensional datasets remains a critical barrier to progress in biological research. In standard analyses of scRNA-seq data, feature selection steps aim to reduce the dimensionality of the data by focusing on a subset of genes that are the most biologically variable across a set of cells. Ideally, these features provide the genes that are the most informative for partitioning groups of transcriptionally distinct cells, each representing a different cell type or identity. In this work, we propose a simple feature selection model where a binomial sampling process for each mRNA species produces a null model of technical variation. To compare our model to existing methods, we use scRNA-seq data where cell identities have been established a priori for each cell, and characterize whether different feature sets retain biologically varying genes, distort neighborhood structures, and allow popular clustering algorithms to partition groups of cells into their established classes. We find that our model of biological variation, which we term "Differentially Distributed Genes" or DDGs, outperforms existing methods, and enables dimensionality reduction without loss of critical structure within the data set.


title: An integrated analysis tool reveals intrinsic biases in gene set enrichment

abstract: Generating meaningful interpretations of gene lists remains a challenge for all large-scale studies. Many approaches exist, often based on evaluating gene enrichment among pre-determined gene classes. Here, we conceived and implemented yet another analysis tool (YAAT), specifically for data from the widely-used model organism C. elegans. YAAT extends standard enrichment analyses, using a combination of co-expression data and profiles of phylogenetic conservation, to identify groups of functionally-related genes. It additionally allows class clustering, providing inference of functional links between groups of genes. We give examples of the utility of YAAT for uncovering unsuspected links between genes and show how the approach can be used to prioritise genes for in-depth study. Our analyses revealed several limitations to the meaningful interpretation of gene lists, specifically related to data sources and the "universe" of gene lists used. We hope that YAAT will represent a model for integrated analysis that could be useful for large-scale exploration of biological function in other species.

category: bioinformatics


title: We are not ready yet: limitations of transfer learning for Disease Named Entity Recognition

abstract: Intense research has been done in the area of biomedical natural language processing. Since the breakthrough of transfer learning-based methods, BERT models are used in a variety of biomedical and clinical applications. For the available data sets, these models show excellent results - partly exceeding the inter-annotator agreements. However, biomedical named entity recognition applied on COVID-19 preprints shows a performance drop compared to the results on available test data. The question arises how well trained models are able to predict on completely new data, i.e. to generalize. Based on the example of disease named entity recognition, we investigate the robustness of different machine learning-based methods - thereof transfer learning - and show that current state-of-the-art methods work well for a given training and the corresponding test set but experience a significant lack of generalization when applying to new data. We therefore argue that there is a need for larger annotated data sets for training and testing.


title: Critical Assessment of Metagenome Interpretation - the second round of challenges

abstract:
Evaluating metagenomic software is key for optimizing metagenome interpretation and focus of the community-driven initiative for the Critical Assessment of Metagenome Interpretation (CAMI). In its second challenge, CAMI engaged the community to assess their methods on realistic and complex metagenomic datasets with long and short reads, created from [~]1,700 novel and known microbial genomes, as well as [~]600 novel plasmids and viruses. Altogether 5,002 results by 76 program versions were analyzed, representing a 22x increase in results.

Substantial improvements were seen in metagenome assembly, some due to using long-read data. The presence of related strains still was challenging for assembly and genome binning, as was assembly quality for the latter. Taxon profilers demonstrated a marked maturation, with taxon profilers and binners excelling at higher bacterial taxonomic ranks, but underperforming for viruses and archaea. Assessment of clinical pathogen detection techniques revealed a need to improve reproducibility. Analysis of program runtimes and memory usage identified highly efficient programs, including some top performers with other metrics. The CAMI II results identify current challenges, but also guide researchers in selecting methods for specific analyses.


title: SurvBenchmark: comprehensive benchmarking study of survival analysis methods using both omics data and clinical data

abstract:
Survival analysis is a branch of statistics that deals with both, the tracking of time and of the survival status simultaneously as the dependent response. Current comparisons of survival model performance mostly center on clinical data with classic statistical survival models, with prediction accuracy often serving as the sole metric of model performance. Moreover, survival analysis approaches for censored omics data have not been thoroughly investigated. The common approach is to binarise the survival time and perform a classification analysis.

Here, we develop a benchmarking framework, SurvBenchmark, that evaluates a diverse collection of survival models for both clinical and omics datasets. SurvBenchmark not only focuses on classical approaches such as the Cox model, but it also evaluates state-of-art machine learning survival models. All approaches were assessed using multiple performance metrics, these include model predictability, stability, flexibility and computational issues. Our systematic comparison framework with over 320 comparisons (20 methods over 16 datasets) shows that the performances of survival models vary in practice over real-world datasets and over the choice of the evaluation metric. In particular, we highlight that using multiple performance metrics is critical in providing a balanced assessment of various models. The results in our study will provide practical guidelines for translational scientists and clinicians, as well as define possible areas of investigation in both survival technique and benchmarking strategies.

Contactjean.yang@sydney.edu.au


title: Learning cell communication from spatial graphs of cells

abstract: Tissue niches are sources of cellular variation and key to understanding both single-cell and tissue phenotypes. The interaction of a cell with its niche can be described through cell communication events. These events cannot be directly observed in molecular profiling assays of single cells and have to be inferred. However, computational models of cell communication and variance attribution defined on data from dissociated tissues suffer from multiple limitations with respect to their ability to define and to identify communication events. We address these limitations using spatial molecular profiling data with node-centric expression modeling (NCEM), a computational method based on graph neural networks which reconciles variance attribution and communication modeling in a single model of tissue niches. We use these models in varying complexity across spatial assays, such as immunohistochemistry and MERFISH, and biological systems to demonstrate that the statistical cell-cell dependencies discovered by NCEM are plausible signatures of known molecular processes underlying cell communication. We identify principles of tissue organisation as cell communication events across multiple datasets using interpretation mechanisms. In the primary motor cortex, we found gene expression variation that is due to niche composition variation across cortical depth. Using the same approach, we also identified niche-dependent cell state variation in CD8 T cells from inflamed colon and colorectal cancer. Finally, we show that NCEMs can be extended to mixed models of explicit cell communication events and latent intrinsic sources of variation in conditional variational autoencoders to yield holistic models of cellular variation in spatial molecular profiling data. Altogether, this graphical model of cellular niches is a step towards understanding emergent tissue phenotypes.


title: Conditional ERK3 overexpression cooperates with PTEN deletion to promote lung adenocarcinoma formation in mice

abstract: Extracellular signal-regulated kinase 3 (ERK3), also known as MAPK6, belongs to the atypical mitogen-activated protein kinases (MAPKs) subfamily. In comparison with the well-studied classical MAPKs ERK1 and ERK2, much less is known about the cellular and molecular actions of ERK3. Accumulating studies have revealed the upregulation of ERK3 expression and suggested an important role for ERK3 in promoting tumor cell growth and invasion in multiple cancers, in particular lung cancer. However, it is unknown whether or not ERK3 plays a role in spontaneous tumorigenesis. To determine the role of ERK3 in lung tumorigenesis, we created a conditional ERK3 transgenic mouse line in which ERK3 transgene expression is controlled by Cre recombinase. By crossing with a lung tissue-specific CCSP-iCre mouse line, we have found that conditional ERK3 overexpression cooperates with PTEN deletion to induce the formation of lung adenocarcinomas (LUADs). Mechanistically, ERK3 overexpression stimulates activating phosphorylations of ERBB3 and ERBB2 by upregulating SP1-mediated gene transcription of NRG1, a potent ligand for ERBB3/ERBB2. To our knowledge, our study is the first revealing a bona fide tumor-promoting role for ERK3 using genetically engineered mouse models. Together with previous findings showing important roles of ERK3 in cultured cells and in xenograft lung tumor model, our findings corroborate that ERK3 acts as an oncoprotein in promoting LUAD development and progression.

category: cancer biology


title: Variation in root system architecture among the founder parents of two 8-way MAGIC wheat populations for selection in breeding

abstract: Root system architecture (RSA) is a target for breeding because of the interest to develop crops with roots that use nutrients and water more effectively. Breeding for RSA requires phenotypic diversity in populations amenable to QTL identification to provide markers for large breeding programs. This study examined the variation for root traits across the parents of two multi-parent advanced generation inter-cross (MAGIC) wheat populations from NIAB and CSIRO for 16 days in an upgraded version of the non-invasive, germination paper-based phenotyping platform, GrowScreen-PaGe. Across all parents, total root length varied up to 1.90 fold, root biomass 2.25 fold and seminal root angle 1.16 fold. The CSIRO parents grew faster, exhibited slightly wider seminal root angle and produced larger root systems compared to NIAB parents. Lateral root lengths, leaf lengths and biomass contrasted most between fastest (Robigus - NIAB and AC Barrie - CSIRO) and slowest growing parents (Rialto - NIAB and G204 Xiaoyan54 - CSIRO). Lengths of lateral and total root, and leaf number and length had moderate to high heritability (0.30-0.67) and repeatability. Lengths of lateral roots and leaves are good targets for enhancing wheat crop establishment, a critical stage for crop productivity.

category: plant biology


title: Sex Identification in Cattle, based on Amelogenin Gene

abstract: Sex identification is considered an important step in the field of forensic sciences, wildlife and livestock breeding management. In the current experiment we used Amelogenin gene as a biological marker for polymerase chain reaction test to identify the sex of cattle from blood remnants, collected at slaughter house. Due to the conserved region of the gene on both sex chromosomes (X and Y) a single primers pair was employed to amplify the gene in a single polymerase chain reaction. In case of band patterns, a 178 base pair fragment for AMELY and a 241 base pair fragment for AMELX genes were produced. The primers competence and exactness was initially checked on known gender cattle samples and then applied to unknown cattle samples for the validation of the experiment. PCR amplicons of unknown gender showed only one band (241-bp) for female DNA and two bands (241-bp, 178-bp) for male DNA, on the platform of agarose gel upon electrophoresis. Our findings showed that the PCR protocol based on AMELX/Y gene is a reliable technique for the identification of cattle sex.

category: zoology


title: Loss of muscarinic M5 receptor function manifests disparate impairments in exploratory behavior in male and female mice despite common dopamine regulation

abstract:
There are five cloned muscarinic acetylcholine receptors (M1-M5). Of these, the muscarinic type 5 receptor (M5) is the only one localized to dopamine neurons in the ventral tegmental area and substantia nigra. Unlike M1-M4, the M5 receptor has relatively restricted expression in the brain, making it an attractive therapeutic target. Here we performed an in-depth characterization of M5-dependent potentiation of dopamine transmission in the nucleus accumbens and accompanying exploratory behaviors in male and female mice. We show that M5 receptors potentiate dopamine transmission by acting directly on the terminals within the nucleus accumbens. Using the agonist oxotremorine, we revealed a unique concentration response curve and a sensitivity to repeated stressor exposure. We found that constitutive deletion of M5 receptors reduced exploration of the center of an open field while at the same time impairing normal habituation only in male mice. In addition, M5 deletion reduced exploration of salient stimuli, especially under conditions of high novelty, yet had no effect on hedonia. We conclude that M5 receptors are critical for both engaging with the environment and updating behavioral output in responses to the environment cues, specifically in male mice. A cardinal feature of mood and anxiety disorders is a withdrawal from the environment. These data indicate that boosting M5 receptor activity may be a useful therapeutic target for ameliorating these symptoms of depression and anxiety.

Significance StatementThe basic physiological and behavioral functions of the muscarinic M5 receptor remain understudied. Furthermore, its presence on dopamine neurons, relatively restricted expression in the brain, and recent crystallization make it an attractive target for therapeutic development. Yet, most preclinical studies of M5 receptor function have primarily focused on substance use disorders in male rodents. Here we characterized the role of M5 receptors in potentiating dopamine transmission in the nucleus accumbens, finding impaired functioning after stress exposure. Furthermore, we show that M5 receptors can modulate exploratory behavior in a sex-specific manner, without impacting hedonic behavior. These findings further illustrate the therapeutic potential of the M5 receptor, warranting further research in the context of treating mood disorders.

category: neuroscience


title: A corollary discharge mediates saccade related inhibition of single units in mnemonic structures of the human brain

abstract: Despite the critical link between visual exploration and memory, little is known about how single-unit activity (SUA) in the human mesial temporal lobe (MTL) is modulated by saccadic eye movements (SEMs). Here we characterize SEM associated SUA modulations, unit-by-unit, and contrast them to image onset, and to occipital lobe SUA. We reveal evidence for a corollary discharge (CD)-like modulatory signal that accompanies SEMs, inhibiting/exciting a unique population of broad/narrow spiking units, respectively, before and during SEMs, and with directional selectivity. These findings comport well with the timing, directional nature, and inhibitory circuit implementation of a CD. Additionally, by linking SUA to event-related potentials (ERPs), which are directionally modulated following SEMs, we recontextualize the ERP associated with SEM as a proxy for both the strength of inhibition and saccade direction, providing a mechanistic underpinning for the more commonly recorded SEM-related ERP in the human brain.


title: Molecular Mechanisms for Frequency Specificity in a Drosophila Hearing Organ

abstract: Discrimination for sound frequency is essential for auditory communications in animals. Here, by combining in vivo calcium imaging and behavioral assay, we found that Drosophila larvae can sense a wide range of sound frequency and the behavioral specificity is mediated with the selectivity of the lch5 chordotonal organ neurons to sounds that forms a combinatorial coding of frequency. We also disclosed that Brivido1 (Brv1) and Piezo-like (Pzl), each expresses in a subset of lch5 neurons and mediate hearing sensation to certain frequency ranges. Intriguingly, mouse Piezo2 can rescue pzl-mutants phenotypes, suggesting a conserved role of the Piezo family proteins in high-frequency hearing.


title: The presence of corticomuscular coherence during unipedal stance

abstract: Standing in unipedal stance requires higher effort to maintain posture balance within narrow base-of-support. Although changes in cortical activity are known to occur as standing task difficulty increased, it is unclear whether it indicates a change in cortical control of muscle activity. To elucidate this point, this study examined corticomuscular coherence (CMC) between the sensorimotor cortex and ankle joint muscles during three tasks such as bipedal stance, unipedal stance, and isometric contraction tasks. For twenty-one healthy participants, we measured the maximal peak of CMC (CMCmax) between electroencephalograms overlying the foot representation area and surface electromyograms from the tibialis anterior (TA), medial gastrocnemius (MG), lateral gastrocnemius (LG), and soleus (SOL), respectively, for each task. We also measured the center of pressure (COP) during both stance tasks. Although there was no significant CMC during bipedal stance in all participants, most participants (n = 14) showed significant CMC for all muscles during unipedal stance with larger COP fluctuation in most participants. Indeed, there was significant difference in CMCmax between unipedal and bipedal stance tasks. The greater CMC would indicate increased cortical control of muscle activity to compensate for postural instability during unipedal stance.


title: Improved Interpretability of Brain-Behavior CCA with Domain-driven Dimension Reduction

abstract: Canonical Correlation Analysis (CCA) has been widely applied to study correlations between neuroimaging data and behavioral data. Practical use of CCA typically requires dimensionality reduction with, for example, Principal Components Analysis (PCA), however, this can result in CCA results that are difficult to interpret. In this paper, we introduce a Domain-driven Dimension Reduction (DDR) method, reducing the dimensionality of the original datasets combining human knowledge of the structure of the variables studied. We apply the method to the Human Connectome Project S1200 release and compare standard PCA across all variables with DDR applied to individual classes of variables, finding that DDR-CCA results are more stable and interpretable, allowing the contribution of each class of variable to be better understood. By carefully designing the analysis pipeline and cross-validating the results, we offer more insights on the interpretation of CCA applied to brain-behaviour data.


title: Serial dependence tracks objects and scenes independently

abstract: The visual world is made up of objects and scenes. Object perception requires both discriminating an individual object from others and binding together different perceptual samples of that object across time. Such binding manifests by serial dependence, the attraction of the current perception of a visual attribute towards values of that attribute seen in the recent past. Scene perception is subserved by global mechanisms like ensemble perception, the rapid extraction of the average feature value of a group of objects. The current study examined to what extent the perception of single objects in multi-object scenes depended on previous feature values of that object, or on the average previous attribute of all objects in the scene. Results show that serial dependence occurs independently on two simultaneously present objects, that ensemble perception depends only on previous ensembles, and that serial dependence of an individual object occurs only on the features of that particular object. These results suggest that the temporal integration of successive perceptual samples operates simultaneously at independent levels of visual processing.


title: Serial dependence tracks objects and scenes in parallel and independently


title: Nitrous Oxide Enhances Focused Ultrasound Mediated Delivery of Viral Gene Therapy

abstract:
Transcranial Magnetic Resonance Guided Focused Ultrasound (MRgFUS) can be used in conjunction with intravenous microbubbles to open the blood brain barrier (BBB) in discrete brain regions. This method is limited by the microbubble dose, the amount of energy that can be safely delivered across an intact skull, and the inability to target areas of the brain outside of central brain regions. We find that N2O administration substantially decreases the required dose of microbubbles and/or focused ultrasound pressure while still reliably opening the blood brain barrier. Additionally, we observe that the use of nitrous oxide improves the delivery of adeno-associated virus (AAV9) vector when compared to medical air. Our findings may help overcome focused ultrasound limitations and improve targeted delivery of therapeutics in humans.

One Sentence SummaryNitrous Oxide potentiates Blood Brain Barrier opening with Focused Ultrasound and enhances viral vector delivery.


title: Experimental evolution of Vibrio cholerae: Identification of genes involved in motility in presence of polymyxin B

abstract: Vibrio cholerae is the bacteria responsible for the cholera disease and a natural inhabitant of aquatic environments. Biofilm formation is important for human colonization and environmental survival. Motility is essential for adhesion and biofilm formation by V. cholerae. In a previous study, we showed that motility and biofilm formation are altered in the presence of sub-inhibitory concentrations of polymyxin B in V. cholerae. In this study, we performed an experimental evolution to identify genes rescuing the motility in the presence of polymyxin B. Mutations in 5 genes have been identified: ihfA, vacJ (mlaA), mlaF, dacB and ccmH. The details of these mutations, their potential impact on the function of the proteins they encode and on the motility in presence of polymyxin B are discussed.

category: microbiology


title: Disentangling marine microbial networks across space

abstract: Although microbial interactions underpin ocean ecosystem functions, they remain barely known. Different studies have analyzed microbial interactions using static association networks based on omics-data. However, microbial associations are dynamic and can change across physicochemical gradients and spatial scales, which needs to be considered to understand the ocean ecosystem better. We explored associations between archaea, bacteria, and picoeukaryotes along the water column from the surface to the deep ocean across the northern subtropical to the southern temperate ocean and the Mediterranean Sea by defining sample-specific subnetworks. Quantifying spatial association recurrence, we found the lowest fraction of global associations in the bathypelagic zone, while associations endemic of certain regions increased with depth. Overall, our results highlight the need to study the dynamic nature of plankton networks and our approach represents a step forward towards a better comprehension of the biogeography of microbial interactions across ocean regions and depth layers.


title: Pparα and fatty acid oxidation coordinate hepatic transcriptional architecture

abstract:
Fasting requires tight coordination between the metabolism and transcriptional output of hepatocytes to maintain systemic glucose and lipid homeostasis. Genetically-defined deficits in hepatic fatty acid oxidation result in dramatic fasting-induced hepatocyte lipid accumulation and induction of genes for oxidative metabolism, thereby providing a mouse model to interrogate the mechanisms by which the liver senses and transcriptionally responds to fluctuations in lipid levels. While fatty acid oxidation is required for a rise in acetyl-CoA and subsequent lysine acetylation following a fast, changes in histone acetylation (total, H3K9ac, and H3K27ac) associated with transcription do not require fatty acid oxidation. Instead, excess fatty acids prompt induction of lipid catabolic genes largely via ligand-activated Ppar. We observe that active enhancers in fasting mice are enriched for Ppar binding motifs, and that inhibition of hepatic fatty acid oxidation results in elevated enhancer priming and acetylation proximal to Ppar binding sites within regulatory elements largely associated with genes in lipid metabolism. Also, a greater number of Ppar-associated H3K27ac signal changes occur at active enhancers compared to promoters, suggesting a genomic mechanism for Ppar to tune target gene expression levels. Overall, these data demonstrate the requirement for Ppar activation in maintaining transcriptionally permissive hepatic genomic architecture particularly when fatty acid oxidation is limiting.

HIGHLIGHTSO_LIFasting-induced transcription and histone acetylation are largely independent of acetyl-CoA concentration.
C_LIO_LIDeficits in fatty acid oxidation prompt epigenetic changes and Ppar-sensitive transcription.
C_LIO_LIFasting prompts enhancer priming and acetylation proximal to Ppar binding sites independent of Ppar.
C_LIO_LIPatterns of Ppar target genes can be distinguished by epigenetic marks at promoters and enhancers.
C_LI

category: molecular biology


title: Genome-resolved metagenomic analyses reveal the presence of a bacterial endosymbiont in an avian nasal mite (Rhinonyssidae; Mesostigmata)

abstract: Rhinonyssidae (Mesostigmata) is a family of nasal mites only found in birds. All species are hematophagous endoparasites, which may damage the nasal cavities of birds, and also could be potential reservoirs or vectors of other infections. However, the role of members of Rhinonyssidae as disease vectors in wild bird populations remains uninvestigated, with studies of the microbiomes of Rhinonyssidae being almost non-existent. In the nasal mite (Tinaminyssus melloi) from rock doves (Columba livia), a previous study found evidence of a highly abundant putatively endosymbiotic bacteria from Class Alphaproteobacteria. Here, we expanded the sample size of this species, incorporated contamination controls, and increased sequencing depth in shotgun sequencing and genome-resolved metagenomic analyses. Our goal was to increase the information regarding this mite species with its putative endosymbiont. Our results support the endosymbiotic nature of this bacterial taxon, which is the first described for birds nasal mites to date, and improve the overall understanding of the microbiota inhabiting these mites.


title: RNA uridyl transferases TUT4/7 differentially regulate miRNA variants depending on the cancer cell-type

abstract:
The human terminal uridyl transferases TUT4 and TUT7 (TUT4/7) catalyse additions of uridines at the 3' end of RNAs such as the precursors of the tumour suppressor miRNA let-7, upon recruitment by the oncoprotein LIN28A. Consequently, let-7 family miRNAs are downregulated. Disruption of this TUT4/7 activity inhibits tumorigenesis and hence targeting TUT4/7 can be a potential anti-cancer therapy. In this study, we investigate TUT4/7-mediated RNA regulation in two cancer cell lines by establishing catalytic knockout models. Upon TUT4/7 mutation, we observe a significant reduction in miRNA uridylation, which results in defects in cancer cell properties such as cell proliferation and migration. With the loss of TUT4/7-mediated miRNA uridylation, the uridylated miRNA variants are replaced by adenylated isomiRs. Changes in miRNA modification profiles are accompanied by deregulation of expression levels in specific cases. Unlike let-7s, most miRNAs do not depend on LIN28A for TUT4/7-mediated regulation. Additionally, we identify TUT4/7-regulated cell-type-specific miRNA clusters and deregulation in their corresponding mRNA targets. Expression levels of miR-200c-3p and miR-141-3p are regulated by TUT4/7 in a cancer cell type specific manner. Subsequently, BCL2 which is a well-established target of miR-200c is upregulated. Therefore, TUT4/7 loss triggers deregulation of miRNA-mRNA networks in a cell-type-specific manner. Understanding of the underlying biology of such cell-type-specific deregulation will be key when targeting TUT4/7 for cancer therapy.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/451846v2_ufig1.gif" ALT="Figure 1">
View larger version (53K):
org.highwire.dtl.DTLVardef@a18bdcorg.highwire.dtl.DTLVardef@1e5534forg.highwire.dtl.DTLVardef@c08e76org.highwire.dtl.DTLVardef@1c90423_HPS_FORMAT_FIGEXP  M_FIG C_FIG Highlights TUT4/7 catalyse 3' uridylation of most miRNAs
 Loss of uridylated isomiRs leads to the gain of adenylated isomiRs
 TUT4/7-mediated miRNA deregulation and miRNA-mRNA interactions are specific to the cell line type
 Loss of TUT4/7-mediated RNA uridylation inhibits cancer cell function


title: Creating a bottleneck: Robust LC3B lipidation analysis by adjusting autophagic flux with low concentrations of Bafilomycin A1

abstract: Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.

category: cell biology


title: PPARdelta signaling activation improves metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes.

abstract: Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease. A major caveat however is that they remain functionally and structurally immature in culture, limiting their potential for disease modeling and regenerative approaches. Here, we address the question of how different metabolic pathways can be modulated in order to induce efficient hPSC-CM maturation. We show that PPAR signaling acts in an isoform-specific manner to balance glycolysis and fatty acid oxidation (FAO). PPARD activation or inhibition results in efficient respective up- or down-regulation of the gene regulatory networks underlying FAO in hPSC-CMs. PPARD induction further increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation and augments FAO flux. Lastly PPARD activation results in enhanced myofibril organization and improved contractility. Transient lactate exposure, commonly used in hPSC-CM purification protocols, induces an independent program of cardiac maturation, but when combined with PPARD activation equally results in a metabolic switch to FAO. In summary, we identify multiple axes of metabolic modifications of hPSC-CMs and a role for PPARD signaling in inducing the metabolic switch to FAO in hPSC-CMs. Our findings provide new and easily implemented opportunities to generate mature hPSC-CMs for disease modeling and regenerative therapy.


title: Cellular remodeling and JAK inhibition promote zygotic gene expression in the Ciona germline

abstract: During development, remodeling of the cellular transcriptome and proteome underlies cell fate decisions and, in somatic lineages, transcription control is a major determinant of fateful biomolecular transitions. By contrast, early germline fate specification in numerous vertebrate and invertebrate species relies extensively on RNA-level regulation, exerted on asymmetrically inherited maternal supplies, with little-to-no zygotic transcription. However delayed, a maternal-to-zygotic transition is nevertheless poised to complete the deployment of pre-gametic programs in the germline. Here, we focused on early germline specification in the tunicate Ciona to study zygotic genome activation. We first demonstrate that a peculiar cellular remodeling event excludes localized postplasmic mRNAs, including Pem-1, which encodes the general inhibitor of transcription. Subsequently, zygotic transcription begins in Pem-1-negative primordial germ cells (PGCs), as revealed by histochemical detection of elongating RNA Polymerase II (RNAPII), and nascent transcripts from the Mef2 locus. Using PGC-specific Mef2 transcription as a read-out, we uncovered a provisional antagonism between JAK and MEK/BMPRI/GSK3 signaling, which controls the onset of zygotic gene expression, following cellular remodeling of PGC. We propose a 2-step model for the onset of zygotic transcription in the Ciona germline, which relies on successive cellular remodeling and JAK inhibition, and discuss the significance of germ plasm dislocation and remodeling in the context of developmental fate specification.

category: developmental biology


title: Sex-specific association between urinary kisspeptin and pubertal development

abstract: Kisspeptins are critical neuropeptides for puberty onset and progression, playing a pivotal role in the reactivation of the hypothalamus-pituitary-gonadal axis in late childhood. Despite their importance, little is known as to how kisspeptin peripheral concentrations are related to sexual maturation in humans, specially using non-invasive measures that allow more widespread testing. Using a cross-sectional design, we investigated whether peripheral kisspeptin concentrations, measured by enzyme-linked immunosorbent assays (ELISA) in two-hour retention midstream urine, are associated with developmental markers in 209 (120 girls) typically developing, 9 to 15-year-olds. Developmental variables were age, self-reported pubertal status using the Pubertal Development Scale, and saliva concentrations of hormones that indicate gonadal (testosterone) and adrenal (dehydroepiandrosterone sulfate) functioning. We found marked sexual differences in urine kisspeptins (controlled for body mass index and socioeconomic status). While concentrations were similar in both sexes until the age of around 12 years, in males there was a positive linear correlation with all developmental measures thereafter, while in girls, kisspeptin concentrations did not change. Of note, our results are in line with those of previous studies using more invasive methods (e.g. blood samples), which indicates that kisspeptin concentrations from two-hour retention midstream urine have potential in exploring sex-specific peripheral action of these peptides.


title: Are marine biodiversity hotspots still blackspots for barcoding?

abstract: Marine biodiversity underpins ecosystem health and societal well-being. Preservation of biodiversity hotspots is a global challenge. Molecular tools, like DNA barcoding and metabarcoding, hold great potential for biodiversity monitoring, possibly outperforming more traditional taxonomic methods. However, metabarcoding-based biodiversity assessments are limited by the availability of sequences in barcoding reference databases; a lack thereof results in high percentages of unassigned sequences. In this study we (i) present the current status of known vs. barcoded marine species at a global scale based on online taxonomic and genetic databases; and (ii) compare the current status with data from ten years ago. Then we analyzed occurrence data of marine animal species from five Large Marine Ecosystems (LMEs) classified as biodiversity hotspots, to identify any consistent disparities in COI barcoding coverage between geographic regions and at phylum level. Barcoding coverage varied among LMEs (from 36.8% to 62.4% COI-barcoded species) and phyla (from 4.8% to 74.7% COI-barcoded species), with Porifera, Bryozoa and Platyhelminthes being highly underrepresented, compared to Chordata, Arthropoda and Mollusca. We demonstrate that although barcoded marine species increased from 9.5% to 14.2% since the last assessment in 2011, about 15,000 (corresponding to 7.8% increase) new species were described from 2011 to 2021. The next ten years will thus be crucial to enroll concrete collaborative measures and long term initiatives (e.g., Horizon 2030, Ocean Decade) to populate barcoding libraries for the marine realm.

category: ecology


title: Mutation rate and spectrum in Caenorhabditis elegans mutation accumulation lines subjected to RNAi-induced knockdown of the mismatch repair gene msh-2

abstract: DNA mismatch repair (MMR), an evolutionarily conserved repair pathway shared by prokaryotic and eukaryotic species alike, influences molecular evolution by detecting and correcting mismatches that escape DNA polymerase proofreading, thereby protecting genetic fidelity, reducing the mutational load, and preventing lethality. Herein we conduct the first genome-wide evaluation of the alterations to the mutation rate and spectrum under impaired activity of the MutS homolog, msh-2, in Caenorhabditis elegans. We performed mutation accumulation (MA) under RNAi-induced knockdown of msh-2 for 50 generations in obligately outcrossing fog-2(lf) lines, followed by next-generation sequencing of 19 MA lines and the ancestral control. msh-2 impairment substantially increased the frequency of nuclear base substitutions ([~]23x) and small indels ([~]328x) relative to wildtype. However, we observed no increase in the mutation rates of mtDNA, and copy-number changes of single-copy genes. There was a marked increase in copy-number variation of rDNA genes under MMR impairment. In C. elegans, msh-2 repairs transitions more efficiently than transversions as well as increases the AT mutational bias relative to wildtype. The local sequence context, including sequence complexity, G+C-content, and flanking bases influenced the mutation rate. The X chromosome had a lower substitution and higher indel rate than autosomes, which can either result from sex-specific mutation rates or a nonrandom distribution of mutable sites in the genome. Comparison of MMR impairment in C. elegans to that in other species shows that the specificity of the MMR varies between taxa, and is more efficient in detecting and repairing small indels in eukaryotes relative to prokaryotes.

category: evolutionary biology


title: Genome mosaicism in field strains of Mycoplasma bovis as footprints of in-host horizontal chromosomal transfer

abstract:
Horizontal gene transfer was long thought to be marginal in Mollicutes, but the capacity of some of these wall-less bacteria to exchange large chromosomal regions has been recently documented. Mycoplasma chromosomal transfer (MCT) is an unconventional mechanism that relies on the presence of a functional integrative conjugative element (ICE) in at least one partner and involves the horizontal acquisition of small and large chromosomal fragments from any part of the donor genome, which results in progenies composed of an infinitive variety of mosaic genomes. The present study focuses on Mycoplasma bovis, an important pathogen of cattle responsible for major economic losses worldwide. By combining phylogenetic tree reconstructions and detailed comparative genome analyses of 36 isolates collected in Spain (2016-2018) we confirmed the mosaic nature of 16 field isolates and mapped chromosomal transfers exchanged between their hypothetical ancestors. This study provides evidence that MCT can take place in the field, most likely during co-infections by multiple strains. Because mobile genetic elements (MGEs) are classical contributors of genome plasticity, the presence of phages, insertion sequences (ISs) and ICEs was also investigated. Data revealed that these elements are widespread within the M. bovis species and evidenced classical horizontal transfer of phages and ICEs in addition to MCT. These events contribute to wide-genome diversity and reorganization within this species and may have a tremendous impact on diagnostic and disease control.

IMPORTANCEMycoplasma bovis is a major pathogen of cattle with significant detrimental economic and animal welfare on cattle rearing worldwide. Understanding the evolution and the adaptative potential of pathogenic mycoplasma species in the natural host is essential to combating them. In this study, we documented the occurrence of mycoplasma chromosomal transfer, an atypical mechanism of horizontal gene transfer, in field isolates of M. bovis that provide new insights into the evolution of this pathogenic species in their natural host. Despite these events are expected to occur at low frequency, their impact is accountable for genome-wide variety and reorganization within M. bovis species, which may compromise both diagnostic and disease control.


title: Rapid shifts in mitochondrial tRNA import in a plant lineage with extensive mitochondrial tRNA gene loss

abstract: In most eukaryotes, transfer RNAs (tRNAs) are one of the very few classes of genes remaining in the mitochondrial genome, but some mitochondria have lost these vestiges of their prokaryotic ancestry. Sequencing of mitogenomes from the flowering plant genus Silene previously revealed a large range in tRNA gene content, suggesting rapid and ongoing gene loss/replacement. Here, we use this system to test longstanding hypotheses about how mitochondrial tRNA genes are replaced by importing nuclear-encoded tRNAs. We traced the evolutionary history of these gene loss events by sequencing mitochondrial genomes from key outgroups (Agrostemma githago and Silene [=Lychnis] chalcedonica). We then performed the first global sequencing of purified plant mitochondrial tRNA populations to characterize the expression of mitochondrial-encoded tRNAs and the identity of imported nuclear-encoded tRNAs. We also confirmed the utility of high-throughput sequencing methods for the detection of tRNA import by sequencing mitochondrial tRNA populations in a species (Solanum tuberosum) with known tRNA trafficking patterns. Mitochondrial tRNA sequencing in Silene revealed substantial shifts in the abundance of some nuclear-encoded tRNAs in conjunction with their recent history of mt-tRNA gene loss and surprising cases where tRNAs with anticodons still encoded in the mitochondrial genome also appeared to be imported. These data suggest that nuclear-encoded counterparts are likely replacing mitochondrial tRNAs even in systems with recent mitochondrial tRNA gene loss, and the redundant import of a nuclear-encoded tRNA may provide a mechanism for functional replacement between translation systems separated by billions of years of evolutionary divergence.


title: Interspecific gene flow and vicariance in Northern African rear-edge white oak populations revealed by joint analysis of microsatellites and flanking sequences.

abstract: Inference of recent population divergence requires fast evolving markers and necessitates to differentiate shared genetic variation caused by ancestral polymorphism and gene flow. Theoretical research shows that the use of compound marker systems integrating linked polymorphisms with different mutational dynamics, such as a microsatellite and its flanking sequences, can improve estimation of population structure and inference of demographic history, especially in the case of complex population dynamics. However, empirical application in natural populations has so far been limited by lack of suitable methods for data collection. A solution comes from the development of sequence-based microsatellite genotyping which we used to study molecular variation at 36 sequenced nuclear microsatellites in seven Quecus canariensis and four Quercus faginea rear-edge populations across Algeria, to decipher their taxonomic relationship, past evolutionary history and recent demographic trajectory. First, we compare the estimation of population genetics parameters and simulation-based inference of demographic history from microsatellite sequence alone, flanking sequence alone or the combination of linked microsatellite and flanking sequence variation. Second, we measure variable importance from random forest approximate Bayesian computation to identify which of these sequence types is most informative. Whereas the analysis of microsatellite variation alone indicates recent interspecific gene flow, additional information gained from combining with nucleotide variation in flanking sequences, by reducing homoplasy, suggests ancient interspecific gene flow followed by drift in isolation instead. The weight of each polymorphism in the inference also demonstrates the value of linked variations with contrasted mutation dynamic to improve estimation of both demographic and mutational parameters.


title: Joint analysis of microsatellites and flanking sequences enlightens complex demographic history of interspecific gene flow and vicariance in rear-edge oak populations.


title: Genomic and transcriptomic analyses of the subterranean termite Reticulitermes speratus: gene duplication facilitates social evolution

abstract:
Termites are model social organisms characterized by a polyphenic caste system. Subterranean termites (Rhinotermitidae) are ecologically and economically important species, including acting as destructive pests. Rhinotermitidae occupies an important evolutionary position within the clade representing an intermediate taxon between the higher (Termitidae) and lower (other families) termites. Here, we report the genome, transcriptome and methylome of the Japanese subterranean termite Reticulitermes speratus. The analyses highlight the significance of gene duplication in social evolution in this termite. Gene duplication associated with caste-biased gene expression is prevalent in the R. speratus genome. Such duplicated genes encompass diverse categories related to social functions, including lipocalins (chemical communication), cellulases (wood digestion and social interaction), lysozymes (social immunity), geranylgeranyl diphosphate synthase (social defense) and a novel class of termite lineage-specific genes with unknown functions. Paralogous genes were often observed in tandem in the genome, but the expression patterns were highly variable, exhibiting caste biases. Some duplicated genes assayed were expressed in caste-specific organs, such as the accessory glands of the queen ovary and frontal glands in soldier heads. We propose that gene duplication facilitates social evolution through regulatory diversification leading to caste-biased expression and subfunctionalization and/or neofunctionalization that confers caste-specialized functions.

Significance StatementTermites are model social organisms characterized by a sophisticated caste system, where distinct castes arise from the same genome. Our genomics data of Japanese subterranean termite provides insights into the evolution of the social system, highlighting the significance of gene duplication. Gene duplication associated with caste-biased gene expression is prevalent in the termite genome. Many of the duplicated genes were related to social functions, such as chemical communication, social immunity and defense, and they often expressed in caste-specific organs. We propose that gene duplication facilitates social evolution through regulatory diversification leading to caste-biased expression and functional specialization. In addition, since subterranean termites are ecologically and economically important species including destructive pests in the world, our genomics data serves as a foundation for these studies.

category: genomics


title: Cooperative escape in ants and robots

abstract: The solution of complex problems by the collective action of simple agents in both biologically evolved and synthetically engineered systems involves cooperative action. Understanding the resulting emergent solutions requires integrating across the organismal behaviors of many individuals. Here we investigate an ecologically relevant collective task in black carpenter ants Camponotus pennsylvanicus: escape from a soft, erodible confining corral. Individual ants show a transition from individual exploratory excavation at random locations to spatially localized collective exploitative excavation and escape from the corral. A minimal continuum theory that coarse-grains over individual actions and considers their integrated influence on the environment leads to the emergence of an effective phase space of behaviors in terms of excavation strength and cooperation intensity. To test the theory over the range of predicted behaviors, we used custom-built robots (RAnts) that respond to stimuli and show the emergence (and failure) of cooperative excavation and escape. Overall, our approach shows how the cooperative completion of tasks can arise from relatively simple rules that involve the interaction of simple agents with a dynamically changing environment that serves as an enabler and modulator of behavior.

category: animal behavior and cognition


title: Isonicotinamide extends yeast chronological lifespan through a mechanism that diminishes nucleotides

abstract: Chronological lifespan (CLS) of budding yeast, Saccharomyces cerevisiae, is a commonly utilized model for cellular aging of non-dividing cells such as neurons. CLS is strongly extended by isonicotinamide (INAM), a non-metabolized isomer of the NAD+ precursor nicotinamide (NAM), but the underlying mechanisms of lifespan extension remain uncharacterized. To identify potential biochemical INAM targets, we performed a chemical genetic screen with the yeast gene knockout (YKO) strain collection for INAM-hypersensitive mutants. Significantly enriched Gene Ontology terms that emerged included SWR1 and other transcription elongation factors, as well as metabolic pathways converging on one-carbon metabolism and contributing to nucleotide biosynthesis, together suggesting that INAM perturbs nucleotide pools. In line with this model, INAM effects on cell growth were synergistic with mycophenolic acid (MPA), which extends lifespan by reducing guanine nucleotide pools. Direct measurements of nucleotides and precursors by mass spectrometry indicated that INAM reduced nucleotides, including cAMP, at 24- and 96-hour time points post-inoculation. Taken together, we conclude that INAM extends CLS by perturbing nucleotide metabolism, which may be a common functional feature of multiple anti-aging interventions.

category: biochemistry


title: Parallel Framework for Inferring Genome-Scale Gene Regulatory Networks

abstract:
Genome-scale network inference is essential to understand comprehensive interaction patterns. Current methods are limited to the reconstruction of small to moderate-size networks. The most obvious alternative is to propose a novel method or alter existing methods that may leverage parallel computing paradigms. Very few attempts also have been made to re-engineer existing methods by executing selective iterative steps concurrently. We propose a generic framework in this paper that leverages parallel computing without re-engineering the original methods. The proposed framework uses state-of-the-art methods as a black box to infer sub-networks of the segmented data matrix. A simple merger was designed based on preferential attachment to generate the global network by merging the sub-networks.

Fifteen (15) inference methods were considered for experimentation. Qualitative and speedup analysis was carried out using DREAM challenge networks. The proposed framework was implemented on all the 15 inference methods using large expression matrices. The results were auspicious as we could infer large networks in reasonable time without compromising the qualitative aspects of the original (serial) algorithm.

CLR, the top performer, was then used to infer the network from the expression profiles of an Alzheimers disease (AD) affected mouse model consisting of 45,101 genes. We have also highlighted few hub genes from the network that are functionally related to various diseases.


title: Multiplex viral tropism assay in complex cell populations with single-cell resolution

abstract: Gene therapy constitutes one of the most promising modes of disease treatments. Two key properties for therapeutic delivery vectors are the transduction efficiency (how well the vector delivers therapeutic cargo to desired target cells) and specificity (how well it avoids off-target delivery into the other unintended cells within the body). Here we developed a novel technology that enables multiplex measurement of transduction efficiency and specificity, particularly by measuring how libraries of delivery vectors transduce libraries of diverse cell types. We demonstrated that pairing high-throughput measurement of AAV identity with high-resolution single-cell RNA transcriptomic sequencing maps how natural and engineered AAV variants transduce individual cells within human cerebral and ocular organoids. This library- on-library technology is important for determining the safety and efficacy of therapeutic delivery vectors.

category: synthetic biology


title: Custom-made design of metabolite composition in N. benthamiana leaves using CRISPR activators.

abstract: Transcriptional regulators based on CRISPR architecture expand our ability of reprogramming endogenous gene expression in plants. One of their potential applications is the customization of plant metabolome through the activation of selected enzymes in a given metabolic pathway. Using the previously described multiplexable CRISPR activator dCasEV2.1, we assayed the selective enrichment in Nicotiana benthamiana leaves of four different flavonoids, namely naringenin, eriodictyol, kaempferol and quercetin. After careful selection of target genes and guide RNAs combinations, we created successful activation programs for each of the four metabolites, each program activating between three and seven genes, and with individual gene activation levels ranging from 4- to 1500-fold. Metabolic analysis of the flavonoid profiles of each multigene activation program showed a sharp and selective enrichment of the intended metabolites and their glycosylated derivatives. Remarkably, principal component analysis of untargeted metabolic profiles clearly separated samples according to their activation treatment, and hierarchical clustering separated the samples in five groups, corresponding to the expected four highly enriched metabolite groups, plus an un-activated control. These results demonstrate that dCasEV2.1 is a powerful tool for re-routing metabolic fluxes towards the accumulation of metabolites of interest, opening the door for custom-made design of metabolic contents in plants.


title: Age-related differences in encoding-retrieval similarity and their relationship to false memory

abstract: Typical aging is associated with increases in false memory rates among older adults. Such errors are frequently associated with differential neural activity during encoding and retrieval in older compared to younger adults within visual cortices, hippocampus, and front-parietal regions. It remains unknown how pattern similarity reductions relate to false memories in healthy aging. Using encoding-retrieval similarity (ERS) analyses in a sample of younger and older adults, we examined how the similarity of neural patterns between memory phases associated with target and lure objects was impacted by age and contributed to false memory rates. Single-item ERS for targets and lures was reduced by age throughout much of the ventral visual stream and the posterior hippocampus. Furthermore, ERS associated with perceptual lures within the visual stream maintained differential relationships with false memory. Finally, a global ERS metric accounted for age deficits in single-item ERS, but did not contribute to false memory rates. These findings highlight the contribution of age-related reductions in ERS across multiple representational levels to false memories in healthy aging.


title: Pooled screening of CAR T cells identifies non-native signaling domains for next- generation immunotherapies

abstract: Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers, but have shown limited efficacy against solid tumors. Here, we introduce  CAR Pooling, a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with diverse immune costimulatory domains were assessed in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several non-native domains from the TNF receptor family exhibited enhanced proliferation (CD40) or cytotoxicity (BAFF-R and TACI) relative to clinical benchmarks, and fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic and NK-cell-like innate phenotype previously associated with positive clinical outcomes.  CAR Pooling enables efficient exploration of how CAR design affects cell activity and can be applied to optimize receptors across a range of applications and cell types.

category: immunology


title: Novel strains of Klebsiella africana and Klebsiella pneumoniae in Australian Fruit Bats (Pteropus poliocephalus)

abstract: Over the past decade human associated multidrug resistant (MDR) and hypervirulent Klebsiella pneumoniae lineages have been increasingly detected in wildlife. This study investigated the occurrence of K. pneumoniae species complex (KpSC) in grey-headed flying foxes (GHFF), an Australian fruit bat. Thirty-nine KpSC isolates were cultured from 275 GHFF faecal samples (14.2%), comprising K. pneumoniae (sensu stricto) (n=30), Klebsiella africana (n=8) and Klebsiella variicola subsp. variicola (n=1). The majority (79.5%) of isolates belonged to novel sequence types (ST), including two novel K. africana STs. This is the first report of K. africana outside of Africa and in a non-human host. A minority (15.4%) of GHFF KpSC isolates shared STs with human clinical K. pneumoniae strains, of which, none belonged to MDR clonal lineages that cause frequent nosocomial outbreaks, and no isolates were characterised as hypervirulent. The occurrence of KpSC isolates carrying acquired antimicrobial resistance genes in GHFF was low (1.1%), with three K. pneumoniae isolates harbouring both fluoroquinolone and trimethoprim resistance genes. This study indicates that GHFF are not reservoirs for MDR and hypervirulent KpSC strains, but they do carry novel K. africana lineages. The health risks associated with KpSC carriage by GHFF are deemed low for the public and GHFF.


title: Novel insights into pulmonary phosphate homeostasis and osteoclastogenesis emerge from the study of pulmonary alveolar microlithiasis.

abstract: Pulmonary alveolar microlithiasis (PAM) is an autosomal recessive lung disease caused by a deficiency in the pulmonary epithelial Npt2b sodium-phosphate co-transporter that results in accumulation of phosphate and formation of hydroxyapatite microliths in the alveolar space. The single cell transcriptomic analysis of a PAM lung explant showing a robust osteoclast gene signature in alveolar monocytes and the finding that calcium phosphate microliths contain a rich protein and lipid matrix that includes bone resorbing osteoclast enzymes suggested a role for osteoclast-like cells in the defense against microliths. While investigating the mechanisms of microlith clearance, we found that Npt2b modulates pulmonary phosphate homeostasis through effects on alternative phosphate transporter activity and alveolar osteoprotegerin, and that microliths induce osteoclast formation and activity in a receptor activator of nuclear factor-{kappa}B ligand (RANKL) and dietary phosphate dependent manner. This work reveals that Npt2b and pulmonary osteoclast-like cells play key roles in pulmonary homeostasis and suggest potential new therapeutic targets for the treatment of lung disease.


title: The transcription factor Xrp1 orchestrates both reduced translation and cell competition upon defective ribosome assembly or function

abstract: Ribosomal Protein (Rp) gene haploinsufficiency affects overall translation rate, leads to cell elimination by competition with wild type cells in mosaic tissues, and sometimes leads to accumulation of protein aggregates. The changes in ribosomal subunit levels observed are not sufficient for these effects, which all depend on the AT-hook, bZip domain protein Xrp1. In Rp+/- cells, Xrp1 reduced global translation through PERK-dependent phosphorylation of eIF2. eIF2 phosphorylation was sufficient to reduce translation in, and also enable cell competition of, otherwise wild type cells. Unexpectedly, however, many other defects reducing ribosome biogenesis or function (depletion of TAF1B, eIF2, eIF4G, eIF6, eEF2, eEF11, or eIF5A), also increased eIF2 phosphorylation and enabled cell competition. In all cases this was through the Xrp1 expression that was induced, placing Xrp1 as the downstream instigator of cell competition that also contributed to overall translation deficits. In the absence of Xrp1, translation differences between cells were not themselves sufficient to trigger cell competition. Thus, Xrp1, which is shown here to be a sequence-specific transcription factor, is the master regulator that triggers cell competition and other consequences of multiple ribosomal stresses.


title: From telomere to telomere: the transcriptional and epigenetic state of human repeat elements

abstract: Mobile elements and highly repetitive genomic regions are potent sources of lineage-specific genomic innovation and fingerprint individual genomes. Comprehensive analyses of large, composite or arrayed repeat elements and those found in more complex regions of the genome require a complete, linear genome assembly. Here we present the first de novo repeat discovery and annotation of a complete human reference genome, T2T-CHM13v1.0. We identified novel satellite arrays, expanded the catalog of variants and families for known repeats and mobile elements, characterized new classes of complex, composite repeats, and provided comprehensive annotations of retroelement transduction events. Utilizing PRO-seq to detect nascent transcription and nanopore sequencing to delineate CpG methylation profiles, we defined the structure of transcriptionally active retroelements in humans, including for the first time those found in centromeres. Together, these data provide expanded insight into the diversity, distribution and evolution of repetitive regions that have shaped the human genome.


title: Cultural behaviours can be experimentally induced in wild baboons despite constraints on social information transmission

abstract: AO_SCPLOWBSTRACTC_SCPLOWThe formation of culture in animal societies, including humans, relies on the social transmission of information amongst individuals. This spread depends upon the transmission of social information, or social learning, between individuals. However, not all information spreads. To better understand how constraints at the individual-, dyad- and group-level might influence the formation of culture, we experimentally introduced four innovations (novel behaviours) across three troops of wild chacma baboons (Papio ursinus). At the individual-level, different phenotypic traits constrained individuals use of social information about the innovations. At the dyad-level, we found evidence for social reinforcement and directed social learning affecting who learnt and from whom. Group-level characteristics also limited the diffusion of information, which spread more slowly through social networks that showed less mixing across age classes. Nevertheless, despite these multi-level limitations, the four innovations quickly spread through all the social groups in which they were tested, suggesting that the formation of animal cultures can be surprisingly resilient to constraints on information transmission.


title: In situ solid-liquid extraction enhances recovery of taxadiene from engineered S. cerevisiae cell factories

abstract: A novel in situ solid phase adsorption strategy was investigated for enhanced recovery of taxadiene, a precursor to the blockbuster anticancer drug, paclitaxel, from engineered Saccharomyces cerevisiae. A synthetic adsorbent resin (HP-20) was employed to capture taxadiene across a range of cultivation scales. Cultivations from 12 % (w/v) resin concentration resulted in bead fragmentation which were found to be detrimental to cellular growth. After cultivation, the use of acetone for desorption captured intracellular and secreted taxadiene, achieving an integration of the bioprocess. Implementation of the proposed method at microscale (2 mL) and benchtop bioreactor scale (250 mL) resulted in 1.9-fold and 1.4-fold increments in taxadiene titer, respectively, compared to the extraction method using a dodecane overlay. Taxadiene was found to be distributed between resin beads and biomass in a ratio of 50 %. Finally, a maximum taxadiene titer of 76 {+/-} 19 mg/L was achieved in the benchtop bioreactor cultivations.

category: bioengineering


title: Proteome-wide quantitative RNA interactome capture (qRIC) identifies phosphorylation sites with regulatory potential in RBM20

abstract:
RNA-binding proteins (RBPs) are major regulators of gene expression at the post-transcriptional level. While many posttranslational modification sites in RBPs have been identified, little is known about how these modifications regulate RBP function. Here, we developed quantitative RNA-interactome capture (qRIC) to quantify the fraction of cellular RBPs pulled down with polyadenylated mRNAs. Applying qRIC to HEK293T cells quantified pull-down efficiencies of over 300 RBPs. Combining qRIC with phosphoproteomics allowed us to systematically compare pull-down efficiencies of phosphorylated and non-phosphorylated forms of RBPs. Over hundred phosphorylation events increased or decreased pull-down efficiency compared to the unmodified RBPs and thus have regulatory potential. Our data captures known regulatory phosphorylation sites in ELAVL1, SF3B1 and UPF1 and identifies new potentially regulatory sites. Follow-up experiments on the cardiac splicing regulator RBM20 revealed that multiple phosphorylation sites in the C-terminal disordered region affect nucleo-cytoplasmic localization, association with cytosolic RNA granules and alternative splicing. Together, we show that qRIC is a scalable method to identify functional posttranslational modification sites in RBPs.

HighlightsO_LIqRIC globally quantifies the fraction of RNA-binding proteins pulled down with mRNA
C_LIO_LICombining qRIC with phosphoproteomics identifies sites that affect RNA binding
C_LIO_LIPhosphorylation sites in RBM20 regulate its function in splicing
C_LI


title: Substrate recognition determinants of human eIF2α phosphatases

abstract: Phosphorylation of the translation initiation factor eIF2 is a rapid and vital cellular defence against many forms of stress. In mammals, the levels of eIF2 phosphorylation are set through the antagonistic action of four protein kinases and two heterodimeric protein phosphatases. The phosphatases are composed of the catalytic subunit PP1 and one of two related non-catalytic subunits, PPP1R15A or PPP1R15B (R15A or R15B). Attempts at reconstituting recombinant holophosphatases have generated two models, one proposing that substrate recruitment requires the addition of actin, whilst the second proposes that this function is encoded by R15s. The biological relevance of actin in substrate recruitment has not been evaluated. Here we generated a series of truncation mutants and tested their properties in mammalian cells. We show that substrate recruitment is encoded by an evolutionary conserved region in R15s, R15A325-554 and R15B340-639. Actin does not bind these regions establishing that it is not required for substrate recruitment. Activity assays in cells showed that R15A325-674 and R15B340-713, encompassing the substrate-binding region and the PP1 binding-region, exhibit wild-type activity. This study identifies essential regions of R15s and demonstrates they function as substrate receptors. This work will guide the design of future structural studies with biological significance.


title: Zic1 advances epaxial myotome morphogenesis to cover the neural tube via Wnt11r

abstract: The dorsal axial muscles, or epaxial muscles, are a fundamental structure covering the spinal cord and vertebrae, as well as mobilizing the vertebrate trunk. To date, mechanisms underlying the morphogenetic process shaping the epaxial myotome are largely unknown. To address this, we used the medaka zic1/zic4-enhancer mutant Double anal fin (Da), which exhibits ventralized dorsal trunk structures resulting in impaired epaxial myotome morphology and incomplete coverage over the neural tube. In wild type, dorsal dermomyotome (DM) cells, progenitors of myotomal cells, reduce their proliferative activity after somitogenesis and subsequently form unique large protrusions extending dorsally, potentially guiding the epaxial myotome dorsally. In Da, by contrast, DM cells maintain the high proliferative activity and form mainly small protrusions. By combining RNA- and ChIP-sequencing analyses, we revealed direct targets of Zic1 which are specifically expressed in dorsal somites and involved in various aspects of development, such as cell migration, extracellular matrix organization and cell-cell communication. Among these, we identified wnt11r as a crucial factor regulating both cell proliferation and protrusive activity of DM cells. We propose that the dorsal movement of the epaxial myotome is guided by DM cells and that Zic1 empowers this activity via Wnt11r to achieve the neural tube coverage.


title: High throughput and affordable genome-wide methylation profiling of circulating cell-free DNA by Methylated DNA sequencing (MeD-seq) of LpnPI digested fragments

abstract:
BackgroundDNA methylation detection in liquid biopsies provides a highly promising and much needed means for real-time monitoring of disease load in advanced cancer patient care. Compared to the often-used somatic mutations, tissue- and cancer-type specific epigenetic marks affect a larger part of the cancer genome and generally have a high penetrance throughout the tumour. Here we describe the successful application of the recently described MeD-seq assay for genome-wide DNA methylation profiling on cell-free DNA (cfDNA). The compatibility of the MeD-seq assay with different types of blood collection tubes, cfDNA input amounts, cfDNA isolation methods, and vacuum-concentration of samples was evaluated using plasma from both metastatic cancer patients and healthy blood donors (HBDs). To investigate the potential value of cfDNA methylation profiling for tumour load monitoring, we profiled paired samples from 8 patients with resectable colorectal liver metastases (CRLM) before and after surgery.

ResultsThe MeD-seq assay worked on plasma-derived cfDNA from both EDTA and CellSave blood collection tubes when at least 10 ng of cfDNA was used. From the 3 evaluated cfDNA isolation methods, both the manual QIAamp Circulating Nucleic Acid Kit (Qiagen) and the semi-automated Maxwell(R) RSC ccfDNA Plasma Kit (Promega) were compatible with MeD-seq analysis, whereas the QIAsymphony DSP Circulating DNA Kit (Qiagen) yielded significantly fewer reads when compared to the QIAamp kit (P<0.001). Vacuum-concentration of samples before MeD-seq analysis was possible with samples in AVE buffer (QIAamp) or water, but yielded inconsistent results for samples in EDTA-containing Maxwell buffer. Principal component analysis showed that pre-surgical samples from CRLM patients were very distinct from HBDs, whereas post-surgical samples were more similar. Several described methylation markers for colorectal cancer monitoring in liquid biopsies showed differential methylation between pre-surgical CRLM samples and HBDs in our data, supporting the validity of our approach. Results for MSC, ITGA4, GRIA4, and EYA4, were validated by quantitative methylation specific PCR.

ConclusionsThe MeD-seq assay provides a promising new method for cfDNA methylation profiling. Potential future applications of the assay include marker discovery specifically for liquid biopsy analysis as well as direct use as a disease load monitoring tool in advanced cancer patients.


title: Paleoecological implications of Lower-Middle Triassic stromatolites and microbe-metazoan build-ups in the Germanic Basin: Insights into the aftermath of the Permian - Triassic crisis

abstract: The aftermath of the Permian - Triassic crisis is characterized by ubiquitous occurrences of microbial sediments around the world. For instance, Triassic deposits of the Germanic Basin have shown to provide a rich record of stromatolites as well as of microbe-metazoan build-ups with non-spicular demosponges. Despite their paleoecological significance, however, all of these microbialites have only rarely been studied. This study aims to fill this gap by examining and comparing microbialites from the Upper Buntsandstein (Olenekian, Early Triassic) and the lower Middle Muschelkalk (Anisian, Middle Triassic). By combining analytical petrography (optical microscopy, micro X-ray fluorescence, Raman spectroscopy) and geochemistry ({delta}13Ccarb, {delta}18Ocarb), we show that all studied microbialites formed in hypersaline lagoons to sabkha environments. Olenekian deposits in Jena and surroundings and Anisian strata at Werbach contain stromatolites. Anisian successions at Hardheim, in contrast, host microbe-metazoan build-ups. Thus, the key-difference is the absence or presence of non-spicular demosponges in microbialites. After the Permian - Triassic crisis, the widespread microbialites (e.g., stromatolites/microbe-metazoan build-ups) possibly resulted from suppressed ecological competition and occupied the vacant ecological niche. It seems plausible that microbes and non-spicular demosponges had a mutualistic relationship and it is tempting to speculate that the investigated microbial-metazoan build-ups reflect an ancient evolutionary and ecologic association. Furthermore, both microbes and non-spicular demosponges may benefit from elevated salinities. Perhaps it was minor differences in salinities that controlled whether or not non-spicular demosponges could develop.

category: paleontology


title: Drug-free nasal spray as a barrier against SARS-CoV-2 infection: safety and efficacy in human nasal airway epithelia

abstract:
BackgroundFor SARS-CoV-2 and other respiratory viruses, the nasal epithelium is a key portal for infection. Therefore, the nose is an important target of prophylactic and therapeutic interventions against these viruses. We developed a nasal spray (AM-301, a medical device marketed as Bentrio) to protect against infection by SARS-CoV-2 and potentially other viruses.

Aims of the studyTo test the safety and efficacy of AM-301 against SARS-CoV-2 infection.

MethodsAM-301 was tested on an in vitro 3D model of primary human nasal airway epithelium. Safety was assessed in assays for tight junction integrity, cytotoxicity and cilia beating frequency. Efficacy against SARS-CoV-2 infection was evaluated in prophylaxis and infection mitigation assays.

ResultsAM-301 did not have any detrimental effect on the nasal epithelium. Prophylactic treatment with AM-301 reduced viral titer significantly vs. controls over 4 days, reaching a maximum reduction of 99%. When treatment with AM-301 was started 24 or 30 h after infection, epithelia that received the formulation had a 12- or 14-fold lower titer than controls.

ConclusionAM-301 was found to be safe in vitro, and it significantly decelerated viral titer growth in experimental models of prophylaxis and mitigation. Its physical (non-pharmaceutical) mechanism of action, safety and efficacy pave the way for further investigation of its possible use against a broad spectrum of viruses, allergens and pollutants.

category: pharmacology and toxicology


title: Cell adhesion factors during adolescence support amygdalo-cortical connections and flexible action later in life

abstract: Adolescent brain development is characterized by dramatic neuronal remodeling in the prefrontal cortex. This plasticity is presumed to act in part to "set the stage" for prefrontal cortical function in adulthood, but causal relationships have largely not been verified. Integrins are cell adhesion factors that provide a link between the extracellular matrix and the intracellular actin cytoskeleton. We find that {beta}1-integrin presence in the prelimbic subregion of the prefrontal cortex (PL) during adolescence, but not adulthood, is necessary for adult mice to select actions based on reward likelihood and value. These behaviors require coordinated limbic-frontal-striatal circuits. We identified projections from the basolateral amygdala (BLA) to PL as being necessary for mice to express learned response strategies. We then visualized adolescent PL neurons receiving input from the BLA and projecting to the dorsomedial striatum (DMS), a primary striatal output by which the PL controls reward-related behavior. These projection-defined neurons had a more "adult-like" morphology relative to a general population of layer V PL neurons. {beta}1-integrin loss caused the overexpression of stubby-type dendritic spines at the expense of more mature spines, a phenotype not observed when {beta}1-integrins were silenced before or after adolescence. Together, these experiments localize {beta}1-integrin-mediated cell adhesion activity within a developing di-synaptic circuit that coordinates flexible action.


title: Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease

abstract:
The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. Whilst the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the N-terminal FinO domain and the variable C-terminal region required for ProQ activity. Two C-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. By contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of non-functional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria.

SIGNIFICANCEProteins that interact with RNA play a vital role in controlling key functions in pathogenic bacteria. RNA-binding proteins regulate how, when and where bacteria feed, swim or interact with a host, and it is critical that we understand how RNAs associate with these proteins. ProQ is one of the three major RNA-binding proteins (RBPs) in Gram-negative bacteria. In this study, we mapped the amino acid residues of ProQ that are essential for function. We successfully identified residue substitutions that rendered the ProQ RBP both non-functional and unable to interact with RNA. Our findings raise the possibility that the Lon protease mediates a quality control mechanism of ProQ that targets this RBP in the absence of RNA. A posttranslational quality control mechanism of this type could prevent the accumulation of nonfunctional RBPs in the bacterial cytoplasm.


title: Mistreating birth-death models as priors in phylogenetic analysis compromises our ability to compare models

abstract: Time-calibrated phylogenetic trees are fundamental to a wide range of evolutionary studies. Typically, these trees are inferred in a Bayesian framework, with the phylogeny itself treated as a parameter with a prior distribution (a "tree prior"). This prior distribution is often a variant of the stochastic birth-death process, which models speciation events, extinction events, and sampling events (of extinct and/or extant lineages). However, the samples produced by this process are observations, so their probability should be viewed as a likelihood rather than a prior probability. We show that treating the samples as part of the prior results in incorrect marginal likelihood estimates and can result in model-comparison approaches disfavoring the best model within a set of candidate models. The ability to correctly compare the fit of competing tree models is critical to accurate phylogenetic estimates, especially of divergence times, and also to studying the processes that govern lineage diversification. We outline potential remedies, and provide guidance for researchers interested in comparing the fit of competing tree models.


title: C25-modified rifamycin derivatives with improved activity against Mycobacterium abscessus

abstract: Infections caused by Mycobacterium abscessus are difficult to treat due to its intrinsic resistance to most antibiotics. Formation of biofilms and the capacity of M. abscessus to survive inside host phagocytes further complicate eradication. Herein, we explored whether addition of a carbamate-linked group at the C25 position of rifamycin SV blocks enzymatic inactivation by ArrMab, an ADP-ribosyltransferase conferring resistance to rifampicin. Unlike rifampicin, 5j, a benzyl piperidine rifamycin derivative with a morpholino substituted C3 position, is not modified by purified ArrMab. Additionally, we show that the ArrMab D82 residue is essential for catalytic activity. Thermal profiling of ArrMab in the presence of 5j, rifampicin or rifabutin shows that 5j does not bind to ArrMab. We found that the activity of 5j is comparable to amikacin against M. abscessus planktonic cultures and pellicles. Critically, 5j also exerts potent antimicrobial activity against M. abscessus in human macrophages and shows synergistic activity with amikacin and azithromycin.


title: Natural variation in copper tolerance in Drosophila melanogaster is shaped by transcriptional and physiological changes in the gut

abstract: Increases in industrialisation and anthropogenic activity have resulted in an increase in pollutants released into the environment. Of these pollutants, heavy metals such as copper are particularly concerning due to their bio-accumulative nature. Due to its highly heterogeneous distribution and its dual nature as both an essential micronutrient and toxic element, the genetic basis of copper tolerance is likely shaped by a complex interplay of physiological and environmental factors. Drosophila melanogaster, a long-standing sentinel of environmental toxins, is uniquely suited for the study of copper tolerance in arthropods and other more diverse species. In this study, we utilized the natural variation present in multiple populations of D. melanogaster collected across Europe to screen for variation in copper tolerance, which we found to be highly variable both within and between locations. While these collection locations covered a wide range of atmospheric and soil pollution levels, the degree of urbanization at the collection sites, rather than any other combination of environmental factors, was linked to copper tolerance. Moreover, differential expression analysis revealed that metabolism, reproduction, and protease induction contribute to copper response in tolerant and sensitive lines to different degrees. Additionally, the greatest transcriptomic and physiological responses to copper toxicity were seen in the midgut; where preservation of gut acidity is strongly linked to greater tolerance. Overall, our study provides a unique perspective on the genetic and environmental factors that shape copper tolerance in natural D. melanogaster populations and identifies new genes and physiological traits involved in this complex phenotype.


title: The spatial landscape of clonal somatic mutations in benign and malignant tissue

abstract: Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis of cancer. Here, we provide an unsupervised approach to study spatial genome integrity in situ to gain molecular insight into clonal relationships. We employed spatially resolved transcriptomics to infer spatial copy number variations in >120 000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of an unsupervised approach to capture the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.


title: How fragile we are. Influence of STimulator of INterferon Genes, STING, variants on pathogen recognition and immune response efficiency

abstract: The STimulator of INterferon Genes (STING) protein is a cornerstone of the human immune response. Its activation by cGAMP upon the presence of cytosolic DNA stimulates the production of type I interferons and inflammatory cytokines which are crucial for protecting cells from infections. STING signaling pathway can also influence both tumor-suppressive and tumor-promoting mechanisms, rendering it an appealing target for drug design. In the human population, several STING variants exist and exhibit dramatic differences in their activity, impacting the efficiency of the host defense against infections. Understanding the differential molecular mechanisms exhibited by these variants is of utmost importance notably towards personalized medicine treatments against diseases such as viral infections (COVID-19, Dengue...), cancers, or auto-inflammatory diseases. Owing to micro-seconds scale molecular modeling simulations and post-processing by contacts analysis and Machine Learning techniques, we reveal the dynamical behavior of four STING variants (wild type, G230A, R293Q, and G230A-R293Q) and we rationalize the variability of efficiency observed experimentally. Our results show that the decrease of STING activity is linked to a stiffening of key-structural features of the binding cavity, together with changes of the interaction patterns within the protein.


title: TOR signalling is required for host lipid metabolic remodelling and survival following enteric infection in Drosophila

abstract: When infected by enteric pathogenic bacteria, animals need to initiate local and whole-body defence strategies. While most attention has focused on the role of innate immune anti-bacterial responses, less is known about how changes in host metabolism contribute to host defence. Using Drosophila as a model system, we identify induction of intestinal target-of-rapamycin (TOR) kinase signalling as a key adaptive metabolic response to enteric infection. We find TOR is induced independently of the IMD innate immune pathway, and functions together with IMD signalling to promote infection survival. These protective effects of TOR signalling are associated with re-modelling of host lipid metabolism. Thus, we see that TOR switches intestinal metabolism to lipolysis and fatty acid oxidation. In addition, TOR is required to limit excessive infection mediated wasting of adipose lipid stores by promoting an increase in the levels of fat body-expressed de novo lipid synthesis genes. Our data support a model in which induction of TOR represents a host tolerance response to counteract infection-mediated lipid wasting in order to promote survival.

category: physiology


title: A sex chromosome drives the emergence of vocal learning following hormonal manipulation in female zebra finches

abstract: Zebra finches are sexually dimorphic vocal learners. Males learn to sing by imitating mature conspecifics, but females do not. The lack of vocal learning in females is associated with anatomical differences in the neural circuits responsible for vocal learning, including the atrophy of several brain regions during development1. However, this atrophy can be prevented and song learning retained in females after pharmacological estrogen treatment2-4. Little is known about the genetic machinery controlling this sex and estrogen responsive song system development. To screen for drivers, we performed an unbiased analysis of transcriptomes from song control nuclei and surrounding motor regions in zebra finches of either sex treated with 17-{beta}-estradiol or vehicle until sacrifice on day 30, when divergence between the sexes is anatomically apparent. Utilizing the newly assembled autosomes and sex chromosomes from the zebra finch Vertebrate Genomes Project assemblies5, we identified correlated gene modules that were associated to song nuclei in a sex and estradiol dependent manner. Female estradiol treated HVC, in the vocal learning circuit, acquired the smallest of the modular specializations observed in male HVC. This module was enriched for genes governing anatomical development, and its specilization was dispraportionately influenced by the expression of Z sex chromosome transcripts in HVC. We propose that vocal learning may be prevented in female zebra finches via the suppression of an estrogen inducible Z chromosome cis-acting regulatory element.


title: Flight-tone mediated circadian control of audibility in mating swarms of Anopheles mosquitoes

abstract:
Mating swarms of malaria mosquitoes form every day at sunset throughout the tropical world, they typically last less than 30 minutes. Activity patterns must thus be highly synchronized between the sexes. Moreover, males must be able to identify the few sporadically entering females by detecting the females faint flight tones. We here show that the Anopheles circadian clock ensures a tight synchrony of male and female activity and -importantly - also retunes the males acoustic detection system: by lifting their own flight tones at dusk, males actively enhance the audibility of females. The reported phenomenon of  harmonic convergence is a random by-product of the mosquitoes flight tone variance. Intriguingly, flight tones of individual mosquitoes occupy narrow -partly non-overlapping-frequency ranges, suggesting that the audibility of individual females varies across males.

One Sentence SummaryMale mosquitoes sharpen their hearing at sunset.


title: IgG surface mobility promotes antibody-dependent cellular phagocytosis by Syk and Arp2/3 mediated reorganization of Fcγ receptors in macrophages.

abstract:
By visualizing the movements of Rituximab during Antibody dependent cellular phagocytosis (ADCP) of B lymphoma cells by macrophages, we found that Fc{gamma} receptors (Fc{gamma}R) on the macrophage surface microcluster, recruit Syk and undergro large-scale reorganization at the phagocytic synapse prior to and during engulfment of the target cell. Given these dramatic rearrangements, we analyzed how the surface mobility of Rituximab contributes to Fc{gamma}R signal amplification and ADCP efficiency. Depolymerization of the target cell actin cytoskeleton resulted in free diffusion of Rituximab docked to CD20, enhanced microcluster reorganization, Syk recruitment and ADCP. Conversely, immobilization of Rituximab by chemical fixation impaired microcluster formation and diminished Syk recruitment and ADCP. In macrophages lacking Syk, Rituximab accumulated at the base of the phagosome and were trogocytosed, consistent with Syk kinase activity being necessary to trigger redistribution of Rituximab-Fc{gamma}R during engulfment and to prevent antigenic modulation of the target. Total internal reflection fluorescence analysis of Fc{gamma}R-IgG on fluid supported lipid bilayers revealed a membrane topography displaying inward reaching leading edges and protruding contact sites reminiscent of podosomes. This topography was distinct from the closely apposed macrophage/target membranes observed during engagement of IgG displayed on immobile supported lipid bilayers. The organization of this contact, pseudopod extension and the rearrangement of microclusters depended critically on Arp 2/3. Thus, Syk and Arp2/3 coordinate actin rearrangements and Fc{gamma}R-IgG complexes that were of previously unrecognized complexity for the clearance of cells displaying surface-mobile antigens.

Significance StatementADCP is an important effector mechanism for the removal of malignant, immunologically aberrant, and infected cells during treatment with therapeutic antibodies or adaptive immune responses. Most transmembrane protein antigens are mobile with transient confinement from the actin of the target cell. This work demonstrates that macrophage forces overcome these confinements to rearrange Fc{gamma}R-IgG-antigen complexes before and during ADCP. Thus, new paradigms are needed as ADCP has largely been studied using model target particles that display immobile antigens. Moreover, we found that the mobility of the therapeutic antibody, Rituximab, on the surface of B lymphoma cells foretells ADCP efficacy, with lower densities of IgG mediating ADCP on increasingly mobile antigens.


title: An intestinally secreted host factor limits bacterial colonization but promotes microsporidia invasion of C. elegans

abstract: Microsporidia are ubiquitous obligate intracellular pathogens of animals. These parasites often infect hosts through an oral route, but little is known about the function of host intestinal proteins that facilitate microsporidia invasion. To identify such factors necessary for infection by Nematocida parisii, a natural microsporidian pathogen of Caenorhabditis elegans, we performed a forward genetic screen to identify mutant animals that have a Fitness Advantage with Nematocida (Fawn). We isolated four fawn mutants that are resistant to Nematocida infection and contain mutations in T14E8.4, which we renamed aaim-1 (Antibacterial and Aids invasion by Microsporidia). Expression of AAIM-1 in the intestine of aaim-1 animals restores N. parisii infectivity and this rescue of infectivity is dependent upon AAIM-1 secretion. N. parisii spores in aaim-1 animals are improperly oriented in the intestinal lumen, leading to reduced levels of parasite invasion. Conversely, aaim-1 mutants display both increased colonization and susceptibility to the bacterial pathogen Pseudomonas aeruginosa and overexpression of AAIM-1 reduces P. aeruginosa colonization. Competitive fitness assays show that aaim-1 mutants are favoured in the presence of N. parisii but disadvantaged on P. aeruginosa compared to wild type animals. Together, this work demonstrates how microsporidia exploits a secreted protein to promote host invasion. Our results also suggest evolutionary trade-offs may exist to optimizing host defense against multiple classes of pathogens.


title: An intestinally secreted host factor promotes microsporidia invasion of C. elegans


title: A comparison of wild and captive Comal Springs riffle beetle (Heterelmis comalensis) microbiomes

abstract: The gut microbiome is affected by host intrinsic factors, diet, environment, and strongly linked to hosts health. Although fluctuations of microbiome composition are normal, some are due to changes in host environmental conditions. When species are moved into captive environments for conservation, education, or rehabilitation, these new conditions can influence a change in gut microbiome composition. Here, we compared the microbiomes of wild and captive Comal Springs riffle beetles (Heterelmis comalensis) by using amplicon sequencing of the 16S rRNA gene. We found that the microbiome of captive beetles was more diverse than wild beetle microbiomes. We identified 24 Amplicon Sequence Variants (ASVs) with relative abundances significantly different between the wild and captive beetles. Many of the ASVs overrepresented in captive beetle microbiomes belong to taxa linked to nitrogen-rich environments. This is one of the first studies comparing the effects of captivity on the microbiome of an endangered insect species. Our findings provide valuable information for future applications in the management of captive populations of H. comalensis.