title: Random attention can explain apparent object choice behavior in free-walking blowflies

abstract:
Flies are often observed to approach dark objects. To a naive observer they seem to pay selective attention to one out of several objects although previous research identified a reflex-like fixation behavior integrating responses to all objects as possible underlying mechanism. In a combination of behavioral experiments and computational modelling, we investigate the choice behavior of flies freely walking towards an arrangement of two objects placed at a variable distance from each other. The walking trajectories are oriented towards one of the objects much earlier than predicted by a simple reactive model. We show that object choice can be explained by a continuous control scheme in combination with a mechanism randomly responding to the position of each object according to a stochastic process. Although this may be viewed as a special form of an attention-like mechanism, the model does not require an explicit decision mechanism or a memory for the drawn decision.

Summary StatementWalking blowflies apparently choose one of two objects to approach. In model simulations, a fixation scheme combined with random attention replicates this behavior without an explicit decision mechanism.


title: A general design of caging-group free photoactivatable fluorophores for live-cell nanoscopy

abstract: The controlled switching of fluorophores between non-fluorescent and fluorescent states is central to every superresolution fluorescence microscopy (nanoscopy) technique, and the exploration of radically new switching mechanisms remains critical to boosting the performance of established, as well as emerging superresolution methods. Photoactivatable dyes offer significant improvements to many of these techniques, but often rely on photolabile protecting groups that limit their applications. Here we describe a general method to transform 3,6-diaminoxanthones into caging-group free photoactivatable fluorophores. These photoactivatable xanthones (PaX) assemble rapidly and cleanly into highly fluorescent, photo- and chemically stable pyronine dyes upon irradiation with light. The strategy is extendable to carbon- and silicon-bridged xanthone analogs, yielding a new family of photoactivatable labels spanning much of the visible spectrum. Our results demonstrate the versatility and utility of PaX dyes in fixed and live-cell fluorescence microscopy, and both coordinate-targeted stimulated emission depletion (STED) and coordinate-stochastic single-molecule localization superresolution microscopy (SMLM).

category: biochemistry


title: Duesselpore: a full-stack local web server for rapid and simple analysis of Oxford Nanopore Sequencing data

abstract: Genome-wide analysis of transcriptomes offers extensive insights into the molecular mechanisms underlying the physiology of all known species and discover those that are still hidden. Oxford Nanopore Technologies (ONT) has recently been developed as a fast, miniaturized, portable and a cost effective alternative to Next Generation Sequencing. However, RNA-seq data analysis software that exploit ONT portability and allows scientists to easily analyze ONT data everywhere without bioinformatic expertise is not widely available. We developed Duesselpore, an easy-to-follow deep sequencing workflow that runs as a local webserver and allows the analysis of ONT data everywhere without requiring additional bioinformatic tools or internet connection. Duesselpore output includes differentially expressed genes and further downstream analyses, such as variance heatmap, disease and gene ontology plots, gene concept network plots and exports customized pathways for different cellular processes. We validated Duesselpore by analyzing the transcriptomic changes induced by PCB126, a dioxin-like PCB and a potent aryl hydrocarbon receptor (AhR) agonist in human HaCaT keratinocytes, a well characterized model system. Duesselpore was specifically developed to analyze ONT data but we also implemented NGS data analysis. Duesselpore is compatible with Microsoft and Mac operating systems, allows convenient, reliable and cost-effective analysis of ONT and NGS data.

category: bioinformatics


title: Interleukin-6 Drives Key Pathologic Outcomes in Experimental Acetaminophen-induced Liver Failure

abstract:
Background and AimsIn severe cases of acetaminophen (APAP) overdose, acute liver injury rapidly progresses to acute liver failure (ALF), producing life-threatening complications including, hepatic encephalopathy (HE) and multi-organ failure (MOF). Systemic levels of interleukin-6 (IL-6) and IL-10 are highest in ALF patients with the most severe complications and the poorest prognosis. The mechanistic basis for dysregulation of these cytokines, and their association with outcome in ALF, remain poorly defined.

MethodsTo investigate the impact of IL-6 and IL-10 in ALF, we used an experimental setting of failed liver repair after APAP overdose in which a high dose of APAP is administered (i.e., 500-600 mg/kg). Mice were treated with neutralizing antibodies to block IL-6 and IL-10.

ResultsIn mice with APAP-induced ALF, high levels of IL-10 reduced monocyte recruitment and trafficking in the liver resulting in impaired clearance of dead cell debris. Kupffer cells in these mice, displayed features of myeloid-derived suppressor cells, including high level expression of IL-10 and PD-L1, which were increased in an IL-6-dependent manner. Similar to ALF patients with HE, cerebral blood flow was reduced in mice with APAP-induced ALF. Remarkably, although IL-6 is hepatoprotective in mice treated with low doses of APAP (i.e., 300 mg/kg), IL-6 neutralization in mice with APAP-induced ALF fully restored cerebral blood flow and reduced mortality.

ConclusionCollectively, these studies demonstrate that exaggerated production of IL-6 in APAP-induced ALF triggers immune suppression (i.e., high levels of IL-10 and PD-L1), reduces cerebral blood flow (a feature of hepatic encephalopathy), disrupts liver repair (i.e., failed clearance of dead cells), and increases mortality.

category: pathology


title: Cerebellar and subcortical atrophy contribute to psychiatric symptoms in frontotemporal dementia

abstract: Recent studies have suggested that cerebellar and subcortical structures are impacted early in the disease progression of genetic frontotemporal dementia (FTD) due to microtubule-associated protein tau (MAPT), progranulin (GRN) and chromosome 9 open reading frame 72 (C9orf72). However, the clinical contribution of the structures involved in the cerebello-subcortical circuitry has been understudied in FTD given their potentially central role in cognition and behaviour processes. The present study aims to investigate whether there is an association between the atrophy of the cerebellar and subcortical structures, and neuropsychiatric symptoms (using the revised version of the Cambridge Behavioral Inventory, CBI-R) across genetic mutations and whether this association starts during the preclinical phase of the disease. Our study included 983 participants from the Genetic Frontotemporal dementia Initiative (GENFI) including mutation carriers (n=608) and non-carrier first-degree relatives of known symptomatic carriers (n= 375). Voxel-wise analysis of the thalamus, striatum, globus pallidus, amygdala, and the cerebellum was performed using deformation based morphometry (DBM) and partial least squares analyses (PLS) were used to link morphometry and behavioural symptoms. Our univariate results suggest that in this group of primarily presymptomatic subjects, volume loss in subcortical and cerebellar structure was primarily a function of aging, with only the C9orf72 group showing more pronounced volume loss in the thalamus compared to the non-carrier individuals. PLS analyses demonstrated that the cerebello-subcortical circuitry is related to all neuropsychiatric symptoms from the CBI-R, with significant overlap in brain/behaviour patterns, but also specificity for each genetic group. The biggest differences were in the extent of the cerebellar involvement (larger extent in C9orf72 group) and more prominent amygdalar contribution in the MAPT group. Finally, our findings demonstrated that C9orf72 and MAPT brain scores were related to estimated years before the age of symptom onset (EYO) in a second order relationship highlighting a steeper brain score decline 20 years before expected symptom onset, while GRN brain scores were related to age and not EYO. Overall, these results demonstrated the important role of the subcortical structures and especially of the cerebellum in genetic FTD symptom expression.

category: neuroscience


title: Retinal input influences pace of neurogenesis but not cell-type configuration of the visual forebrain

abstract: The brain is assembled during development by both innate and experience-dependent mechanisms1-7, but the relative contribution of these factors is poorly understood. Axons of retinal ganglion cells (RGCs) connect the eye to the brain, forming a bottleneck for the transmission of visual information to central visual areas. RGCs secrete molecules from their axons that control proliferation, differentiation and migration of downstream components7-9. Spontaneously generated waves of retinal activity, but also intense visual stimulation, can entrain responses of RGCs10 and central neurons11-16. Here we asked how the cellular composition of central targets is altered in a vertebrate brain that is depleted of retinal input throughout development. For this, we first established a molecular catalog17 and gene expression atlas18 of neuronal subpopulations in the retinorecipient areas of larval zebrafish. We then searched for changes in lakritz (atoh7-) mutants, in which RGCs do not form19. Although individual forebrain-expressed genes are dysregulated in lakritz mutants, the complete set of 77 putative neuronal cell types in thalamus, pretectum and tectum are present. While neurogenesis and differentiation trajectories are overall unaltered, a greater proportion of cells remain in an uncommitted progenitor stage in the mutant. Optogenetic stimulation of a pretectal area20,21 evokes a visual behavior in blind mutants indistinguishable from wildtype. Our analysis shows that, in this vertebrate visual system, neurons are produced more slowly, but specified and wired up in a proper configuration in the absence of any retinal signals.


title: Apolipoprotein ε4 modifies obesity-related atrophy in the hippocampal formation of cognitively healthy adults.

abstract:
Characterizing age- and risk-related hippocampal vulnerabilities may inform about the neural underpinnings of cognitive decline. We studied the impact of three risk-factors, Apolipoprotein (APOE)-{varepsilon}4, a family history of dementia, and central obesity, on CA1, CA2/3, dentate gyrus (DG) and subiculum in 158 cognitively healthy adults (38-71 years). Subfields were labelled with the Automatic Segmentation of Hippocampal Subfields (ASHS) and FreeSurfer (version 6) protocols. Volumetric and microstructural measurements from quantitative magnetization transfer and Neurite Orientation Density and Dispersion Imaging were extracted for each subfield and reduced to three principal components capturing apparent myelin/neurite packing, size/complexity, and metabolism. Aging was associated with an inverse U-shaped curve on myelin/neurite packing and affected all subfields. Obesity led to reductions in myelin/neurite packing and size/complexity regardless of APOE and FH status. However, amongst individuals with a healthy Waist-Hip-Ratio, APOE {varepsilon}4 carriers showed lower size/complexity than non-carriers. Protocol type did not affect this risk pattern. These findings provide novel evidence for interactive effects between APOE and central obesity on the hippocampal formation of cognitively healthy adults.

HighlightsO_LIAge-related inverted U-shaped curve of hippocampal myelin/neurite packing
C_LIO_LIObesity-related reductions of hippocampal myelin/neurite packing and size/complexity
C_LIO_LIAPOE modifies the effects of obesity on hippocampal size/complexity
C_LIO_LIAge-related slowing of spatial navigation
C_LIO_LINo APOE, family history, or obesity effects on cognition
C_LI


title: The Genomic Prehistory of the Indigenous People of Uruguay

abstract: The prehistory of the people of Uruguay is greatly complicated by the dramatic and severe effects of European contact, as with most of the Americas. After the series of military campaigns that exterminated the last remnants of nomadic peoples, Uruguayan official history masked and diluted the former indigenous ethnic diversity into the narrative of a singular people that all but died out. Here we present the first whole genome sequences of the Indigenous people of the region before the arrival of Europeans, from an archaeological site in eastern Uruguay that dates from 2,000 years before present. We find a surprising connection to ancient individuals from Panama and eastern Brazil, but not to modern Amazonians. This result may be indicative of a distinct migration route into South America that may have occurred along the Atlantic coast. We also find a distinct ancestry previously undetected in South America. Though this work begins to piece together some of the demographic nuance of the region, the sequencing of ancient individuals from across Uruguay is needed to better understand the ancient prehistory and genetic diversity that existed before European contact, thereby helping to rebuild the history of the indigenous population of what is now Uruguay.

category: genomics


title: Using OPM-MEG in contrasting magnetic environments

abstract: Magnetoencephalography (MEG) has been revolutionised in recent years by optically pumped magnetometers (OPMs). "OPM-MEG" offers higher sensitivity, better spatial resolution and lower cost than conventional instrumentation based on superconducting quantum interference devices (SQUIDS). Moreover, OPMs offer the possibility of motion robustness and lifespan compliance, dramatically expanding the range of MEG applications. However, OPM-MEG remains nascent technology; it places stringent requirements on magnetic shielding, and whilst a number of viable systems exist, most are custom made and there have been no cross-site investigations showing the reliability of data. In this paper, we undertake the first cross-site OPM-MEG comparison, using near identical commercial systems scanning the same participant. The two sites are deliberately contrasting, with different magnetic environments: a "green field" campus university site with an OPM-optimised shielded room (low interference) and a city centre hospital site with a "standard" (non-optimised) MSR (high interference). We show that despite a 25-fold difference in background field, and a 30-fold difference in low frequency interference, using dynamic field control and software-based suppression of interference we can generate comparable noise floors at both sites. In human data recorded during a visuo-motor task and a face processing paradigm, we were able to generate similar data, with source localisation showing that brain regions could be pinpointed with just ~10 mm spatial discrepancy and temporal correlations of > 80%. Overall, our study demonstrates that "plug- and-play" OPM-MEG systems exist and can be sited even in challenging magnetic environments.


title: Cbln1 regulates axon growth and guidance in multiple neural regions

abstract:
The accurate construction of neural circuits requires the precise control of axon growth and guidance, which is regulated by multiple growth and guidance cues during early nervous system development. It is generally thought that the growth and guidance cues that control the major steps of axon guidance have been defined. Here, we describe cerebellin-1 (Cbln1) as a novel cue that controls diverse aspects of axon growth and guidance throughout the central nervous system (CNS). Cbln1 has previously been shown to function in late neural development to influence synapse organization. Here we find that Cbln1 has an essential role in early neural development. Cbln1 is expressed on the axons and growth cones of developing commissural neurons and functions in an autocrine manner to promote axon growth. Cbln1 is also expressed in intermediate target tissues and functions as an attractive guidance cue. We find that these functions of Cbln1 are mediated by neurexin-2 (Nrxn2), which functions as the Cbln1 receptor for axon growth and guidance. In addition to the developing spinal cord, we further show that Cbln1 functions in diverse parts of the CNS with major roles in cerebellar parallel fiber growth and retinal ganglion cell axon guidance. Despite the prevailing role of Cbln1 as a synaptic organizer, our study discovers a new and unexpected function for Cbln1 as a general axon growth and guidance cue throughout the nervous system.

Impact statementDespite the prevailing role of Cbln1 as a synaptic organizer, our study discovers a new and unexpected function for Cbln1 as a general axon growth and guidance cue throughout the nervous system.


title: The manipulation of spinal motor neuron axonal growth promotes spinal cord repair

abstract: The loss of motor function in patients with spinal cord injury (SCI) is primarily due to the severing of the corticospinal tract (CST). Spinal motor neurons are located in the anterior horn of the spinal cord, and as the lower neurons of the CST, they control voluntary movement. Furthermore, its intrinsic axonal growth ability is significantly stronger than that of cerebral cortex pyramid neurons, which are the upper CST neurons. Therefore, we established an axonal regeneration model of spinal motor neurons to investigate the feasibility of repairing SCI by promoting axonal regeneration of spinal motor neurons. We demonstrated that conditionally knocking out pten in mature spinal motor neurons drastically enhanced axonal regeneration in vivo, and the regenerating axons of the spinal motor neurons re-established synapses with other cells in the damaged spinal cord. Thus, this strategy may serve as a novel and effective treatment method for SCI.


title: The evolution of color naming reflects pressure for efficiency: Evidence from the recent past

abstract: It has been proposed that semantic systems evolve under pressure for efficiency. This hypothesis has so far been supported largely indirectly, by synchronic cross-language comparison, rather than directly by diachronic data. Here, we directly test this hypothesis in the domain of color naming, by analyzing recent diachronic data from Nafaanra, a language of Ghana and Cote dIvoire, and comparing it with quantitative predictions derived from the mathematical theory of efficient data compression. We show that color naming in Nafaanra has changed over the past four decades while remaining near-optimally efficient, and that this outcome would be unlikely under a random drift process that maintains structured color categories without pressure for efficiency. To our knowledge, this finding provides the first direct evidence that color naming evolves under pressure for efficiency, supporting the hypothesis that efficiency shapes the evolution of the lexicon.

category: animal behavior and cognition


title: Comparative genomics on cultivated and uncultivated, freshwater and marine Candidatus Manganitrophaceae species implies their worldwide reach in manganese chemolithoautotrophy

abstract:
Chemolithoautotrophic manganese oxidation has long been theorized, but only recently demonstrated in a bacterial co-culture. The majority member of the co-culture, Candidatus Manganitrophus noduliformans, is a distinct but not yet isolated lineage in the phylum Nitrospirota (Nitrospirae). Here, we established two additional MnCO3-oxidizing cultures using inocula from Santa Barbara (USA) and Boetsap (South Africa). Both cultures were dominated by strains of a new species, designated Candidatus Manganitrophus morganii. The next abundant members differed in the available cultures, suggesting that while Ca. Manganitrophus species have not been isolated in pure culture, they may not require a specific syntrophic relationship with another species. Phylogeny of cultivated Ca. Manganitrophus and related metagenome-assembled genomes revealed a coherent taxonomic family, Candidatus Manganitrophaceae, from both freshwater and marine environments and distributed globally. Comparative genomic analyses support this family being Mn(II)-oxidizing chemolithoautotrophs. Among the 895 shared genes were a subset of those hypothesized for Mn(II) oxidation (Cyc2 and PCC_1) and oxygen reduction (TO_1 and TO_2) that could facilitate Mn(II) lithotrophy. An unusual, plausibly reverse Complex 1 containing 2 additional pumping subunits was also shared by the family, as were genes for the reverse TCA carbon fixation cycle, which could enable Mn(II) autotrophy. All members of the family lacked genes for nitrification found in Nitrospira species. The results suggest that Ca. Manganitrophaceae share a core set of candidate genes for the newly discovered manganese dependent chemolithoautotrophic lifestyle, and likely have a broad, global distribution.

ImportanceManganese (Mn) is an abundant redox-active metal that cycled in many of Earths biomes. While diverse bacteria and archaea have been demonstrated to respire Mn(III/IV), only recently have bacteria been implicated in Mn(II) oxidation dependent growth. Here, two new Mn(II)-oxidizing enrichment cultures originated from two continents and hemispheres were examined. By comparing the community composition of the enrichments and performing phylogenomic analysis on the abundant Nitrospirota therein, new insights are gleaned on cell interactions, taxonomy, and machineries that may underlie Mn(II)-based lithotrophy and autotrophy.

category: microbiology


title: Host casein kinase 1-mediated phosphorylation modulates phase separation of a rhabdovirus phosphoprotein and virus infection

abstract: Liquid-liquid phase separation (LLPS) plays important roles in forming cellular membraneless organelles. However, how host factors regulate LLPS of viral proteins during negative-sense RNA (NSR) virus infections is largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV) as a model to demonstrate regulation of host casein kinase 1 in phase separation and infection of NSR viruses. We first found that the BYSMV phosphoprotein (P) formed spherical granules with liquid properties and recruited viral nucleotide (N) and polymerase (L) proteins in vivo. Moreover, the P-formed granules were tethered to the ER/actin network for trafficking and fusion. BYSMV P alone formed droplets and incorporated the N protein and genomic RNA in vitro. Interestingly, phase separation of BYSMV P was inhibited by host casein kinase 1 (CK1)-dependent phosphorylation of an intrinsically disordered P protein region. Genetic assays demonstrated that the unphosphorylated mutant of BYSMV P exhibited condensed phase, which promoted virus replication through concentrating the N, L proteins, and genome RNA into viroplasms. Whereas, the phosphorylation-mimic mutant existed in diffuse phase state leading to enhanced virus transcription. Collectively, our results demonstrate that host CK1 modulates phase separation of viral P protein and virus infection.


title: Identification of diverse viruses associated with grasshoppers unveils phylogenetic congruence between hosts and viruses

abstract:
Locusts and grasshoppers are one of the most dangerous agricultural pests. Environmentally benign microbial pesticides are increasingly desirable for controlling locust outbreaks in fragile ecosystems. Here we use metagenomic sequencing to profile the rich viral communities in 34 grasshopper species and report 322 viruses, including 202 novel species. Most of the identified viruses are related to other insect viruses and some are targeted by antiviral RNAi pathway, indicating they infect grasshoppers. Some plant/fungi/vertebrate associated viruses are also abundant in our samples. Our analysis of relationships between host and virus phylogenies suggests that the composition of viromes is closely allied with host evolution, and there is significant phylogenetic relatedness between grasshoppers and viruses from Lispiviridae, Partitiviridae, Orthomyxoviridae, Virgaviridae and Flaviviridae. Overall, this study is a thorough exploration of viruses in grasshoppers and provide an essential evolutionary and ecological context for host-virus interaction in Acridoidea.

Author SummaryLocusts are the most destructive migratory pest in the world and continue to cause massive damages that endanger food security and threaten millions of people in 21st century. While chemical pesticides are still heavily relied on, biological pesticides developed from natural pathogens offer a reliable, less harmful alternative for controlling locust outbreaks in fragile ecosystems. Unfortunately, little is known about natural pathogens infecting this pest. In this study, we profile the viral communities in 34 grasshopper species include some major swarming species. While we identified as many as 202 novel viral species associated with grasshoppers, some of them are of potential to be developed as biocontrol agents. Our analysis of relatedness of phylogenies of grasshoppers and associated viruses helps to shed light on the eco-evolutionary interactions between insects and viruses. This work provides a valuable dataset of both academic and applied interest.


title: A Yeast-Based Screening System for Differential Identification of Poison Inhibitors and Catalytic Inhibitors of Human Topoisomerase I

abstract: 1.Inhibition of human topoisomerase I (TOP1) by camptothecin and topotecan has been shown to reduce excessive transcription of PAMP (Pathogen-Associated Molecular Pattern) - induced genes in prior studies, preventing death from sepsis in animal models of bacterial and SARS-CoV-2 infections. The TOP1 catalytic activity likely resolves the topological constraints on DNA that encodes these genes to facilitate the transcription induction that leads to excess inflammation. The increased accumulation of TOP1 covalent complex (TOP1cc) following DNA cleavage is the basis for the anticancer efficacy of the TOP1 poison inhibitors developed for anticancer treatment. The potential cytotoxicity and mutagenicity of TOP1 targeting cancer drugs pose serious concerns for employing them as therapies in sepsis prevention. The aim of this study is to develop a novel yeast-based screening system that employs yeast strains expressing wild-type or a dominant lethal mutant recombinant human TOP1. This yeast-based screening system can identify human TOP1 poison inhibitors for anticancer efficacy as well as catalytic inhibitors that can inhibit TOP1 DNA binding or cleavage activity in steps prior to the formation of the TOP1cc. In addition to distinguishing between such TOP1 catalytic inhibitors and TOP1 poison inhibitors, results from this yeast-based screening system will also allow elimination of compounds that are likely to be cytotoxic based on their effect on yeast cell growth that is independent of recombinant human TOP1 overexpression.

category: molecular biology


title: Reversible regulation of Cas12a activities by AcrVA5-mediated acetylation and CobB-mediated deacetylation

abstract: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated (Cas) system protects bacteria and archaea from the invasion of foreign genetic elements. To cope with the host CRISPR systems, phages have evolved many strategies, including the anti-CRISPR (Acr) proteins, to inactivate the Cas nucleases. Recently, it has been reported that the type V-A Cas12a effector can be acetylated and inactivated by AcrVA5, which is a GNAT-family acetyltransferase. However, it is unclear whether the host has any coping strategies to reactivate the defense system. Here we show that the AcrVA5-acetylated Cas12a can be deacetylated by bacterial deacetylase CobB, reactivating Cas12a for both in vitro cleavage of target DNA sequences and in vivo protection of the host from invasion of foreign nucleic acids. Therefore, this study not only shows the reversible regulation of Cas12a activities by post-translational modification but also reveals CobB as a secondary safeguard to bacterial CRISPR defense systems. In addition, we demonstrate that AcrVA5 is a wide-spectrum acetyltransferase, acetylating a large number of target proteins besides Cas12a, and the AcrVA5-acetylated targets can also be deacetylated by CobB.


title: A Predictive Energy Landscape Model of MetamorphicProtein Conformational Specificity

abstract:
"Metamorphic" proteins challenge state-of-the-art structure prediction methods reliant on amino acid similarity. Unfortunately, this obviates a more effective thermodynamic approach necessary to properly evaluate the impact of amino acid changes on the stability of two different folds. A vital capability of such a thermodynamic approach would be the quantification of the free energy differences between 1) the energy landscape minima of each native fold, and 2) each fold and the denatured state. Here we develop an energetic framework for conformational specificity, based on an ensemble description of protein thermodynamics. This energetic framework was able to successfully recapitulate the structures of high-identity enginerered sequences experimentally shown to adopt either Streptococcus protein GA or GB folds, demonstrating that this approach indeed reflected the energetic determinants of fold. Residue-level decomposition of the conformational specificity suggested several testable hypotheses, notably among them that fold-switching could be affected by local de-stabilization of the populated fold at positions sensitive to equilibrium perturbation. Since this ensemble-based compatibility framework is applicable to any structure and any sequence, it may be practically useful for the future targeted design, or large-scale proteomic detection, of novel metamorphic proteins.

Impact StatementMetamorphic proteins are single amino acid sequences capable of adopting more than one structure at equilibrium. Detection and design of these molecules hold great promise for biological understanding and materials engineering, but to do so requires a thermodynamic framework capable of estimating the free energy differences between the two structures and the denatured state. We present such a framework, show it to be effective for the well-studied metamorphic protein GA/GB system, and suggest testable hypotheses for engineering novel fold-switch proteins.


title: Electrochemical biosensors based on peptide-kinase interactions at the kinase docking site

abstract:
Kinases are important cancer biomarkers and are conventionally detected based on their catalytic activity. Kinases regulate cellular activities by phosphorylation of motif-specific multiple substrate proteins, resulting in lack of selectivity of activity-based kinase biosensors. We present an alternative approach of sensing kinases based on the interactions of their allosteric docking sites with a specific partner protein. The new approach was demonstrated for the ERK2 kinase and its substrate ELK-1. A peptide derived from ELK-1 was bound to a gold electrode and ERK2 sensing was performed by electrochemical impedance spectroscopy. The sensors showed high level of target selectivity for ERK2 when compared with p38{gamma} kinase and BSA. ERK2 was detected in its cellular concentration range, 0.2-8.0 M. Using the flexibility of peptide design, our method is generic for developing sensitive and substrate-specific biosensors and other disease-related enzymes based on their interactions.

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title: Mutant KRAS modulates colorectal cancer cells invasive response to fibroblast-secreted factors through the HGF/C-MET axis

abstract:
Genetic alterations influence the malignant potential of cancer cells, and so does the tumor microenvironment. Herein, we combined the study of KRAS oncogenic effects in colorectal cancer cells with the influence of fibroblasts-derived factors. Results revealed that mutant KRAS regulates cell fate through both autonomous and non-autonomous signaling mechanisms. Specifically, processes such as proliferation and cell-cell aggregation were autonomously controlled by mutant KRAS independently of the stimulation with fibroblasts conditioned media. However, cancer cell invasion revealed to be a KRAS-dependent non-autonomous effect, resulting from the cooperation between fibroblasts-derived HGF and mutant KRAS regulation of C-MET expression. C-MET downregulation upon KRAS silencing rendered cells less responsive to HGF and thus less invasive. Yet, in one cell line, KRAS inhibition triggered invasion upon stimulation with fibroblasts conditioned media. Inhibition of PIK3CA oncogene did not promoted invasion, thus showing a KRAS-specific effect. Moreover, the invasive capacity also depended on the HGF-C-MET axis. Overall, our study awards oncogenic KRAS an important role in modulating the response to fibroblast-secreted factors either by promoting or impairing invasion, and depicts the HGF-C-MET axis as a putative therapeutic target to impair the invasive properties of mutant KRAS cancer cells.

SignificanceTargeting mutant KRAS cancers is an urgent clinical need. HGF-C-MET axis inhibition arises as a possible strategy to target mutant KRAS CRC, both primary and metastatic tumors.

Additional informationO_ST_ABSFinancial supportC_ST_ABSThis work was supported through FEDER funds through the Operational Programme for Competitiveness Factors (COMPETE 2020), Programa Operacional de Competitividade e Internacionalizacao (POCI), Programa Operacional Regional do Norte (Norte 2020), European Regional Development Fund (ERDF), and by National Funds through the Portuguese Foundation for Science and Technology (FCT) (PTDC/MED-ONC/31354/2017). PDC is a PhD student from Doctoral Program in Pathology and Molecular Genetics from the Institute of Biomedical Sciences Abel Salazar (ICBAS, University of Porto) and she is funded through a PhD fellowship (SFRH/BD/131156/2017) awarded by the FCT. FM is a PhD student from Doctoral Program in Biomedicine from the Faculty of Medicine of the University of Porto and she is funded through a PhD fellowship (SFRH/BD/143669/2019) awarded by the FCT. SM is a PhD student from Doctoral Program in Biomedicine from the Faculty of Medicine of the University of Porto and she is funded through a PhD fellowship (SFRH/BD/143642/2019) awarded by the FCT. AR is a junior researcher hired by IPATIMUP under the CaTCh project funded by FEDER and FCT (POCI-01-0145-FEDER-031354). ALM is a PhD student from Doctoral Program in Biomedicine from the Faculty of Medicine of the University of Porto and she is funded through a PhD fellowship (2020.08932.BD) awarded by the FCT. MJO is principal researcher at INEB. SV is hired by IPATIMUP under norma transitoria do DL n.{o} 57/2016 alterada pela lei n.{o} 57/2017.

category: cancer biology


title: Building an RNA-based Toggle Switch using Inhibitory RNA Aptamers

abstract:
Synthetic RNA systems offer unique advantages such as faster response, increased specificity, and programmability compared to conventional protein-based networks. Here, we demonstrate an in-vitro RNA-based toggle switch using RNA aptamers capable of inhibiting the transcriptional activity of T7 or SP6 RNA polymerases. The activities of both polymerases are monitored simultaneously by using Broccoli and Malachite green light-up aptamer systems. In our toggle switch, a T7 promoter drives the expression of SP6 inhibitory aptamers, and an SP6 promoter expresses T7 inhibitory aptamers. We show that the two distinct states originating from the mutual inhibition of aptamers can be toggled by adding DNA sequences to sequester the RNA inhibitory aptamers. Finally, we assessed our RNA-based toggle switch in cell-like conditions by introducing controlled degradation of RNAs using a mix of RNases. Our results demonstrate that the RNA-based toggle switch could be used as a control element for nucleic acid networks in synthetic biology applications.

Graphical TOC Entry

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category: synthetic biology


title: Whole-brain fluorescence-MRI coregistration for precise anatomical mapping of virus infection

abstract: Neurotropic virus infections cause tremendous disease burden. Methods visualizing infection in the whole brain remain unavailable which greatly impedes understanding of viral neurotropism and pathogenesis. We devised an approach to visualize the distribution of neurotropic virus infection in whole mouse brain ex vivo. Optical projection tomography (OPT) signal was coregistered with a unique magnetic resonance imaging (MRI) brain template, enabling precise anatomical mapping of viral distribution, and the effect of type I interferon on distribution of infection was analyzed. Guided by OPT-MR, we show that Langat virus specifically targets sensory brain systems and the lack of type I interferon response results in an anatomical shift in infection patterns in the brain. We confirm this regional tropism, observed with whole brain OPT-MRI, by confocal and electron microscopy to provide unprecedented insight into viral neurotropism. This approach can be applied to any fluorescently labeled target in the brain.


title: Structure of the rabies virus glycoprotein trimer bound to a pre-fusion specific neutralizing antibody

abstract:
Rabies infection is nearly 100% lethal if untreated and kills over 50,000 people annually, many of them children. Existing rabies vaccines target the rabies virus glycoprotein (RABV-G) but generate short-lived immune responses, likely because the protein is heterogeneous under physiological conditions. Here, we report the 3.39[A] cryo-EM structure of trimeric, pre-fusion RABV-G complexed with RVA122, a potently neutralizing human antibody. RVA122 binds to a quaternary epitope at the top of RABV-G, bridging domains and stabilizing RABV-G protomers in a prefusion state. RABV-G trimerization involves side-to-side interactions between the central -helix and adjacent loops, rather than contacts between central helices, and interactions among the fusion loops at the glycoprotein base. These results provide a basis to develop improved rabies vaccines based on RABV-G stabilized in the prefusion conformation.

One sentence summaryWe report the structure and trimeric interface of pre-fusion rabies virus glycoprotein bound to the neutralizing antibody RVA122.


title: Effects of Chronic Intermittent Hypoxia and Chronic Sleep Fragmentation on Gut Microbiome, Serum Metabolome, Liver and Adipose Tissue Morphology

abstract: Chronic intermittent hypoxia (CIH) and chronic sleep fragmentation (CSF) are two cardinal pathological features of obstructive sleep apnea (OSA). Dietary obesity is a crucial risk intermediator for OSA and metabolic disorders. Gut microbiota affect hepatic and adipose tissue morphology under conditions of CIH or CSF through downstream metabolites. However, the exact relationship is unclear. Herein, chow and high-fat diet (HFD)-fed mice were subjected to CIH or CSF for 10 weeks each and compared to normoxia (NM) or normal sleep (NS) controls. 16S rRNA amplicon sequencing, untargeted liquid chromatography-tandem mass spectrometry, and histological assessment of liver and adipose tissues were used to investigate the correlations between the microbiome, metabolome, and lipid metabolism under CIH or CSF condition. Our results demonstrated that CIH and CSF regulate the abundance of intestinal microbes (such as Akkermansia mucinphila, Clostridium spp., Lactococcus spp., and Bifidobacterium spp.) and functional metabolites, such as tryptophan, free fatty acids, branched amino acids, and bile acids, which influence adipose tissue and hepatic lipid metabolism, and the level of lipid deposition in tissues and peripheral blood. In conclusion, CIH and CSF adversely affect fecal microbiota composition and function, and host metabolism; these findings provide new insight into the independent and synergistic effects of CIH, CSF, and HFD on lipid disorders.


title: Antenna modification leads to enhanced nitrogenase activity in a high light tolerant cyanobacterium

abstract:
Biological nitrogen fixation is an energy intensive process that contributes significantly towards supporting life on this planet. Among nitrogen-fixing organisms, cyanobacteria remain unrivaled in their ability to fuel the energetically expensive nitrogenase reaction with photosynthetically harnessed solar energy. In heterocystous cyanobacteria light-driven, photosystem I (PSI)-mediated ATP synthesis plays a key role in propelling the nitrogenase reaction. Efficient light transfer to the photosystems rely on phycobilisomes (PBS), the major antenna protein complexes. PBS undergo degradation as a natural response to nitrogen starvation. Upon nitrogen availability, these proteins are resynthesized back to normal levels in vegetative cells, but their occurrence and function in heterocysts remains inconclusive. Anabaena 33047 is a heterocystous cyanobacterium that thrives under high light, harbors higher amounts of PBS in its heterocysts and fixes nitrogen at higher rates compared to other heterocystous cyanobacteria. To assess the relationship between PBS in heterocysts and nitrogenase function, we engineered a strain that retains high amounts of the antenna proteins in its heterocysts. Intriguingly, under high light intensities the engineered strain exhibited unusually high rates of nitrogenase activity compared to the wild type. Spectroscopic analysis revealed altered PSI kinetics in the mutant, with increased cyclic electron flow around PSI, a route that contributes to ATP generation and nitrogenase activity in heterocysts. Retaining higher levels of PBS in heterocysts appears to be an effective strategy to enhance nitrogenase function in cyanobacteria that are equipped with the machinery to operate under high light intensities.

ImportanceThe function of phycobilisomes, the large antenna protein complexes in heterocysts has long been debated. This study provides direct evidence of the involvement of these proteins in supporting nitrogenase activity in Anabaena 33047, a heterocystous cyanobacterium that has affinity for very high light intensities. This strain was previously known to be recalcitrant to genetic manipulation and hence despite its many appealing traits, remained largely unexplored. We developed a genetic modification system for this strain and generated a {Delta}nblA mutant that exhibited resistance to phycobilisome degradation upon nitrogen starvation. Physiological characterization of the strain indicated that PBS degradation is not essential for acclimation to nitrogen deficiency and retention of PBS is advantageous for nitrogenase function.


title: Bacterial LomR Induces the Vibriophage VP882 VqmA-Directed Quorum-Sensing Lysogeny-Lysis Transition

abstract:
The bacterial cell-cell communication process called quorum sensing enables groups of bacteria to synchronously alter behavior in response to changes in cell population density. Quorum sensing relies on the production, release, accumulation, and detection of extracellular signal molecules called autoinducers. Here, we investigate a mechanism employed by a vibriophage to surveil host quorum sensing and tune its lysogeny-lysis decision to host cell density. The phage possesses a gene called vqmAPhage encoding a quorum-sensing receptor homologous to vibrio VqmA. Both VqmA receptors can detect the host bacteria-produced autoinducer called DPO. DPO-bound VqmAPhage launches the phage lysis process. We discover that the bacterial host produces an inducer of the VqmAPhage-directed quorum-sensing lysogeny-lysis transition. Production of the inducer appears to be widespread among bacteria. A screen of the Escherichia coli Keio collection for mutants impaired for inducer production revealed lomR, located in a prophage, and encoding a poorly understood protein. In the E. coli screening strain, lomR is interrupted by DNA encoding an insertion element. The 3 domain of this LomR protein is sufficient to induce VqmAPhage-directed lysis. Alanine-scanning mutagenesis showed that substitution at either of two key residues abrogates inducer activity. Full-length LomR is similar to the outer membrane porin OmpX in E. coli and Vibrio parahaemolyticus O3:K6, and OmpT in Vibrio cholerae C6706, and indeed, OmpX and OmpT can induce VqmAPhage-directed activity. Possibly, development of the LomR, OmpX, or OmpT proteins as tools to direct phage lysis of host cells could be used to control bacteria in medical or industrial settings.

ABSTRACT IMPORTANCEBacteria communicate with chemical signal molecules using a process called quorum sensing. Quorum sensing allows bacteria to track their cell numbers and orchestrate collective behaviors. Recently, we discovered that a virus that infects and kills bacteria "eavesdrops" on its hosts quorum-sensing process. Specifically, the virus monitors host cell growth by detecting the accumulation of host quorum-sensing signal molecules. In response to the garnered quorum-sensing information, the virus kills the host bacterial cells when the bacterial population has reached a high cell density. This strategy presumably enhances transmission of viruses to new host cells. Here, we discover and characterize three closely-related bacterial host-produced proteins called LomR, OmpX, and OmpT that are capable of inducing the viral quorum-sensing-mediated killing program. Development of this class of inducer proteins as tools to drive "on demand" virus-mediated lysis of pathogenic host bacterial cells could be used to control bacteria in medical or industrial settings.


title: Pseudomonas synergizes with fluconazole against Candida during treatment of polymicrobial infection

abstract: Polymicrobial infections are challenging to treat because we dont fully understand how pathogens interact during infection and how these interactions affect drug efficacy. Candida albicans and Pseudomonas aeruginosa are opportunistic pathogens that can be found in similar sites of infection such as in burn wounds and most importantly in the lungs of CF and mechanically ventilated patients. C. albicans is particularly difficult to treat because of the paucity of antifungal agents, some of which lack fungicidal activity. In this study, we investigated the efficacy of anti-fungal treatment during C. albicans-P. aeruginosa co-culture in vitro and co-infection in the mucosal zebrafish infection model analogous to the lung. We find that P. aeruginosa enhances the activity of fluconazole (FLC), an anti-fungal drug that is fungistatic in vitro, to promote both clearance of C. albicans during co-infection in vivo and fungal killing in vitro. This synergy between FLC treatment andbacterial antagonism is partly due to iron piracy, as it is reduced upon iron supplementation and knockout of bacterial siderophores. Our work demonstrates that FLC has enhanced activity in clinically relevant contexts and highlights the need to understand antimicrobial effectiveness in the complex environment of the host with its associated microbial communities.


title: Mfsd2b and Spns2 are essential for maintenance of blood vessels during development and protection of anaphylaxis

abstract: Sphingosine-1-phosphate (S1P) is a potent lipid mediator that is secreted by several cell types to induce signaling. We recently showed that Mfsd2b is an S1P transporter from hematopoietic cells, which contributes approximately 50% plasma S1P. To further determine the sources of plasma S1P, here, we report the characterizations of compound deletions of Mfsd2b and Spns2, another S1P transporter from endothelial cells. Global deletion of Mfsd2b and Spns2 (gDKO) resulted in embryonic lethality between E13.5 and E14.5 with severe hemorrhage that largely recapitulated the phenotypes from global S1P1 knockout mice, indicating that together with Mfsd2b, Spns2 also provides embryonic source of S1P for S1P1 stimulation. The hemorrhagic phenotypes in gDKO embryos were accompanied by increased angiogenesis and defects of tight junction proteins, indicating that S1P from Mfsd2b and Spns2 is essential for blood vessel integrity and maturation. The various sources of S1P in postnatal stages are yet to be fully understood. Postnatal ablation of S1P synthesis enzymes using Mx1Cre shows that Mx1Cre-sensitive cells provide most of plasma S1P. Interestingly, we showed that compound postnatal deletion of Mfsd2b and Spns2 using Mx1Cre (ctDKO-Mx1Cre) resulted in maximal reduction of 80% plasma S1P. Thus, a small amount of plasma S1P is supplied from other sources independent of Mfsd2b and Spns2. Nevertheless, the vasculature in the lung of ctDKO-Mx1Cre mice was compromised. Furthermore, ctDKO-Mx1Cre mice also exhibited severe susceptibility to anaphylaxis, indicating that S1P from Mfsd2b and Spns2 is indispensable during vascular stress. Together, our results show that Mfsd2b and Spns2 provide a critical source of S1P for embryonic development and they also provide a majority of plasma S1P for vascular homeostasis.


title: Stimulation of immunity-linked genes by membrane disruption is linked to Golgi function and the ARF-1 GTPase

abstract: Immune-linked genes (ILGs) are activated in response to pathogens but can also be activated by lipid imbalance. Why pathogen attack and metabolic changes both impact ILG activation is unclear. Organelles in the secretory pathway have distinct protein and lipid components and genetically separable stress programs. These stress pathways activate restorative transcriptional programs when lipid ratios become unbalanced or during dysregulated protein folding and trafficking. We find that ILGs are specifically activated when membrane phosphatidylcholine ratios change in the secretory pathway. Consistent with this result, disruption of Golgi function after RNAi targeting the ADP-ribosylation factor ARF-1 also activates ILG expression. Since increased protein secretion is altered by metabolic changes and pathogen responses, our data argue that ILG upregulation is a conserved, coordinated response to changes in trafficking resulting from intrinsic cues (lipid changes) or extrinsic stimulation (during the immune response). These findings uncover important and previously unexplored links between metabolism and the stress response.

category: cell biology


title: Binding stoichiometry and structural model of the HIV-1 Rev/Importin beta complex

abstract: HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein Importin {beta} (Imp{beta}), but how Rev associates with Imp{beta} is poorly understood. Here we report biochemical, biophysical and structural studies of the Imp{beta}/Rev complex. Gel shift, native mass spectrometry and isothermal titration calorimetry data reveal that Imp{beta} binds two Rev monomers through independent binding sites. Small-angle X-ray scattering (SAXS) data suggest that the HEAT repeats of Imp{beta} retain an extended conformation upon binding Rev, which according to NMR data is primarily recognized through its helical hairpin domain. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix 2 within Revs Arginine-Rich Motif (ARM) as a primary Imp{beta} binding epitope. Crosslinking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Imp{beta} with Rev helix 2 roughly parallel to the HEAT-repeat superhelical axis while the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Imp{beta} and highlight an atypical binding behaviour that distinguishes Rev from canonical cellular Imp{beta} cargos.


title: Rigid DNA nanotube tethers suppress high frequency noise in dual-trap optical tweezers systems

abstract: Dual trap optical tweezers are a powerful tool to trap and characterize the biophysical properties of single biomolecules such as the folding pathways of proteins and nucleic acids, and the chemomechanical activity of molecular motors. Despite its vastly successful application, noise from drift and fluctuation of the optics, and Brownian motion of the trapped beads still hinder the techniques ability to directly visualize folding of small biomolecules or the single nucleotide stepping of polymerases, especially at low forces (<10 pN) and sub-millisecond timescales. Rigid DNA nanotubes have been used to replace the conventional dsDNA linker to reduce optical tweezers noise in the low force range. However, optical tweezers are used to study a wide range of biophysical events, with timescales ranging from microseconds to seconds, and length changes ranging from sub nanometers to tens of nanometers. In this study, we systematically evaluate how noise is distributed across different frequencies in dual trap optical tweezers systems-and show that rigid DNA nanotube tethers suppress only high frequency noise (kHz), while the low frequency noise remains the same when compared to that of dsDNA tethers.

category: biophysics


title: A nuclear cAMP microdomain suppresses tumor growth by Hippo pathway inactivation

abstract: cAMP signaling pathways are critical for both oncogenesis and tumor suppression. cAMP signaling is localized to multiple spatially distinct microdomains, but the role of cAMP microdomains in cancer cell biology is poorly understood. We developed a tunable genetic system that allows us to activate cAMP signaling in specific microdomains. We uncovered a previously unappreciated nuclear cAMP microdomain that functionally activates a tumor suppressive pathway in a broad range of cancers by inhibiting YAP, a key effector protein of the Hippo pathway, inside the nucleus. We show that nuclear cAMP induces a LATS-dependent pathway leading to phosphorylation of nuclear YAP solely at serine 397, export of YAP from the nucleus, without YAP protein degradation. Thus, nuclear cAMP inhibition of nuclear YAP is distinct from other known mechanisms of Hippo regulation. Pharmacologic targeting of specific cAMP microdomains remains an untapped therapeutic approach for cancer, and since Hippo pathway deregulation can lead to oncogenesis and chemotherapeutic resistance, drugs directed at the nuclear cAMP microdomain may provide new avenues for the treatment of cancer.


title: Disruption of vacuolin microdomains in the host Dictyostelium discoideum increases resistance to Mycobacterium marinum-induced membrane damage and infection

abstract: Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, manipulates the host phagosome maturation pathway to replicate intracellularly. Mycobacterium marinum, a closely-related species, and Dictyostelium discoideum, a social amoeba and alternative phagocytic host, have been used as models to study host-pathogen interactions occurring during mycobacterial infections. Vacuolins, functional homologues of the mammalian flotillins, organize membrane microdomains and play a role in vesicular trafficking. Various pathogens have been reported to manipulate their membrane association and function. During infection of D. discoideum with M. marinum, Vacuolin C was specifically and highly induced and all three vacuolin isoforms were enriched at the mycobacteria-containing-vacuole (MCV). In addition, absence of vacuolins reduced escape from the MCV and conferred resistance to M. marinum infection. Moreover, ESAT-6, the membrane-disrupting virulence factor of M. marinum, was less associated with membranes when vacuolins were absent. Together, these results suggest that vacuolins are important host factors that are manipulated by mycobacteria to inflict membrane damage and escape from their compartment.


title: Birnaviridae virus factories show features of liquid-liquid phase separation, and are distinct from paracrystalline arrays of virions observed by electron microscopy

abstract:
To gain more information about the nature of Birnaviridae virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the polymerase (VP1) that we have previously shown is a marker for VFs in infected cells expressing GFP1-10. We found that VFs co-localized with 5-ethynyl uridine in the presence of actinomycin D, confirming they were the site of de novo RNA synthesis, and VFs were visible in infected cells that were fixed and permeabilized with digitonin, demonstrating that they were not membrane bound. Fluorescence recovery after photobleaching (FRAP) a region of interest within the VFs occurred rapidly, recovering from approximately 25% to 87% the original intensity over 146 seconds, and VFs were dissolved by 1,6-hexanediol treatment, demonstrating they showed properties consistent with liquid-liquid phase separation. There was a lower co-localization of the VF GFP signal with the capsid protein VP2 (Manders coefficient (MC) 0.6), compared to VP3 (MC, 0.9), which prompted us to investigate the VF ultrastructure by transmission electron microscopy (TEM). In infected cells, paracrystalline arrays (PAs) of virions were observed in the cytoplasm, as well as discrete electron dense regions. Using correlative light and electron microscopy (CLEM), we observed that the electron dense regions correlated with the GFP signal of the VFs, which were distinct from the PAs. In summary, Birnaviridae VFs are sites of de novo RNA synthesis, are not bound by a membrane, show properties consistent with liquid-liquid phase separation, and are distinct from the PAs observed by TEM.

ImportanceMembers of the Birnaviridae infect birds, fish and insects, and are responsible for diseases of significant economic importance to the poultry industry and aquaculture. Despite their importance, how they replicate in cells remains poorly understood. Here, we show that the Birnaviridae virus factories are not membrane bound, demonstrate properties consistent with liquid-liquid phase separation, and are distinct from the paracrystalline arrays of virions observed by transmission electron microscopy, enhancing our fundamental knowledge of virus replication that could be used to develop strategies to control disease, or optimize their therapeutic application.


title: Direct regulation of cell cycle regulatory gene expression by NtrX to promote Sinorhizobium meliloti cell division

abstract:
Cell division of the alfalfa symbiont, Sinorhizobium meliloti, is regulated by the CtrA signaling network. The gene expression of regulatory proteins in the network is affected by nutrient signaling. In this study, we found that NtrX, one of the regulators of nitrogen metabolic response, can directly regulate the expression of several regulatory genes from the CtrA signaling network. Three sets of S. meliloti ntrX mutants, including the plasmid insertion strain, the depletion strain and the substitution of the 53rd aspartate (ntrXD53E) from a plasmid in the wild-type strain (Sm1021), showed similar cell division defects, such as slow growth, abnormal morphology of partial cells and delayed DNA synthesis. Transcript quantitative evaluation indicated that the transcription of genes such as ctrA and gcrA was up-regulated, while the transcription of genes such as dnaA and ftsZl was down-regulated in the insertion mutant and the strain of Sm1021 expressing ntrXD53E. Correspondingly, inducible transcription of ntrX activates the expression of dnaA and ftsZ1, but represses ctrA and gcrA in the depletion strain. The expression levels of CtrA and GcrA were confirmed by western blotting, which were consistent with the transcription data. The transcriptional regulation of these genes requires phosphorylation of the conserved 53rd aspartate in the NtrX protein. The NtrX protein binds directly to the promoter regions of ctrA, gcrA, dnaA and ftsZ1 by recognizing the characteristic sequence CAAN2-5TTG Our findings reveal that NtrX is a novel transcriptional regulator of the CtrA signaling pathway genes, and positively affects bacterial cell division, associated with nitrogen metabolism.

IMPORTANCESinorhizobium meliloti infects the host alfalfa and induces formation of nitrogen-fixing nodules. Proliferation of rhizobia in plant tissues and cells is strictly controlled in the early stage of symbiotic interactions. However, the control mechanism is not very clear. Cell division of S. meliloti in the free-living state is regulated by the CtrA signaling network, but molecular mechanisms by which the CtrA system is associated with environmental nutrient signals (e.g., ammonia nitrogen) need to be further explored. This study demonstrates that NtrX, a regulator of nitrogen metabolism, required for symbiotic nodulation and nitrogen fixation by S. meliloti 1021, can act as a transcriptional regulator of the CtrA signaling system. It may link nitrogen signaling to cell cycle regulation in Rhizobium species.


title: The Nerve Growth Factor IB-like Receptor Nurr1 (NR4A2) recruits CoREST transcription repressor complexes to silence HIV following proviral reactivation in microglial cells

abstract:
Human immune deficiency virus (HIV) infection of microglial cells in the brain leads to chronic neuroinflammation, which is antecedent to the development of HIV-associated neurocognitive disorders (HAND) in the majority of patients. Productively HIV infected microglia release multiple neurotoxins including proinflammatory cytokines and HIV proteins such as envelope glycoprotein (gp120) and transactivator of transcription (Tat). However, powerful counteracting silencing mechanisms in microglial cells result in the rapid shutdown of HIV expression to limit neuronal damage. Here we investigated whether the Nerve Growth Factor IB-like nuclear receptor Nurr1 (NR4A2), which is a repressor of inflammation in the brain, acts to directly restrict HIV expression. HIV silencing was substantially enhanced by Nurr1 agonists in both immortalized human microglial cells (h{micro}glia) and induced pluripotent stem cells (iPSC)-derived human microglial cells (iMG). Overexpression of Nurr1 led to viral suppression, whereas by contrast, knock down (KD) of endogenous Nurr1 blocked HIV silencing. Chromatin immunoprecipitation (ChIP) assays showed that Nurr1 mediates recruitment of the CoREST/HDAC1/G9a/EZH2 transcription repressor complex to HIV promoter resulting in epigenetic silencing of active HIV. Transcriptomic studies demonstrated that in addition to repressing HIV transcription, Nurr1 also downregulated numerous cellular genes involved in inflammation, cell cycle, and metabolism, thus promoting HIV latency and microglial homoeostasis. Thus, Nurr1 plays a pivotal role in modulating the cycles of proviral reactivation by cytokines and potentiating the proviral transcriptional shutdown. These data highlight the therapeutic potential of Nurr1 agonists for inducing HIV silencing and microglial homeostasis and amelioration of the neuroinflammation associated with HAND.

AUTHOR SUMMARYHIV enters the brain almost immediately after infection where it infects perivascular macrophages, microglia and, to a less extent, astrocytes. In previous work using an immortalized human microglial cell model, we observed that integrated HIV constantly underwent cycles of reactivation and subsequent silencing. In the present study, we found that the Nurr1 nuclear receptor is a key mediator of HIV silencing. The functional activation of Nurr1 by specific agonists, or the over expression of Nurr1, resulted in rapid silencing of activated HIV in microglial cells. Global gene expression analysis confirmed that Nurr1 not only repressed HIV expression but also regulated numerous genes involved in microglial homeostasis and inflammation. Thus, Nurr1 is pivotal for HIV silencing and repression of inflammation in the brain and is a promising therapeutic target for treatment of HAND.


title: Looking for lipases and lipolytic organisms in low-temperature anaerobic reactors treating domestic wastewater

abstract: Poor lipid degradation limits low-temperature anaerobic treatment of domestic wastewater even when psychrophiles are used. We combined metagenomics and metaproteomics to find lipolytic bacteria and their potential, and actual, cold-adapted extracellular lipases in anaerobic membrane bioreactors treating domestic wastewater at 4{degrees}C and 15{degrees}C. Of the 40 recovered putative lipolytic metagenome-assembled genomes (MAGs), only three (Chlorobium, Desulfobacter, and Mycolicibacterium) were common and abundant (relative abundance [≥] 1%) in all reactors. Notably, some MAGs that represented aerobic autotrophs contained lipases. Therefore, we hypothesised that the lipases we found are not always associated with exogenous lipid degradation and can have other roles such as polyhydroxyalkanoates (PHA) accumulation/degradation and interference with the outer membranes of other bacteria. Metaproteomics did not provide sufficient proteome coverage for relatively lower abundant proteins such as lipases though the expression of fadL genes, long-chain fatty acid transporters, was confirmed for four genera (Dechloromonas, Azoarcus, Aeromonas and Sulfurimonas), none of which were recovered as putative lipolytic MAGs. Metaproteomics also confirmed the presence of 15 relatively abundant ([≥]1%) genera in all reactors, of which at least 6 can potentially accumulate lipid/polyhydroxyalkanoates. For most putative lipolytic MAGs, there was no statistically significant correlation between the read abundance and reactor conditions such as temperature, phase (biofilm and bulk liquid), and feed type (treated by ultraviolet light or not). Results obtained by metagenomics and metaproteomics did not confirm each other and further work is required to identify the true lipid degraders in these systems.


title: RNA-guided AsCas12a- and SpCas9-catalyzed knockout and homology directed repair of the omega-1 locus of the human blood fluke, Schistosoma mansoni

abstract: We compared the efficiency of gene knockout (KO) and precision of insertion (knock-in, KI) of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. with that of SpCas9 from Streptococcus pyogenes, aiming to enhance the functional genomics toolkit for Schistosoma mansoni. Programmed DNA cleavages catalyzed by Cas12a and Cas9 result in staggered and blunt ended strand breaks, respectively. TTTV, the optimal protospacer adjacent motif for AsCas12a would occur frequently within the AT-rich genome of this platyhelminth. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key enzyme termed omega-1 that is secreted by the schistosome egg. AsCas12a was more efficient than SpCas9 for gene knockout of omega-1 as determined by tracking of indels by decomposition (P < 0.001). Resulting from CRISPREsso2 analysis, most mutations were deletions; SpCas9 induced short deletions of 3 nt in length whereas AsCas12a induced deletions of 2 to 26 nt. Knockout efficiency of both nucleases markedly increased in the presence of short, single stranded oligodeoxynucleotide (ssODN) donor templates. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands of the targeted gene were tested, resulting in KO efficiencies of 15.67, 28.71 and 21.43% in the SpCas9 plus donor ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans cleavage activity against the ssODNs by activated AsCas12a was not apparent in vitro. Programmed SpCas9 editing led to more precise transgene insertion than AsCas12a, with KI efficiencies of 17.07% for the KI_SpCas9 group, 14.58% for KI_AsCas12a-NT-ssODN and 12.37% for KI_AsCas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases. These findings revealed that AsCas12a and SpCas9 both provide tractable routes for RNA-guided programmed mutation of the genome of the schistosome egg.


title: RNA-guided Cas12a and Cas9 knockout and knockin of the omega-1 locus of Schistosoma mansoni


title: Structures of PKA-phospholamban complexes reveal a mechanism of familial dilated cardiomyopathy

abstract:
Several mutations identified in phospholamban (PLN) have been linked to familial dilated cardiomyopathy (DCM) and heart failure, yet the underlying molecular mechanism remains controversial. PLN interacts with sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and regulates calcium uptake, which is modulated by the protein kinase A (PKA)-dependent phosphorylation of PLN during the fight-or-flight response. Here, we present the crystal structures of the catalytic domain of PKA in complex with wild-type and DCM-mutant PLNs. Our structures, combined with the results from other biophysical and biochemical assays, reveal a common disease mechanism: the mutations in PLN reduce its phosphorylation level by changing its conformation and weakening its interactions with PKA. In addition, we demonstrate that another more ubiquitous SERCA-regulatory peptide, called another-regulin (ALN), shares a similar mechanism mediated by PKA in regulating SERCA activity.

SignificanceDilated cardiomyopathy (DCM) is a common type of heart disease. Familial DCM is associated with mutations on phospholamban (PLN), but the mechanism remains elusive. Phosphorylation of PLN is known to influence its physiological function. We hypothesize that the connection between such mutations and DCM may involve decreased PLN phosphorylation levels due to less efficient binding to protein kinase A. We utilize x-ray crystallography, SPR, enzyme kinetic assays, thermal melt assays, and NMR to examine the structural and energetic consequences for PKA-catalyzed phosphorylation of PLN variants containing DCM-associated mutations. Our results provide a foundation to understand the general working mechanism of PKA and the physiological regulation of PLN by PKA, and also provide important insight into the pathological mechanism of DCM.


title: piCRISPR: Physically Informed Features Improve Deep Learning Models for CRISPR/Cas9 Off-Target Cleavage Prediction

abstract: CRISPR/Cas programmable nuclease systems have become ubiquitous in the field of gene editing. With progressing development, applications in in vivo therapeutic gene editing are increasingly within reach, yet limited by possible adverse side effects from unwanted edits. Recent years have thus seen continuous development of off-target prediction algorithms trained on in vitro cleavage assay data gained from immortalised cell lines. Here, we implement novel deep learning algorithms and feature encodings for off-target prediction and systematically sample the resulting model space in order to find optimal models and inform future modelling efforts. We lay emphasis on physically informed features, hence terming our approach piCRISPR, which we gain on the large, diverse crisprSQL off-target cleavage dataset. We find that our best-performing model highlights the importance of sequence context and chromatin accessibility for cleavage prediction and outperforms state-of-the-art prediction algorithms in terms of area under precision-recall curve.


title: Generic Approach For Mathematical Model of Multi-Strain Pandemics

abstract: Pandemics with multi-strain have become a major concern. We introduce a new model for assessing the connection between multi-strain pandemic and the mortality rate, basic reproduction number, and the maximum of infected individuals. The proposed model provides a general mathematical approach for representing multi-strain pandemics, generalizing for an arbitrary number of strains. We show the proposed model fits well with epidemiological historical data world health over a long period. From a theoretical point of view, we show that the increasing number of strains increases logarithmically the maximum number of infected individuals and the mean mortality rate. Moreover, the mean basic reproduction number is statistically identical to the single, most aggressive strain pandemic for multi-strain pandemics.


title: Environment of origin and domestication affect morphological, physiological, and agronomic response to water deficit in chile pepper (Capsicum sp.)

abstract: Global climate change is having a significant effect on agriculture by causing greater precipitation variability and an increased risk of drought. To mitigate these effects, it is important to identify specific traits, adaptations, and germplasm that improve tolerance to soil water deficit. Local varieties, known as landraces, have undergone generations of farmer-mediated selection and can serve as sources of variation, specifically for tolerance to abiotic stress. Landraces can possess local adaptations, where accessions adapted to a particular environment will outperform others grown under the same conditions. We explore adaptations to water deficit in chile pepper landraces from across an environmental gradient in Mexico, a center of crop domestication and diversity, as well in improved varieties bred for the US. In the present study, we evaluated 25 US and Mexico accessions in a greenhouse experiment under well-watered and water deficit conditions and measured morphological, physiological, and agronomic traits. Accession and irrigation regime influenced biomass and height, while branching, CO2 assimilation, and fruit weight were all influenced by an interaction between accession and irrigation. A priori group contrasts revealed possible adaptations to water deficit for primary branching, CO2 assimilation, and plant height associated with geographic origin, domestication level, and pepper species. Additionally, within the Mexican landraces, the number of primary branches had a strong relationship with precipitation from the environment of origin. This work provides insight into chile pepper response to water deficit and adaptation to drought and identifies possibly tolerant germplasm.

category: plant biology


title: Ly-6A-Induced Growth Inhibition and Cell death in a Transformed CD4+ T cell line: Role of Tumor Necrosis Factor-α (TNF-α)

abstract: Engaging the Ly-6A protein, an inhibitory signaling protein, on CD4+ T cell lines triggers apoptosis. Signaling through Ly-6A activates cell-intrinsic apoptotic cell death pathway as indicated by release of cytochrome C, activation of caspase 3 and 9. In addition Ly-6A induces cytokine production and growth inhibition. The mechanism underlying simultaneous distinct cellular responses has remained unknown. To examine the relatedness of distinct responses generated by engaging Ly-6A, we have quantified the secretion of TNF, TGF{beta} and a related protein GDF10, the three pro-apoptotic, growth inhibitory and tumor suppressive cytokines. While low levels of TGF{beta} and GDF10 were detected after engaging Ly-6A, the production of TNF- was elevated in cell cultures stimulated the Ly-6A protein. Blocking the biological activity of TNF resulted in reduced apoptosis induced by engaging Ly-6A. In contrast, growth inhibition/apoptosis in response to antigen receptor complex stimulation was not observed. Engaging the antigen receptor through activating the epsilon ({varepsilon}) chain of CD3 generated high levels of TGF-{beta} and GDF10 while decreasing TNF. These results suggest that the TNF- cytokine contributes to the Ly-6A-induced growth inhibitory and pro-apoptotic response in CD4+ T cells and provides mechanistic explanation of the observed biologically distinct responses initiated after engaging Ly-6A protein. These findings aid in understanding the inhibitory signaling initiated by Ly-6A protein, especially in the context of its potential immune checkpoint inhibitory role in T cells.

category: immunology


title: Plasmodium-encoded murine IL-6 impairs liver stage infection and elicits long-lasting sterilizing immunity

abstract: Plasmodium sporozoites inoculated by Anopheles mosquitoes into the skin of the mammalian host migrate to the liver before infecting hepatocytes. Previous work demonstrated that early production of IL-6 in the liver was found to be detrimental for the parasite growth, leading to the acquisition of a long-lasting immune protection. Considering IL-6 as a critical pro-inflammatory signal, we explored a novel approach whereby the parasite itself encodes for the murine IL-6 gene. We generated transgenic P.berghei parasites that express murine IL-6 during liver stage development. Though IL-6 transgenic sporozoites develop into exo-erythrocytic forms in cultured hepatocytes in vitro, these parasites were not capable of inducing a blood stage infection in mice. Furthermore, immunization of mice with transgenic IL-6 sporozoites elicited a long-lasting CD8+ T cell-mediated protective immunity against a subsequent infectious sporozoite challenge. Collectively, this study demonstrates that parasite-encoded IL-6 impairs Plasmodium infection at the liver stage, forming the basis of a novel suicide vaccine strategy to elicit protective antimalarial immunity.


title: Occurrence, antimicrobial resistance pattern and molecular characterization of Listeria monocytogenes isolated from bovines milk and meat in Mekelle City, Ethiopia

abstract: Listeria monocytogenes is an opportunistic and emerging foodborne zoonotic pathogen that encompasses a diversity of strains with varied virulence and can cause serious human and animal infections worldwide with low incidence but high hospitalization and case fatality rates. A cross-sectional study was conducted from December 2016 to June 2017 to estimate the molecular epidemiology of L. monocytogenes and its serotypes, and antimicrobial resistance pattern of isolates in Mekelle City. A total of 768 (384 of milk and 384 meat) samples of bovine origin were collected using a purposive random sampling technique. Isolation and identification of L. monocytogenes was done according to standard and recommended bacteriological procedures. Genome-based confirmation of each isolate was performed at species and serovar levels by targeting Iap, Imo0737, ORF2819 and ORF2110 genes using specific primers. In vitro antimicrobial susceptibility testing was performed using agar plate antibiotic disk diffusion method. The overall prevalence of L. monocytogenes was 26 (3.39%). Sample type prevalence rates of L. monocytogenes were 4.17 % and 2.6% in meat and milk samples, respectively. There was a statically significant difference (p<0.05) on the prevalence rates of the organism in meat samples collected from abattoir (1.67%), butcher shops (8.33%), and restaurants (8.33%). Serovars that were identified were belonged to 1/2b and 4b. Large proportions of isolates were highly susceptible to Ampicillin (88.46%) and Vancomycin (84.62%). However, the isolates had shown the highest level of resistance against Nalidixic Acid (96.15%). The highest intermediate was observed to Amoxicillin (57.69%). Moreover, 42.31% of the isolates were developed resistance for more than two drugs. Hence, both its occurrence and development of a multi-drug resistance indicated that, a coordinated effort is imperative to reduce or eliminate the risk posed by this pathogen in food chains and on controlled and careful use of antimicrobials both in veterinary and human treatment regimes.


title: Optimal information loading into working memory in prefrontal cortex

abstract: Working memory involves the short-term maintenance of information and is critical in many tasks. The neural circuit dynamics underlying working memory remain poorly understood, with different aspects of prefrontal cortical (PFC) responses explained by different putative mechanisms. By mathematical analysis, numerical simulations, and using recordings from monkey PFC, we investigate a critical but hitherto ignored aspect of working memory dynamics: information loading. We find that, contrary to common assumptions, optimal information loading involves inputs that are largely orthogonal, rather than similar, to the persistent activities observed during memory maintenance. Using a novel, theoretically principled metric, we show that PFC exhibits the hallmarks of optimal information loading and we find that such dynamics emerge naturally as a dynamical strategy in task-optimized recurrent neural networks. Our theory unifies previous, seemingly conflicting theories of memory maintenance based on attractor or purely sequential dynamics, and reveals a normative principle underlying the widely observed phenomenon of dynamic coding in PFC.


title: A recurrent circuit links antagonistic cerebellar modules during associative motor learning

abstract: The neural architecture of the cerebellum is thought to be specialized for performing supervised learning: specific error-related climbing fiber inputs are used to teach sensorimotor associations to small ensembles of Purkinje cells located in functionally distinct modules that operate independently of each other in a purely feedforward manner. Here, we test whether the basic operation of the cerebellum complies with this basic architecture in mice that learned a simple sensorimotor association during eyeblink conditioning. By recording Purkinje cells in different modules and testing whether their responses rely on recurrent circuits, our results reveal three operational principles about the functional organization of the cerebellum that stand in stark contrast to the conventional view: (1) Antagonistic organization, (2) Recurrent network dynamics, and (3) Intermodular communication. We propose that the neural architecture of the cerebellum implements these three operational principles to achieve optimal performance and solve a number of problems in motor control.


title: Consonance perception in congenital amusia: behavioral and brain responses to harmonicity and beating cues

abstract: Congenital amusia is a neurodevelopmental disorder characterized by difficulties in the perception and production of music, including the perception of consonance and dissonance, or the judgment of certain combinations of pitches as more pleasant than others. Two perceptual cues for dissonance are inharmonicity (the lack of a common fundamental frequency between components) and beating (amplitude fluctuations produced by close, interacting frequency components). In the presence of inharmonicities or beats, amusics have previously been reported to be insensitive to inharmonicity, but to exhibit normal sensitivity to beats. In the present study, we measured adaptive discrimination thresholds in amusic participants and found elevated thresholds for both cues. We recorded EEG and measured the mismatch negativity (MMN) in evoked potentials to consonance and dissonance deviants in an oddball paradigm. The amplitude of the MMN response was similar overall for amusics and controls, but while control participants showed a stronger MMN to harmonicity cues than to beating cues, amusic participants showed a stronger MMN to beating cues than to harmonicity cues. These findings suggest that initial encoding of consonance cues may be intact in amusia despite impaired behavioral performance, but that the relative weight of non-spectral cues may be increased for amusic individuals.


title: Uncovering the architecture of selection in two Bos taurus cattle breeds

abstract: Selection alters the genome via hard sweeps, soft sweeps, and polygenic selection. However, mapping polygenic selection is difficult because it does not leave clear signatures on the genome like a selective sweep. In populations with temporally-stratified genotypes, the Generation Proxy Selection Mapping (GPSM) method identifies variants associated with generation number (or appropriate proxy) and thus variants undergoing directional allele frequency changes. Here, we use GPSM on two large datasets of beef cattle to detect associations between an animals generation and 11 million imputed SNPs. Using these datasets with high power and dense mapping resolution, GPSM detected a total of 294 unique loci actively under selection in two cattle breeds. We observed that GPSM has a high power to detect selection in the very recent past (< 10 years), even when allele frequency changes are small. Variants identified by GPSM reside in genomic regions associated with known breed characteristics, such as fertility and maternal ability in Red Angus and carcass merit and coat color in Simmental. Over 60% of the selected loci reside in or near (<50 kb) annotated genes. Additionally, 36% of selected loci overlap known epigenetic marks or putative functional genomic regions. Using RAiSD and nSL, we identify hundreds of putative selective sweeps; however, these sweeps have little overlap with polygenic selected loci. This makes GPSM a complementary approach to sweep detection methods when temporal genotype data are available. The selected loci that we identify across methods demonstrate the complex architecture of selection in domesticated cattle.

category: evolutionary biology


title: The sequences of 150,119 genomes in the UK biobank

abstract: We describe the analysis of whole genome sequences (WGS) of 150,119 individuals from the UK biobank (UKB). This constitutes a set of high quality variants, including 585,040,410 SNPs, representing 7.0% of all possible human SNPs, and 58,707,036 indels. The large set of variants allows us to characterize selection based on sequence variation within a population through a Depletion Rank (DR) score for windows along the genome. DR analysis shows that coding exons represent a small fraction of regions in the genome subject to strong sequence conservation. We define three cohorts within the UKB, a large British Irish cohort (XBI) and smaller African (XAF) and South Asian (XSA) cohorts. A haplotype reference panel is provided that allows reliable imputation of most variants carried by three or more sequenced individuals. We identified 895,055 structural variants and 2,536,688 microsatellites, groups of variants typically excluded from large scale WGS studies. Using this formidable new resource, we provide several examples of trait associations for rare variants with large effects not found previously through studies based on exome sequencing and/or imputation.

category: genetics


title: Evaluation of methods for the inference of ancestral recombination graphs

abstract: The ancestral recombination graph (ARG) is a structure that describes the joint genealogies of sampled DNA sequences along the genome. Recent computational methods have made impressive progress towards scalably estimating whole-genome genealogies. In addition to inferring the ARG, some of these methods can also provide ARGs sampled from a defined posterior distribution. Obtaining good samples of ARGs is crucial for quantifying statistical uncertainty and for estimating population genetic parameters such as effective population size, mutation rate, and allele age. Here, we use standard neutral coalescent simulations to benchmark the estimates of pairwise coalescence times from three popular ARG inference programs: ARGweaver, Relate, and tsinfer+tsdate. We compare 1) the true coalescence times to the inferred times at each locus; 2) the distribution of coalescence times across all loci to the expected exponential distribution; 3) whether the sampled coalescence times have the properties expected of a valid posterior distribution. We find that inferred coalescence times at each locus are most accurate in ARGweaver, and often more accurate in Relate than in tsinfer+tsdate. However, all three methods tend to overestimate small coalescence times and underestimate large ones. Lastly, the posterior distribution of ARGweaver is closer to the expected posterior distribution than Relates, but this higher accuracy comes at a substantial trade-off in scalability. The best choice of method will depend on the number and length of input sequences and on the goal of downstream analyses, and we provide guidelines for the best practices.


title: Evaluation of methods for estimating coalescence times using ancestral recombination graphs


title: insideOutside: an accessible algorithm for classifying interior and exterior points, with applications in embryology

abstract: A crucial aspect of embryology is relating the position of individual cells to the broader geometry of the embryo. A classic example of this is the first cell-fate decision of the mouse embryo, where interior cells become inner cell mass and exterior cells become trophectoderm. Fluorescent labelling, imaging, and quantification of tissue-specific proteins have advanced our understanding of this dynamic process. However instances arise where these markers are either not available, or not reliable, and we are left only with the cells spatial locations. Therefore, a simple, robust method for classifying interior and exterior cells of an embryo using spatial information is required. Here, we describe a simple mathematical framework and an unsupervised machine learning approach, termed insideOutside, for classifying interior and exterior points of a three-dimensional point-cloud, a common output from imaged cells within the early mouse embryo. We benchmark our method against other published methods to demonstrate that it yields greater accuracy in classification of nuclei from the pre-implantation mouse embryos and greater accuracy when challenged with local surface concavities. The code is freely available as a well-documented, easy-to-use MATLAB script. This method should prove useful within and beyond embryology, with broader applications to similar data from the biological and life sciences.

category: developmental biology


title: Emotional ego- and altercentric biases in high-functioning autism spectrum disorder: Behavioral and neurophysiological evidence

abstract: Self-other distinction is a crucial aspect of social cognition, as it allows us to differentiate our own mental and emotional states from those of others. Research suggests that this ability might be impaired in individuals with autism spectrum disorder (ASD), but convincing evidence of self-other distinction deficits in the emotional domain is lacking. Here we aimed at evaluating emotional self-other distinction abilities in adults with and without ASD, in two behavioral pilot studies and one fMRI study. By using a newly developed virtual ball-tossing game that induced simultaneous positive and negative emotional states in each participant and another person, we were able to measure emotional egocentric and altercentric biases (namely the tendency to ascribe self-/other-related emotions to others/ourselves, respectively). Despite no behavioral differences, individuals with ASD showed decreased activation 1) in the right temporoparietal junction (rTPJ) during active overcoming of the emotional egocentric bias vs. passive game viewing, and 2) in the right supramarginal gyrus (rSMG) during ego-vs. altercentric biases, compared to neurotypical participants. These results suggest a different recruitment of these two regions in ASD when dealing with conflicting emotional states of oneself and another person. Furthermore, they highlight the importance of considering different control conditions when interpreting the involvement of rTPJ and rSMG during self-other distinction processes.


title: A Post-hurricane Quantitative Assessment of the Red-bellied Racer (Alsophis rufiventris) on Saba and Comparison with St. Eustatius

abstract: We estimated occupancy, abundance (lambda), detection probability, density/ha and abundance of a regionally endemic snake in the Colubrid family on the Dutch Caribbean island of Saba in 2021, four years after hurricanes Irma and Maria impacted the island. Line transect surveys were conducted at 74 sites covering 6.7 ha. The proportion of sites occupied was estimated at 0.74 (min 0.48, max 0.90), with occupancy varying between vegetation types and across elevational gradients. Similarly, lambda was estimated at 1.61 (min 0.7, max 3.7) but varied between vegetation types and elevational gradients. Detection probability was estimated at 0.15 (min 0.10, max 0.21). Using Distance sampling, we estimated 10.9 (min 7.3, max 16.2) racers/ha, with a total population estimate of 4,917 (min 2,577, max 6,362) across the entire study region (438.6 ha.) Based on anecdotal observations from Saban residents and prior literature describing the pre-hurricane population as "abundant" (at least 2.0 racers/hour), we posit that the population experienced a hurricane-induced decline but may have since recovered, though not to previous levels (1.28 racers/hour). Nevertheless, our results suggest that racer densities on Saba are currently higher than those on St. Eustatius. Despite this, given the species extremely limited extant range and the presence of invasive species on both islands, prevention of local extirpation should be a high conservation priority.

category: ecology


title: Whokaryote: distinguishing eukaryotic and prokaryotic contigs in metagenomes based on gene structure

abstract: Metagenomics has become a prominent technology to study the functional potential of all organisms in a microbial community. Most studies focus on the bacterial content of these communities, while ignoring eukaryotic microbes. Indeed, many metagenomics analysis pipelines silently assume that all contigs in a metagenome are prokaryotic. However, because of marked differences in gene structure, prokaryotic gene prediction tools fail to accurately predict eukaryotic genes. Here, we developed a classifier that distinguishes eukaryotic from prokaryotic contigs based on foundational differences between these taxa in gene structure. We first developed a random forest classifier that uses intergenic distance, gene density and gene length as the most important features. We show that, with an estimated accuracy of 97%, this classifier with principled features grounded in biology can perform almost as well as the classifiers EukRep and Tiara, which use k-mer frequencies as features. By re-training our classifier with Tiara predictions as additional feature, weaknesses of both types of classifiers are compensated; the result is an enhanced classifier that outperforms all individual classifiers, with an F1-score of 1.00 on precision, recall and accuracy for both eukaryotes and prokaryotes, while still being fast. In a reanalysis of metagenome data from a disease-suppressive plant endosphere microbial community, we show how using Whokaryote to select contigs for eukaryotic gene prediction facilitates the discovery of several biosynthetic gene clusters that were missed in the original study. Our enhanced classifier, which we call  Whokaryote, is wrapped in an easily installable package and is freely available from https://git.wageningenur.nl/lotte.pronk/whokaryote.

category: bioinformatics


title: BamToCov: an efficient toolkit for sequence coverage calculations

abstract:
Many genomics applications requires the calculation of nucleotide coverage of a reference or counting how many reads maps in a reference region.

Here we present BamToCov, a suite of tools for rapid and flexible coverage calculations relying on a memory efficient algorithm and designed for flexible integration in bespoke pipelines. The tools of the suite will process sorted BAM or CRAM files, allowing to extract coverage information using different filtering approaches.

BamToCov tools, unlike existing tools already available, have been developed to require a minimum amount of memory, to be easily integrated in workflows, and to allow for strand-specific coverage analyses. The unique coverage calculation algorithm makes it the ideal choice for the analysis of long reads alignments. The programs and their documentation are freely available at https://github.com/telatin/bamtocov.


title: A copula based topology preserving graph convolution network for clustering of single-cell RNA-seq data

abstract:
Annotation of cells in single-cell clustering requires a homogeneous grouping of cell populations. There are various issues in single cell sequencing that effect homogeneous grouping (clustering) of cells, such as small amount of starting RNA, limited per-cell sequenced reads, cell-to-cell variability due to cell-cycle, cellular morphology, and variable reagent concentrations. Moreover, single cell data is susceptible to technical noise, which affects the quality of genes (or features) selected/extracted prior to clustering.

Here we introduce sc-CGconv (copula based graph convolution network for single cell clustering), a stepwise robust unsupervised feature extraction and clustering approach that formulates and aggregates cell-cell relationships using copula correlation (Ccor), followed by a graph convolution network based clustering approach. sc-CGconv formulates a cell-cell graph using Ccor that is learned by a graph-based artificial intelligence model, graph convolution network. The learned representation (low dimensional embedding) is utilized for cell clustering. sc-CGconv features the following advantages. a. sc-CGconv works with substantially smaller sample sizes to identify homogeneous clusters. b. sc-CGconv can model the expression co-variability of a large number of genes, thereby outperforming state-of-the-art gene selection/extraction methods for clustering. c. sc-CGconv preserves the cell-to-cell variability within the selected gene set by constructing a cell-cell graph through copula correlation measure. d. sc-CGconv provides a topology-preserving embedding of cells in low dimensional space.

The source code and usage information are available at https://github.com/Snehalikalall/CopulaGCN Contact: sumanta.ray@cwi.nl