title: Quantitative analysis of C. elegans transcripts by Nanopore direct-cDNA sequencing reveals terminal hairpins in non trans-spliced mRNAs

abstract:
Nematode mRNA processing involves a trans-splicing step through which a 21nt sequence from a snRNP replaces the original 5 end of the primary transcript. It has long been held that 70% of C. elegans mRNAs are submitted to trans-splicing. Our recent work suggested that the mechanism is more pervasive but not fully captured by mainstream transcriptome sequencing methods.

In this study, we used Oxford Nanopores long-read amplification-free sequencing technology to perform a comprehensive analysis of trans-splicing in worms.

We demonstrated that spliced leader (SL) sequences presence at the 5 end of the messengers affected library preparation and generated sequencing artefacts due to their self-complementarity. Consistent with our previous observations, we found evidence of trans-splicing for most genes. However, a subset of genes appears to be only marginally trans-spliced. These messengers all share the capacity to generate a 5 terminal hairpin structure mimicking the SL structure providing a mechanistic explanation for their non conformity. Altogether, our data provides the most comprehensive quantitative analysis of SL usage to date in C. elegans.


title: Quantitative Cell Proteomic Atlas: Pathway-scale targeted mass spectrometry for high-resolution functional profiling of cell signaling

abstract: In spite of extensive studies of cellular signaling, many fundamental processes such as pathway integration, cross-talk and feedback remain poorly understood. To enable integrated and quantitative measurements of cellular biochemical activities, we have developed the Quantitative Cell Proteomics Atlas (QCPA). QCPA consists of panels of targeted mass spectrometry assays to determine the abundance and stoichiometry of regulatory post-translational modifications of sentinel proteins from most known physiologic and pathogenic signaling pathways in human cells. QCPA currently profiles 1,913 peptides from 469 effectors of cell surface signaling, apoptosis, stress response, gene expression, quiescence, and proliferation. For each protein, QCPA includes triplets of isotopically labeled peptides covering known post-translational regulatory sites to determine their stoichiometries and unmodified protein regions to measure total protein abundance. The QCPA framework incorporates analytes to control for technical variability of sample preparation and mass spectrometric analysis, including TrypQuant, a synthetic substrate for accurate quantification of proteolysis efficiency for proteins containing chemically modified residues. The ability to precisely and accurately quantify most known signaling pathways should enable improved chemoproteomic approaches for the comprehensive analysis of cell signaling and clinical proteomics of diagnostic specimens. QCPA is openly available at https://qcpa.mskcc.org.

category: biochemistry


title: A silk-based platform to stabilize phenylalanine ammonia-lyase (PAL) for orally administered enzyme replacement therapy

abstract: Phenylalanine ammonia-lyase (PAL) has gained attention in recent years for the treatment of phenylketonuria (PKU), a genetic disorder that affects [~]1 in 15,000 individuals globally. However, the enzyme is easily degraded by proteases, unstable at room temperature, and is currently administered in PKU patients as daily subcutaneous injections. We report here the stabilization of the PAL from Anabaena variabilis, which is currently used to formulate pegvaliase, through incorporation in a silk fibroin matrix. The combination with silk stabilizes PAL at 37 {degrees}C. In addition, in vitro studies showed that inclusion in a silk matrix preserves the biological activity of the enzyme in simulated intestinal fluid, enabling the oral administration of pegvaliase for the treatment of PKU.

category: bioengineering


title: ProFeatMap: a customizable tool for 2D feature representation of protein sets

abstract:
SummaryHere, we present ProFeatMap, an intuitive Python-based website allowing to quickly display protein features such as domains, repeats, post-translational modifications location and so forth, into a highly customizable graphical 2D map. Starting from a user-defined protein list, ProFeatMap automatically extracts the main protein features from the Uniprot database. The resulting high-quality maps can help to gain insights, e.g. feature redundancy, that were previously overlooked but which may be useful for the research project. ProFeatMap is freely accessible on the web at: https://profeatmap.pythonanywhere.com/

AvailabilitySource code is freely accessible at https://github.com/profeatmap/ProFeatMap under the GPL license.

Contactbichg@igbmc.fr, yves.nomine@igbmc.fr

Supplementary informationdetailed user guide of ProFeatMap

category: bioinformatics


title: Generative Moment Matching Networks for Genotype Simulation

abstract: The generation of synthetic genomic sequences using neural networks has potential to ameliorate privacy and data sharing concerns and to mitigate potential bias within datasets due to under-representation of some population groups. However, there is not a consensus on which architectures, training procedures, and evaluation metrics should be used when simulating single nucleotide polymorphism (SNP) sequences with neural networks. In this paper, we explore the use of Generative Moment Matching Networks (GMMNs) for SNP simulation, we present some architectural and procedural changes to properly train the networks, and we introduce an evaluation scheme to qualitatively and quantitatively assess the quality of the simulated sequences.


title: Reward Bases: Instantaneous reward revaluation with temporal difference learning

abstract: AO_SCPLOWBSTRACTC_SCPLOWAn influential theory posits that dopaminergic neurons in the mid-brain implement a model-free reinforcement learning algorithm based on temporal difference (TD) learning. A fundamental assumption of this model is that the reward function being optimized is fixed. However, for biological creatures the  reward function can fluctuate substantially over time depending on the internal physiological state of the animal. For instance, food is rewarding when you are hungry, but not when you are satiated. While a variety of experiments have demonstrated that animals can instantly adapt their behaviour when their internal physiological state changes, under current thinking this requires model-based planning since the standard model of TD learning requires retraining from scratch if the reward function changes. Here, we propose a novel and simple extension to TD learning that allows for the zero-shot (instantaneous) generalization to changing reward functions. Mathematically, we show that if we assume the reward function is a linear combination of reward basis vectors, and if we learn a value function for each reward basis using TD learning, then we can recover the true value function by a linear combination of these value function bases. This representational scheme allows instant and perfect generalization to any reward function in the span of the reward basis vectors as well as possesses a straightforward implementation in neural circuitry by parallelizing the standard circuitry required for TD learning. We demonstrate that our algorithm can also reproduce behavioural data on reward revaluation tasks, predict dopamine responses in the nucleus accumbens, as well as learn equally fast as successor representations while requiring much less memory.

category: neuroscience


title: The EGFR/ErbB inhibitor neratinib modifies the neutrophil phosphoproteome and promotes apoptosis and clearance by airway macrophages

abstract: Dysregulated neutrophilic inflammation can be highly destructive in chronic inflammatory diseases due to prolonged neutrophil lifespan and continual release of histotoxic mediators in inflamed tissues. Therapeutic induction of neutrophil apoptosis, an immunologically silent form of cell death, may be beneficial in these diseases, provided that the apoptotic neutrophils are efficiently cleared from the tissue. Our previous research identified ErbB inhibitors as able to induce neutrophil apoptosis and reduce neutrophilic inflammation both in vitro and in vivo (Rahman et al., 2019). Here we extend that work using a clinical ErbB inhibitor, neratinib, which has the potential to be repurposed in inflammatory diseases. We show that neratinib reduces neutrophilic migration to an inflammatory site in zebrafish larvae. Neratinib upregulates efferocytosis and reduces the number of persisting neutrophil corpses in mouse models of acute, but not chronic, lung injury, suggesting the drug may have therapeutic benefits in acute inflammatory settings. Phosphoproteomics analysis of human neutrophils shows that neratinib modifies the phosphorylation of proteins regulating apoptosis, migration and efferocytosis. This work identifies a potential mechanism for neratinib in treating acute lung inflammation by upregulating the clearance of dead neutrophils and, through examination of the neutrophil phosphoproteome, provides important insights into the mechanisms by which this may be occurring.

category: immunology


title: The sphingolipid inhibitor myriocin increases susceptibility of Candida auris to amphotericin B

abstract: The emerging human fungal pathogen Candida auris can cause of hospital outbreaks by transmission between patients and / or medical care staff. It is known for a fast adaption to environmental stress and antifungal treatment. Besides resistance to fluconazole in most isolates, C. auris frequently shows limited susceptibility to echinocandins and even to amphotericin B. Therefore, multidrug resistance (MDR) is frequently found in clinical isolates, leading to a need for new therapeutic targets and compounds. One of those targets could be sphingolipids which are an integral part of the cell membrane and are important for membrane fluidity and the formation of lipid rafts. Here, we show that myriocin-mediated inhibition of the de novo sphingolipid biosynthesis caused severe growth defects in C. auris. Sublethal myriocin concentrations increase fungal susceptibility to amphotericin B and anidulafungin. While the effect is limited for anidulafungin, even isolates that show phenotypic resistance to amphotericin B became susceptible in presence of myriocin. A large-scale screening of 59 clinical isolates validated these initial results and showed that inhibition of the de novo sphingolipid biosynthesis increases C. auris susceptibility to amphotericin B. These observations open new options for future therapeutic strategies and illustrate that sphingolipids might play a pivotal role for the ability of C. auris to develop resistance against amphotericin B.

category: microbiology


title: Mutual inhibition of airway epithelial responses supports viral and fungal co-pathogenesis during coinfection

abstract: Awareness that fungal coinfection complicates viral respiratory infections causing worse disease outcome has recently emerged. The environmental fungus Aspergillus fumigatus (Af) has been reported as the main driver of fungal coinfection in patients suffering from viral infections caused by Cytomegalovirus, Influenza or more recently SARS-CoV2. The airway epithelium is the first common point of contact between inhaled pathogens and the host. Aberrant airway epithelial cell (AEC) responses against fungal challenge have been described in patients susceptible to aspergillosis. Therefore, it is likely that a dysregulation of AEC responses during fungal-viral coinfection represents a potent driver for the development of fungal disease. Here we used an in vitro model of Af-viral infection of AECs to determine outcomes of spore internalisation, killing and viral replication during coinfection. Our data indicate that viral stimulation, while boosting Af uptake by AECs, limits Af spore killing by those cells, favouring fungal persistence and growth. Type I viral-induced interferon release was significantly decreased in the presence of Af hyphal forms suggesting a possible role of Af secreted factors in modulating viral pathogenicity. We next explored the impact of Af challenge in SARS-CoV2 replication within airway epithelial cells using nano-luciferase as a measure of viral replication. We found that Af increased SARS-CoV2 pathogenicity in a strain-dependent manner. Collectively, our findings demonstrate a mutual inhibition of antifungal and antiviral AEC responses during Af-viral coinfection and also suggest that some fungal factors might be key regulators of co-pathogenicity during in lung infection.


title: Combined nanometric and phylogenetic analysis of unique endocytic compartments in Giardia lamblia sheds light on the evolution of endocytosis in Fornicata

abstract: Giardia lamblia, a parasitic protist of the metamonada supergroup, has evolved one of the most diverged endocytic compartment systems investigated so far. Peripheral endocytic compartments, currently known as peripheral vesicles or vacuoles (PVs), perform bulk uptake of fluid phase material which is then digested and sorted either to the cell cytosol or back to the extracellular space. Here, we present a quantitative morphological characterization of these organelles using volumetric electron microscopy and super-resolution microscopy (SRM). We defined a morphological classification for the heterogenous population of PVs and performed a comparative analysis of PVs and endosome-like organelles in representatives of phylogenetically-related taxa, Spironucleus spp. and Tritrichomonas foetus. To investigate the as-yet insufficiently understood connection between PVs and clathrin assemblies in G. lamblia, we further performed an in-depth search for two key elements of the endocytic machinery, clathrin heavy chain (CHC) and clathrin light chain (CLC) across different lineages in Metamonada. Our data point to the loss of a bona fide CLC in the last Fornicata common ancestor (LFCA) with the emergence of a protein analogous to CLC (GlACLC) in the Giardia genus. Taken together, this provides the first comprehensive nanometric view of Giardias endocytic system architecture and sheds light on the evolution of GLACLC analogues in the Fornicata supergroup and, specific to Giardia, as a possible adaptation to the formation and maintenance of stable clathrin assemblies at PVs.

category: cell biology


title: Sustained plumage divergence despite weak genomic differentiation and broad sympatry in sister species of Australian woodswallows (Artamus spp.)

abstract: Plumage divergence can function as a strong premating barrier when species come into secondary contact. When it fails to do so, the results are often genome homogenization and phenotypic hybrids at the zone of contact. This is not the case in the largely sympatric masked- and white-browed woodswallow species (Passeriformes: Artamidae: Artamus spp) complex in Australia where phenotypic integrity is sustained despite no discernible mitochondrial structure. This lack of structure may suggest recent divergence, ongoing gene flow or both, and phenotypic hybrids are reported albeit rarely. Here, we further assessed the population structure and differentiation across the species nuclear genomes using ddRAD-seq. As found in the mitochondrial genome, no structure or divergence within or between the two species is detected in the nuclear genome. This coarse sampling of the genome nonetheless revealed peaks of differentiation around the genes SOX5 and Axin1. Both are involved in the Wnt/{beta}-catenin signaling pathway, which regulates feather development. Reconstruction of demographic history supports a scenario with ongoing gene flow during divergence but involving shifts in direction and magnitude. Our study informs how divergent plumage morphs may arise and be sustained despite whole-genome homogenization and reveals new candidate genes potentially involved in plumage divergence.

category: evolutionary biology


title: Linkage mapping and genome annotation give novel insights into gene family expansions and regional recombination rate variation in the painted lady (Vanessa cardui) butterfly

abstract: Gene family expansions and crossing over are two main mechanisms for the generation of novel genetic variants that can be picked up by natural selection. Here, we developed a high-density, pedigree-based linkage map of the painted lady butterfly (Vanessa cardui) - a non-diapausing, highly polyphagous species famous for its long-distance migratory behavior. We also performed detailed annotations of genes and interspersed repetitive elements for a previously developed genome assembly, characterized species-specific gene family expansions and the relationship between recombination rate variation and genomic features. Identified expanded gene families consisted of clusters of tandem duplications with functions associated with protein and fat metabolism, detoxification, and defense against infection - key functions for the painted ladys unique lifestyle. The detailed assessment of recombination rate variation demonstrated a negative association between recombination rate and chromosome size. Moreover, the recombination landscape along the holocentric chromosomes was bimodal. The regional recombination rate was positively associated with the proportion of short interspersed elements (SINEs), but not the other repeat classes, potentially a consequence of SINEs hijacking the recombination machinery for proliferation. The detailed genetic map developed here will contribute to the understanding of the mechanisms and evolutionary consequences of recombination rate variation in Lepidoptera in general. We conclude that the structure of the painted lady genome has been shaped by a complex interplay between recombination, gene duplications and TE-activity and that specific gene family expansions have been key for the evolution of long-distance migration and the ability to utilize a wide range of host plants.

category: genomics


title: Asymmetric survival in single-cell lineages of cyanobacteria in response to photodamage

abstract: Summary paragraphOxygenic photosynthesis is driven by the coupled action of the light-dependent pigment protein complexes, photosystem I and II, located within the internal thylakoid membrane system. However, photosystem II is known to be prone to photooxidative damage. Thus, photosynthetic organisms have evolved a repair cycle to continuously replace the damaged proteins in photosystem II. However, it has remained difficult to deconvolute the damage and repair processes using traditional ensemble approaches. Here we demonstrate an automated approach using time-lapse fluorescence microscopy and computational image analysis to study the dynamics and effects of photodamage in single cells at sub-cellular resolution in cyanobacteria. By growing cells in a two-dimensional layer, we avoid shading effects, thereby generating uniform and reproducible growth conditions. Using this platform, we analyzed the growth and physiology of multiple strains simultaneously under defined photoinhibitory conditions stimulated by UV-A light. Our results reveal an asymmetric cellular response to photodamage between sibling cells and the generation of an elusive subcellular structure, here named a  photoendosome, derived from the thylakoid which could indicate the presence of a previously unknown photoprotective mechanism. We anticipate these results to be a starting point for further studies to better understand photodamage and repair at the single-cell level.


title: Redox-controlled structural reorganization and flavin strain within the ribonucleotide reductase R2b-NrdI complex monitored by serial femtosecond crystallography

abstract: Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b-NrdI complex, a di-metal carboxylate- flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free-electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. We show that the flavin in the hydroquinone state is under steric strain in the R2b-NrdI protein complex, presumably tuning its redox potential to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O2-interacton site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b- NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.


title: This is GlycoQL

abstract:
MotivationWe have previously designed and implemented a tree-based ontology to represent glycan structures with the aim of searching these structures with a glyco-driven syntax. This resulted in creating the GlySTreeM knowledge-base as a linchpin of the matching procedure and we now introduce a query language, called GlycoQL, for the actual implementation of a glycan structure search.

ResultsThe methodology is described and illustrated with a use-case focused on SARS-CoV-2 spike protein glycosylation. We show how to enhance site annotation with federated queries involving UniProt and GlyConnect, our glycoprotein database.

Availabilitycurrently only available for reviewers at: https://beta.glyconnect.expasy.org/glycoql/

Contactcatherine.hayes@unige.ch; frederique.lisacek@sib.swiss

Supplementary informationSupplementary data are available at https://glyconnect.expasy.org/glystreem/wiki.


title: Biochemical characterization of Pectin Methylesterase Inhibitor 3 from Arabidopsis thaliana

abstract: The Arabidopsis thaliana PECTIN METHYLESTERASE INHIBITOR 3 (PMEI3) gene is frequently used as a tool to manipulate PME activity in vivo, in studies assessing the role of pectin de-methylesterification in the control of cell expansion. One limitation of these studies is that the exact biochemical activity of this protein has not yet been determined. In this manuscript we produced the protein in Pichia pastoris and characterized its activity in vitro. Like other PMEIs, PMEI3 inhibits PME activity in acidic pH conditions for a variety of cell wall extracts and for purified PME preparations, but doesnt affect PME activity at neutral pH. This suggests that the previously observed in vivo effects reflect the inhibition of PME activity at low pH. The protein is remarkable heat stable and shows higher activity against PME3 than against PME2, illustrating how different members of the large PMEI family can differ in their specificities towards PME targets. Finally, application of purified PMEI3 on Arabidopsis thaliana seedlings showed a dose-dependent inhibition of homogalacturonan de-methylesterification and root growth. Purified recombinant PMEI3 is therefore a powerful tool to study the connection between pectin methylesterification and cell expansion.

category: plant biology


title: Whole-body metabolic modelling predicts isoleucine dependency of SARS-CoV-2 replication

abstract: We aimed at investigating host-virus co-metabolism during SARS-CoV-2 infection. Therefore, we extended comprehensive sex-specific, whole-body organ resolved models of human metabolism with the necessary reactions to replicate SARS-CoV-2 in the lung as well as selected peripheral organs. Using this comprehensive host-virus model, we obtained the following key results: 1. The predicted maximal possible virus shedding rate was limited by isoleucine availability. 2. The supported initial viral load depended on the increase in CD4+ T-cells, consistent with the literature. 3. During viral infection, the whole-body metabolism changed including the blood metabolome, which agreed well with metabolomic studies from COVID-19 patients and healthy controls. 4. The virus shedding rate could be reduced by either inhibition of the guanylate kinase 1 or availability of amino acids, e.g., in the diet. 5. The virus variants achieved differed in their maximal possible virus shedding rates, which could be inversely linked to isoleucine occurrences in the sequences. Taken together, this study presents the metabolic crosstalk between host and virus and emphasis the role of amino acid metabolism during SARS-CoV-2 infection, in particular of isoleucine. As such, it provides an example of how computational modelling can complement more canonical approaches to gain insight into host-virus crosstalk and to identify potential therapeutic strategies.

category: systems biology


title: Dysfunctional glymphatic system with disrupted aquaporin-4 expression pattern on astrocytes causes bacterial product accumulation in the CSF during pneumococcal meningitis

abstract:
BackgroundPneumococcal meningitis, inflammation of the meninges due to an infection of the Central Nervous System caused by Streptococcus pneumoniae (the pneumococcus), is the most common form of community-acquired bacterial meningitis globally. The brain is separated from the systemic circulation by the blood-brain barrier (BBB), and meningitis triggers the host immune response increasing the BBB permeability, allowing peripheral immune cells to reach the cerebrospinal fluid (CSF), and increasing debris production. The glymphatic system is a glial-dependent clearance pathway that drives the exchange of compounds between the brain parenchyma and the CSF regulating the waste clearance away from the brain. Aquaporin-4 (AQP4)-water channels on astrocytic end feet regulate the solute transport of the glymphatic system.

MethodsWistar rats, either subjected to pneumococcal meningitis or to artificial-CSF (sham), received Evans blue albumin (EBA) intracisternal. Overall, the meningitis group presented a significant impairment of the glymphatic system by retaining the EBA in the brain without consistently releasing the EBA into the bloodstream compared to the sham non-infected group. Through western blot and immunofluorescence microscopy analysis using rat CSF and brain tissue sections, an increased accumulation of pneumococci was detected over time in the CSF, and because of a loss of drainage between CSF and brain interstitial space, such bacterial accumulation was not observed in the brain parenchyma. Western blot analysis for Iba1, TMEM119 and IFN-{gamma} in rat brain homogenates and NSE in serum showed increased neuroinflammation and neuronal damage in the brain over time during pneumococcal infection. Neurological impairment upon neuronal cell damage caused by meningitis with a malfunctioning glymphatic system was also demonstrated through open-field behavioral tests comparing rats from sham and meningitis groups. Lastly, protein expression analysis of AQP4 revealed no differences in AQP4 between the brains of the rats from the meningitis group and those from the sham non-infected rats. Importantly, confocal microscopy analysis showed a detachment of the astrocytic end feet from the BBB vascular endothelium with consequent misplacement of AQP4-water channels.

ConclusionsThese findings clearly indicate that pneumococcal meningitis decreases the glymphatic systems functionality, increasing the neurotoxic waste debris in the brain ultimately leading to brain-wide neuroinflammation and neuronal damage. Finally, our results clearly showed that during pneumococcal meningitis, the glymphatic system does not function because of a detachment of the astrocytic end feet from the BBB vascular endothelium, which leads to a misplacement of AQP4 with consequent the loss of the AQP4-water channels functionality.


title: Antigen and IL-33 synergistically promote chromatin accessibility at the regulatory regions of pro-inflammatory genes of human skin mast cells

abstract: Antigenic stimulation through cross-linking the IgE receptor and epithelial cell-derived cytokine IL-33 are potent stimuli of mast cell (MC) activation. However, target genes induced by the combined antigen and IL-33 stimulation have not been investigated in human skin mast cells in a genome-wide manner. Furthermore, epigenetic mechanisms by which IL-33 acts synergically with antigenic stimulation to induce gene transcription have not been investigated. We identified the target genes that responded to the combined antigen and IL-33 stimulation using RNA-seq. These genes can be divided into three groups. Genes in the first group are target genes where antigenic stimulation and IL-33 stimulation achieve high levels of synergy in promoting these gene transcription (high synergy target genes). Genes in the second group are target genes for low levels of synergy mediated by the combined antigen and IL-33 stimulation (low synergy target genes). And genes in the third group are not targeted for the antigen and IL-33 synergy (non-synergy target genes). We found that pro-inflammatory cytokine and chemokine genes were enriched among the high synergy target genes. We demonstrate that the combined antigen and IL-33 stimulation-induced or -increased chromatin accessible areas in the regulatory regions of the high synergy target genes but not of the low synergy target genes. Transcription factor binding motif analysis revealed more binding sites for NF-{kappa}B, AP-1, GABPA, and RAP1 in the induced or increased chromatin accessible regions of the high synergy target genes. Our study shines a light on an epigenetic mechanism by which how epithelial cell-derived cytokine IL-33 might cause exacerbation of skin MC-mediated allergic inflammation.


title: Architecture of the biofilm-associated archaic CupE pilus from Pseudomonas aeruginosa

abstract: Chaperone-Usher Pathway (CUP) pili are major adhesins in Gram-negative bacteria, mediating bacterial adherence to biotic and abiotic surfaces. While classical CUP pili have been extensively characterized, little is known about so-called archaic CUP pili, which are phylogenetically widespread and promote biofilm formation by several human pathogens. In this study, we present the electron cryomicroscopy structure of the archaic CupE pilus from the opportunistic human pathogen Pseudomonas aeruginosa. We show that CupE pili consist of CupE1 subunits arranged in a zigzag architecture, with an N-terminal donor {beta}-strand extending from each subunit into the next, where it is anchored by hydrophobic interactions, resulting in an overall flexible pilus arrangement. Imaging CupE pili on the surface of P. aeruginosa cells using electron cryotomography shows that CupE pili adopt variable curvatures in response to their environment, which may facilitate their role in promoting cohesion between bacterial cells. Finally, bioinformatic analysis shows the widespread abundance of cupE genes in isolates of P. aeruginosa and the co-occurrence of cupE with other cup clusters, suggesting interdependence of cup pili in regulating bacterial adherence within biofilms. Taken together, our study provides insights into the architecture of archaic CUP pili and their role in promoting cellular adhesion and biofilm formation in P. aeruginosa.


title: TNFα-induced metabolic reprogramming drives an intrinsic anti-viral state

abstract: Cytokines induce an anti-viral state, yet many of the functional determinants responsible for limiting viral infection are poorly understood. Here, we find that TNF induces significant metabolic remodeling that is critical for its anti-viral activity. Our data demonstrate that TNF activates glycolysis through the induction of muscle-specific hexokinase (HK2). Further, we show that glycolysis is broadly important for TNF-mediated anti-viral defense, as its inhibition attenuates TNFs ability to limit the replication of evolutionarily divergent viruses. Stable-isotope tracing revealed that TNF-mediated glycolytic activation promotes the biosynthesis of UDP-sugars (essential precursors of protein glycosylation) and that inhibition of glycolysis prevents the accumulation of several glycosylated anti-viral proteins. Consistent with the importance of glucose-driven glycosylation, glycosyl-transferase inhibition also attenuated TNFs ability to promote the anti-viral cell state. Collectively, our data indicate that cytokine-mediated metabolic remodeling is an essential component of the anti-viral response.


title: Charting functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders

abstract: Targeted protein degradation is a new pharmacologic paradigm established by drugs that recruit target proteins to E3 ubiquitin ligases via a ternary ligase-degrader-target complex. Based on the structure of the degrader and the neosubstrate, different E3 ligase interfaces are critically involved in this process, thus forming defined "functional hotspots". Understanding disruptive mutations in functional hotspots informs on the architecture of the underlying assembly, and highlights residues prone to cause drug resistance. Until now, their identification was driven by structural methods with limited scalability. Here, we employ haploid genetics to show that hotspot mutations cluster in the substrate receptors of the hijacked ligases and find that type and frequency of mutations are shaped by the essentiality of the harnessed ligase. Intersection with deep mutational scanning data revealed hotspots that are either conserved, or specific for chemically distinct degraders or recruited neosubstrates. Biophysical and structural validation suggest that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we identified and validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and experimentally widely accessible methodology that empowers the characterization of small-molecule degraders and informs on associated resistance mechanisms.

category: molecular biology


title: Specific targeting of plasmids with Argonaute enables genome editing

abstract:
Prokaryotic Argonautes (pAgos) are programmable nucleases involved in cell defense against invading DNA. Recent studies showed that pAgos can bind small single-stranded guide DNAs (gDNAs) to recognize and cleave complementary DNA in vitro. In vivo pAgos preferentially target plasmids, phages and multicopy genetic elements. Here, we reveal that CbAgo nuclease from Clostridium butyricum can be used for genomic DNA cleavage and engineering in bacteria. CbAgo-dependent targeting of genomic loci with plasmid-derived gDNAs promotes recombination between plasmid and chromosomal DNA. Efficient genome cleavage and recombineering depends on the catalytic activity of CbAgo, its interactions with gDNAs, and the extent of homology between plasmid and chromosomal sequences. Specific targeting of plasmids with Argonautes can be used to integrate plasmid-encoded sequences into the chromosome thus enabling genome editing.

One-Sentence SummaryProkaryotic Argonaute nuclease induces DNA interference between plasmid and chromosomal DNA to promote genome recombineering.


title: A congenital hydrocephalus causing mutation in Trim71 results in stem cell differentiation defects through inhibiting Lsd1 mRNA translation

abstract: Congenital hydrocephalus (CH) is a major cause of childhood morbidity. Mono-allelic mutations in Trim71, a conserved stem-cell-specific RNA-binding protein, cause CH, however, molecular basis for pathogenesis mediated by these mutations remains unknown. Here, using mouse embryonic stem cells as a model, we reveal that the mouse R783H mutation (R796H in human) significantly alters Trim71s mRNA substrate specificity and leads to accelerated stem-cell differentiation and neural lineage commitment. The mutant Trim71, but not the wild-type Trim71, binds Lsd1 (Kdm1a) mRNA and represses its translation. Specific inhibition of this repression or a slight increase of Lsd1 in the mutant cells alleviates the defects in stem cell differentiation and neural lineage commitment. These results determine a functionally relevant target of the CH-causing Trim71 mutant that can potentially be a therapeutic target and provide molecular mechanistic insights into the pathogenesis of this disease.


title: RNA localization mechanisms transcend cell morphology

abstract: RNA molecules are localized to specific subcellular regions through interactions between RNA regulatory elements and RNA binding proteins (RBPs). Generally, our knowledge of the mechanistic details behind the localization of a given RNA is restricted to a particular cell type. Here, we show that RNA/RBP interactions that regulate RNA localization in one cell type predictably regulate localization in other cell types with vastly different morphologies. To determine transcriptome-wide RNA spatial distributions across the apicobasal axis of human intestinal epithelial cells, we used our recently developed RNA proximity labeling technique, Halo-seq. We found that mRNAs encoding ribosomal proteins (RP mRNAs) were strongly localized to the basal pole of these cells. Using reporter transcripts and single molecule RNA FISH, we found that pyrimidine-rich TOP motifs in the 5' UTRs of RP mRNAs were su[ffi]cient to drive basal RNA localization. Interestingly, the same TOP motifs were also su[ffi]cient to drive RNA localization to the neurites of mouse neuronal cells. In both cell types, the regulatory activity of the TOP motif was dependent on it being at the extreme 5' end of the transcript, was abolished upon perturbation of the TOP-binding protein LARP1, and was reduced upon inhibition of kinesins. To extend these findings, we compared subcellular RNAseq data from neuronal and epithelial cells. We found that the basal compartment of epithelial cells and the projections of neuronal cells were enriched for highly similar sets of RNAs, indicating that broadly similar mechanisms may be transporting RNAs in both cell types. These findings identify the first RNA element known to regulate RNA localization across the apicobasal axis of epithelial cells, establish LARP1 as an RNA localization regulator, and demonstrate that RNA localization mechanisms cut across cell morphologies.


title: Control of UV damage repair during S phase via Dyrk1A-dependent regulation of cyclin D1/p21waf1cip1 stability

abstract: Nucleotide Excision Repair (NER) eliminates highly-genotoxic solar UV-induced DNA photoproducts that otherwise stimulate malignant melanoma development. Here, a genome-wide CRISPR/Cas9 screen was exploited to uncover novel genes required for efficient NER in human cells. The screen identified Dyrk1A, a dual specificity kinase that phosphorylates the cell cycle regulator and proto-oncoprotein cyclin D1 on threonine 286 (T286), which in turn promotes its timely cytoplasmic relocalization and proteasomal degradation. Failure to modify cyclin D1 on T286 leads to nuclear accumulation of the protein, premature S phase entry, and enhanced cellular proliferation that drives tumourigenesis. We now demonstrate that Dyrk1A knockdown in UV-exposed cells causes significant inhibition of NER uniquely in S phase and reduced cell survival; moreover, both of these aberrant phenotypes were rescued upon concomitant depletion of cyclin D1. Additionally, we show (i) that expression of nonphosphorylatable cyclin D1 (T286A) interferes with NER specifically during S in a CDK-independent manner, and (ii) that this repair defect depends upon co-stabilization/upregulation of the CDK inhibitor p21waf1cip1. Our data indicate that inhibition of NER during S phase might represent a heretofore unappreciated mechanism by which oncogenic nuclear cyclin D1 fosters melanomagenesis.


title: Single Nucleus Sequencing of Human Colon Visceral Smooth Muscle Cells, PDGFRα Cells, and Interstitial Cells of Cajal

abstract:
Background and AimsSmooth muscle cells (SMCs), Interstitial cells of Cajal (ICCs), and PDGFR+ cells (PCs) form a functional syncytium in the bowel known as the  SIP syncytium. The SIP syncytium works in concert with the enteric nervous system (ENS) to coordinate bowel motility. However, our understanding of individual cell types that form this syncytium and how they interact with each other remains limited, with no prior single cell RNAseq analyses focused on human SIP syncytium cells.

MethodsWe analyzed single-nucleus RNA sequencing data from 10,749 human colon SIP syncytium cells (5572 SMC, 372 ICC, and 4805 PC nuclei) derived from 15 individuals.

ResultsConsistent with critical contractile and pacemaker functions and with known ENS interactions, SIP syncytium cell types express many ion channels including mechanosensitive channels in ICCs and PCs. PCs also prominently express ECM-associated genes and the inhibitory neurotransmitter receptor for vasoactive intestinal peptide (VIPR2), a novel finding. We identified two PC clusters that differ in expression of many ion channels and transcriptional regulators. Interestingly, SIP syncytium cells co-express 6 transcription factors (FOS, MEIS1, MEIS2, PBX1, SCMH1, and ZBTB16) that may be part of a combinatorial signature that specifies these cells. Bowel region-specific differences in SIP syncytium gene expression may correlate with regional differences in function, with right (ascending) colon SMCs and PCs expressing more transcriptional regulators and ion channels than SMCs and PCs in left (sigmoid) colon.

ConclusionThese studies provide new insights into SIP syncytium biology that may be valuable for understanding bowel motility disorders and lead to future investigation of highlighted genes and pathways.

SynopsisIn this first single nucleus RNASeq analysis of human SIP syncytium, we identify novel features of SIP syncytium cells, including two types of PDGFR+ cells, a SIP-specific combinatorial transcription factor signature, and colon region differences in gene expression.


title: Cell wound repair requires the coordinated action of linear and branched actin nucleation factors

abstract:
Cells are subjected to a barrage of daily insults that often lead to its cortex being ripped open and requiring immediate repair. An important component of the cell s repair response is the formation of an actomyosin ring at the wound periphery to mediate its closure. Inhibition of linear actin nucleation factors and myosin result in a disrupted contractile apparatus and delayed wound closure. Here we show that branched actin nucleators function as a scaffold to assemble and maintain this contractile actomyosin cable. Removing branched actin leads to the formation of smaller circular actin-myosin structures at the cell cortex and slow wound closure. Removing linear and branched actin results in failed wound closure. Surprisingly, removal of branched actin and myosin results in the formation of parallel linear actin filaments that undergo a chiral swirling movement to close the wound. These results provide insight into actin organization in contractile actomyosin rings and uncover a new mechanism of wound closure.

SummaryHui et al. find that branched actin is required during cell wound repair to serve as a scaffold to anchor the contractile actomyosin cable at the wound periphery. Inhibition of branched actin and myosin results in parallel linear filaments that swirl to close the wound, uncovering a new mechanism for cell wound repair.


title: DMT1 bridges endosomes and mitochondria to modulate mitochondrial iron translocation

abstract:
Transient "kiss-and-run" endosome-mitochondria interactions can mediate mitochondrial iron translocation (MIT) but the associated mechanisms are still elusive. We show that Divalent Metal Transporter 1 (DMT1) modulates MIT via endosome-mitochondria interactions in invasive MDA-MB-231, but not in non-invasive T47D breast cancer cells. CRISPR/Cas9-based DMT1 knockout (KO) stable cells were used to demonstrate that DMT1 regulates MIT, endosomal speed, and labile iron pool (LIP) levels only in MDA-MB-231. DMT1 silencing increases PINK1/Parkin mitophagy markers, the autophagy marker LC3B, as well as mitochondrial ferritin in MDA-MB-231, but not in T47D. Strikingly, re-expression of DMT1 in MDA-MB-231 DMT KO cells rescues all protein levels evaluated. DMT1 silencing decreases Tom20 colocalization with PMPCB, a DMT1 interactor that regulates mitophagy hyperactivation. In MDA-MB-231 both mitochondrial metabolism and invasion were impaired by DMT1 silencing and rescued by DMT1 re-expression. DMT1 acts as a bridge between endosomes and mitochondria to support higher MIT/lower LIP levels, which are necessary for sustaining mitochondrial bioenergetics and invasive cancer cell migration.

SummaryCellular iron metabolism is tightly regulated, and cancer cells rely on mitochondrial iron for malignancy. Here, we report that the divalent metal transporter DMT1 serves as a bridge between endosomes and mitochondria regulating mitochondrial iron translocation in breast cancer cells.


title: Infection with Listeria monocytogenes alters the placental transcriptome and eicosanome

abstract:
IntroductionPlacental infection and inflammation are risk factors for adverse pregnancy outcomes, including preterm labor. However, the mechanisms underlying these outcomes are poorly understood.

MethodsTo study this response, we have employed a pregnant mouse model of placental infection caused by the bacterial pathogen Listeria monocyogenes, which infects the human placenta. Through in vivo bioluminescence imaging, we confirm the presence of placental infection and quantify relative infection levels. Infected and control placentas were collected on embryonic day 18 for RNA sequencing to evaluate gene expression signatures associated with infection by Listeria.

ResultsWe identified an enrichment of genes associated with eicosanoid biosynthesis, suggesting an increase in eicosanoid production in infected tissues. Because of the known importance of eicosanoids in inflammation and timing of labor, we quantified eicosanoid levels in infected and uninfected placentas using semi-targeted mass spectrometry. We found a significant increase in the concentrations of several key eicosanoids: leukotriene B4, lipoxin A4, prostaglandin A2, prostaglandin D2, and eicosatrienoic acid.

DiscussionOur study provides a likely explanation for dysregulation of the timing of labor following placental infection. Further, our results suggest potential biomarkers of placental pathology and targets for clinical intervention.

category: developmental biology


title: Incomplete activation of developmentally required genes Alyref1 and Gabpb1 leads to preimplantation arrest in cloned mouse embryos

abstract: Differentiated cell nuclei can be reprogrammed after nuclear transfer (NT) to oocytes and the produced NT embryos can give rise to cloned animals. However, development of NT embryos is often hampered by recurrent reprogramming failures, including the incomplete activation of developmental genes, yet specific genes responsible for the arrest of NT embryos are not well understood. Here, we searched for developmentally important genes among the reprogramming-resistant H3K9me3-repressed genes, and identified Alyref and Gabpb1 by siRNA screening. Gene knockout of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. Single embryo RNA-seq revealed that Alyref is needed for the formation of inner cell mass. The supplement of Alyref and Gabpb1 by mRNA injection supported efficient preimplantation development of cloned embryos. Thus, our study shows that the H3K9me3-repressed genes contain developmentally required genes and the incomplete activation of such genes results in preimplantation arrest of cloned embryos.


title: Metabolic compatibility and the rarity of prokaryote endosymbioses

abstract: The evolution of the mitochondria was a significant event that gave rise to the eukaryotic lineage and most large complex life. Central to the origins of the mitochondria was an endosymbiosis between prokaryotes. Yet, despite the potential benefits that can stem from a prokaryotic endosymbiosis, their modern occurrence is exceptionally rare. While many factors may contribute to their rarity, we lack methods for estimating the extent to which they constrain the appearance of a prokaryotic endosymbiosis. Here, we address this knowledge gap by examining the role of metabolic compatibility between a prokaryotic host and endosymbiont. We use genome-scale metabolic models from three different databases (AGORA, KBase, and CarveMe) to assess the viability, fitness, and adaptability of potential prokaryotic endosymbioses. We find that while more than half of host-endosymbiont pairings are metabolically viable, the resulting endosymbioses have reduced growth rates compared to their ancestral metabolisms and are unlikely to gain mutations to overcome these fitness differences. In spite of these challenges, we do find that they may be more robust in the face of environmental perturbations at least in comparison with the ancestral host metabolism lineages. Our results provide a critical set of null models and expectations for understanding the forces that shape the structure of prokaryotic life.


title: Human genomic DNA is widely interspersed with i-motif structures

abstract: DNA i-motif structures are formed in the nucleus of human cells and are believed to provide critical genomic regulation. While the existence of i-motif structures in human cells has been demonstrated by immunofluorescent staining and by characterisation of select model genes, the abundance and distribution of such structures in the human genome has remained unclear. Here we utilize high affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in human genomic DNA. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach identified over 650,000 i-motif structures in human genomic DNA. The i-motif structures are widely distributed throughout the human genome and are common among highly expressed genes and in genes upregulated in G0/G1 cell cycle phase. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA.

category: genetics


title: Cell division drives DNA methylation loss in late-replicating domains in primary human cells

abstract: DNA methylation undergoes dramatic age-related changes, first described more than four decades ago1-4. Loss of DNA methylation at late-replicating regions of the genome attached to the nuclear lamina advances with age in normal tissues, and is further exacerbated in cancer5-7. We present here the first experimental evidence that this DNA hypomethylation is directly driven by proliferation-associated DNA replication. Loss of DNA methylation at low-density CpGs in A:T-rich, partially methylated domains (PMD solo-WCGWs), tracks cumulative population doublings in primary cell culture. Cell cycle deceleration resulted in a proportional decrease in the rate of DNA hypomethylation. Blocking DNA replication via Mitomycin C treatment halted methylation loss. Loss of methylation continued unabated after TERT immortalization until finally reaching a severely hypomethylated equilibrium. Ambient oxygen culture conditions increased the rate of methylation loss compared to low-oxygen conditions, suggesting that some methylation loss may occur during unscheduled, oxidative damage repair-associated DNA synthesis. Finally, we present and validate a model to estimate the relative cumulative replicative histories of human cells, which we call "RepliTali" (Replication Times Accumulated in Lifetime).


title: Investigating CRISPR spacer targets and their impact on genomic diversification of Streptococcus mutans

abstract:
CRISPR-Cas is a bacterial immune system that restricts the acquisition of mobile DNA elements. These systems provide immunity against foreign DNA by encoding CRISPR spacers that help target DNA if it re-enters the cell. In this way, CRISPR spacers are a type of molecular tape recorder of foreign DNA encountered by the host microorganism. Here, we extracted [~]8,000 CRISPR spacers from a collection of over three hundred Streptococcus mutans genomes. Phage DNA is a major target of S. mutans spacers. Strains have also generated immunity against mobile DNA elements such as plasmids and integrative and conjugative elements. There may also be considerable immunity generated against bacterial DNA, although the relative contribution of self-targeting versus bona fide intra- or inter-species targeting needs to be investigated further. While there was clear evidence that these systems have acquired immunity against foreign DNA, there appeared to be minimal impact on horizontal gene transfer (HGT) constraints on a species-level. There was little or no impact on genome size, GC content and  openness of the pangenome when comparing between S. mutans strains with low or high CRISPR spacer loads. In summary, while there is evidence of CRISPR spacer acquisition against self and foreign DNA, CRISPR-Cas does not act as a barrier on the expansion of the S. mutans accessory genome.

Impact StatementCRISPR-Cas is a widespread bacterial immune system that has been repurposed as a molecular biology tool. This study investigates the role of these systems in the biology and evolution of the dental caries pathogen Streptococcus mutans. CRISPR spacers, that encode immunity against foreign DNA, were extracted from over three hundred S. mutans isolates. Sequence analysis showed that the CRISPR spacers match against phage, mobile element, and bacterial DNA. This shows that S. mutans is actively acquiring immunity against horizontally acquired DNA. However, additional analysis revealed little to no impact of CRISPR-Cas systems on diversification of the S. mutans genome. This suggests that while these systems are actively acquiring CRISPR spacers to defend against foreign DNA, the overall impact on the S. mutans genome might be small.

Data SummarySupporting data provided on the Github platform: https://github.com/theshieldslab/Streptococcus-mutans-CRISPR-Spacers-Analysis

The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.


title: Genomic surveillance unfolds the dynamics of SARS-CoV-2 transmission and divergence in Bangladesh over the past two years

abstract:
The highly pathogenic virus SARS-CoV-2 has shattered the healthcare system of the world causing the COVID-19 pandemic since first detected in Wuhan, China. Therefore, scrutinizing the genome structure and tracing the transmission of the virus has gained enormous interest in designing appropriate intervention strategies to control the pandemic. In this report, we examined 4622 sequences from Bangladesh and found that they belonged to thirty-five major PANGO lineages, while Delta alone accounted for 39%, and 78% were from just four primary lineages. Our research has also shown Dhaka to be the hub of viral transmission and observed the virus spreading back and forth across the country at different times by building a transmission network. The analysis resulted in 7659 unique mutations, with an average of 24.61 missense mutations per sequence. Moreover, our analysis of genetic diversity and mutation patterns revealed that eight genes were under negative selection pressure to purify deleterious mutations, while three genes were under positive selection pressure.

ImportanceWith 29,122 deaths, 1.95 million infections and a shattered healthcare system from SARS-CoV-2 in Bangladesh, the only way to avoid further complications is to break the transmission network of the virus. Therefore, it is vital to shedding light on the transmission, divergence, mutations, and emergence of new variants using genomic data analyses and surveillance. Here, we present the geographic and temporal distribution of different SARS-CoV-2 variants throughout Bangladesh over the past two years, and their current prevalence. Further, we have developed a transmission network of viral spreads, which in turn will help take intervention measures. Then we analyzed all the mutations that occurred and their effect on evolution as well as the currently present mutations that could trigger a new variant of concern. In short, together with an ongoing genomic surveillance program, these data will help to better understand SARS-CoV-2, its evolution, and pandemic characteristics in Bangladesh.


title: High-throughput proteomics of nanogram-scale samples with Zeno SWATH DIA

abstract: The ability to conduct high-quality proteomic experiments in high throughput has opened new avenues in clinical research, drug discovery, and systems biology. Next to an increase in quantitative precision, recent developments in high-throughput proteomics have also gained proteomic depth, to the extent that earlier gaps between classic and high-throughput experiments have significantly narrowed. Here we introduce and benchmark Zeno SWATH, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) in order to increase proteomic depth and dynamic range in proteomic experiments. Combined with the high acquisition speed, these gains in sensitivity are particularly attractive for conducting high-throughput proteomics experiments with high chromatographic flow rates and fast gradients. We demonstrate that when combined with either micro-flow- or analytical-flow-rate chromatography, Zeno SWATH increases protein identification in complex samples 5- to 10-fold when compared to current SWATH acquisition methods on the same instrument. Using 20-min micro-flow chromatography, Zeno SWATH identified > 6,000 proteins from a 62.5 ng load of human cell lysate with more than 5,000 proteins consistently quantified in triplicate injections with a median CV of 6%. Using 5-min analytical-flow-rate chromatography (800 {micro}l/min), Zeno SWATH identified 4,907 proteins from a triplicate injection of 2 {micro}g of a human cell lysate; or more than 3,000 proteins from 250 ng tryptic digest. Zeno SWATH hence facilitates precise proteomic experiments with small sample amounts using a fast and robust high flow-rate chromatographic method, broadening the application space that requires precise proteomic experiments on a large scale.


title: AlphaFold2 can predict structural and phenotypic effects of single mutations

abstract: AlphaFold2 (AF2) is a promising tool for structural biology, but is it sufficiently accurate to predict the effect of missense mutations? We find that structural variation between closely related (1-3 mutations) protein pairs is correlated across experimental and AF2-predicted structures ([~]90,000 pairs). Analysis of [~]10,000 predicted structures from three high-throughput studies linking sequence and phenotype (fluorescence, foldability, and catalysis) demonstrates that AF2 can predict the phenotypic effect of missense mutations.


title: p66Shc is an apoptotic rheostat whose targeted ROS inhibition improves MI outcomes

abstract: p66Shc is an oxidoreductase that responds to cell stress by translocating to mitochondria, where p66Shc produces pro-apoptotic reactive oxygen species (ROS). This study identifies ROS-active p66Shc as a monomer that produces superoxide anion independent of metal ions, inhibits cytochrome c peroxidase, and is regulated by environmental condition-induced structural changes. p66Shc anti-apoptotic functions, including: cytochrome c reduction, increased electron transport chain activity, and caspase cascade inhibition were also discovered. This study also demonstrates that p66Shc is a stress-dependent rheostat of apoptosis, regulated by p66Shc-mortalin complexes. These complexes decrease pro-apoptotic ROS production, without blocking p66Shc-mediated cytochrome c reduction. However, stress disrupts p66Shc-mortalin interactions, promoting apoptosis. Tipping p66Shcs apoptotic balance toward anti-apoptotic functions by genetic knockdown or p66Shc-selective ROS inhibition decreased pro-apoptotic effects and improved outcomes in zebrafish myocardial infarction models, representing a potential new myocardial infarction treatment with promising results.


title: Adaptive immunity is required for durable responses to alectinib in murine models of EML4-ALK lung cancer

abstract:
PurposeLung cancers bearing oncogenic EML4-ALK fusions respond to targeted tyrosine kinase inhibitors (TKIs; e.g. alectinib), with variation in the degree of shrinkage and duration of treatment (DOT). We previously demonstrated a positive association of a TKI-induced interferon gamma (IFN{gamma}) transcriptional response with DOT in EGFR-mutant lung cancers. Herein, we used murine models of EML4-ALK lung cancer to test a role for host immunity in the therapeutic response to alectinib.

Experimental DesignThree murine EML4-ALK cell lines (EA1, EA2, EA3) were implanted orthotopically into the lungs of immunocompetent and immunodeficient mice and treated with alectinib. Tumor volumes were serially measured by CT. Immune cell content was measured by flow cytometry, multispectral immunofluorescence and CyTOF. Transcriptional responses to alectinib were assessed by RNAseq and secreted chemokines were measured by ELISA.

ResultsAll cell lines were sensitive to alectinib in vitro. EA1 and EA3 tumors retained residual disease that rapidly progressed upon termination of treatment while EA2 tumors were eliminated by TKI treatment. Alectinib induced inflammatory transcriptional programs and multiple chemokines in all cell lines while untreated tumors exhibited distinct baseline chemokine expression patterns and content of CD8+ T cells and myeloid subsets. When propagated in immune-deficient mice, all three cell line-derived lung tumor models exhibited significant shrinkage followed by prompt progression despite continuous alectinib treatment.

ConclusionsThe findings support an hypothesis that host and TKI-stimulated production of chemokines by tumor cells promotes functional engagement of adaptive immune cells within the tumor microenvironment that enhances the durability and depth of TKI response.

Statement of Translational RelevancePatients with metastatic lung cancer harboring ALK fusions are treated with targeted tyrosine kinase inhibitors (TKI) in the first line setting. Despite bearing the same driver oncogene, patients experience a range of tumor burden reduction and variable amounts of residual disease. Residual disease burden associates with patient survival and contributes to the emergence of drug resistance yielding treatment failure. The factors mediating this differential response to TKI and residual disease are incompletely understood. Our group has developed a panel of murine ALK driven lung cancer cell lines that reproducibly show differences in the depth and duration of response when implanted into immunocompetent mice. Data using this model indicate that the presence of CD8+ T cells is a major contributor to the depth and duration of response. These models will be critical in developing rational combination therapies to augment the immune microenvironment engagement along with TKIs to improve outcomes for these patients.

category: cancer biology


title: The evolution of a counter-defense mechanism in a virus constrains its host range

abstract:
Bacteria use diverse immunity mechanisms to defend themselves against their viral predators, bacteriophages. In turn, phages can acquire counter-defense systems, but it remains unclear how such mechanisms arise and what factors constrain viral evolution. Here, we experimentally evolved T4 phage to overcome a phage-defensive toxin-antitoxin system, toxIN, in E. coli. Through recombination, T4 rapidly acquires segmental amplifications of a previously uncharacterized gene, now named tifA, encoding an inhibitor of the toxin, ToxN. These amplifications subsequently drive large deletions elsewhere in T4s genome to maintain a genome size compatible with capsid packaging. The deleted regions include accessory genes that help T4 overcome defense systems in alternative hosts. Thus, our results reveal a trade-off in viral evolution; the emergence of one counter-defense mechanism can lead to loss of other such mechanisms, thereby constraining host range. We propose that the accessory genomes of viruses reflect the integrated evolutionary history of the hosts they infected.

HighlightsO_LIExperimentally evolved T4 to overcome E. coli toxIN, a phage-defensive TA system
C_LIO_LIDiscovered TifA, a phage-encoded protein inhibitor of ToxN
C_LIO_LIAmplification of the tifA locus drives large deletions elsewhere in the T4 genome
C_LIO_LIDeleted genes in evolved T4 clones include those necessary to infect alternative hosts
C_LI


title: Targeted Sequencing of Alzheimers Disease Genes in Gujarat Cohort

abstract: Alzheimers Disease (AD) is the most common type of dementia affecting cognitive and behavioural functions. Its a complex disease that results from the modest effect of gene interaction and environmental factors, as a result of which its exact pathogenesis is still unknown. In the present study, we have performed an association study of exonic regions of 94 genes with AD using the Haloplex target enrichment method and genotyping of 4 genes NOTCH4(rs367398), NEP(rs61760379), APOE (rs121918398), and ATP7B(rs1801243) using PCR RFLP technique. In this study, we screened 32 AD cases and 11 control samples. We have identified 19 variants assigned to 16 genes that were significantly associated with AD with a p-value < 0.05. However, no variants have passed multiple test corrections except NOTCH4/rs367398 genotyped using the PCR-RFLP approach. The functional enrichment analysis of the significant genes for the disease indicates that the genes are significantly involved in AD. Also, functional enrichment analysis for biological processes using ClueGo and network analysis using the STRING database indicated that the genes are directly and indirectly playing a role in AD pathogenesis. The eQTL analysis of the variants with GTEx dataset indicate that out of 19 variants ACHE/rs1799806 and NOTCH4/rs367398 variants is showing significant eQTL for the neighbouring genes for different brain regions and whole blood. Regardless, additional studies with a large cohort are required to establish the role and prevalence of these variants in the local population of Gujarat.


title: Regulation of inflammation and protection against invasive pneumococcal infection by the long pentraxin PTX3

abstract: Streptococcus pneumoniae is a major pathogen in children, elderly subjects and immunodeficient patients. PTX3 is a fluid phase pattern recognition molecule (PRM) involved in resistance to selected microbial agents and in regulation of inflammation. The present study was designed to assess the role of PTX3 in invasive pneumococcal infection. In a murine model of invasive pneumococcal infection, PTX3 was strongly induced in non-hematopoietic (particularly, endothelial) cells. The IL-1{beta}/MyD88 axis played a major role in regulation of the Ptx3 gene expression. Ptx3-/- mice were more susceptible to invasive pneumococcal infection. Although high concentrations of PTX3 had opsonic activity in vitro, no evidence of PTX3-enhanced phagocytosis was obtained in vivo. In contrast, Ptx3-deficient mice showed enhanced recruitment of neutrophils and inflammation. Using P-selectin deficient mice, we found that protection against pneumococcus was dependent upon PTX3-mediated regulation of neutrophil inflammation. In humans, PTX3 genetic polymorphisms were associated with invasive pneumococcal infections. Thus, this fluid phase PRM plays an important role in tuning inflammation and resistance against invasive pneumococcal infection.


title: A genetic model for in vivo proximity labeling of the mammalian secretome

abstract: Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ communication. Enzyme-catalyzed proximity labeling enables the identification of proteins within a specific cellular compartment. Here, we report a BirA*G3 mouse strain that enables CRE-dependent promiscuous biotinylation of proteins trafficking through the endoplasmic reticulum. When broadly activated throughout the mouse, widespread labeling of proteins was observed within the secretory pathway. Streptavidin affinity purification and peptide mapping by quantitative mass spectrometry (MS) proteomics revealed organ-specific secretory profiles and serum trafficking. As expected, secretory proteomes were highly enriched for signal peptide-containing proteins, highlighting both conventional and non-conventional secretory processes, and ectodomain shedding. Lower-abundance proteins with hormone-like properties were recovered and validated using orthogonal approaches. Hepatocyte-specific activation of BirA*G3 highlighted liver-specific biotinylated secretome profiles. The BirA*G3 mouse model demonstrates enhanced labeling efficiency and tissue specificity over viral transduction approaches and will facilitate a deeper understanding of secretory protein interplay in development, and healthy and diseased adult states.


title: A nanobody toolbox to investigate localisation and dynamics of Drosophila titins

abstract: Measuring the positions and dynamics of proteins in intact tissues or whole animals is key to understand protein function. However, to date this is still a challenging task, as accessibility of large antibodies to dense tissues is often limited and fluorescent proteins inserted close to a domain of interest may affect function of the tagged protein. These complications are particularly present in the muscle sarcomere, arguably one of the most protein dense structures in nature, which makes studying morphogenesis at molecular resolution challenging. Here, we have employed an efficient pipeline to generate a nanobody toolbox specifically recognising various domains of two large Drosophila titin homologs, Sallimus and Projectin. We demonstrate the superior labelling qualities of our nanobodies compared to conventional antibodies in intact muscle tissue. Applying our nanobody toolbox to larval muscles revealed a gigantic Sallimus isoform stretched more than 2 {micro}m to bridge the sarcomeric I-band. Furthermore, N- and C-terminal nanobodies against Projectin identified an unexpected polar orientation of Projectin covering the myosin filaments in larval muscles. Finally, expression of a Sallimus nanobody in living larval muscles confirmed the high affinity binding of nanobodies to target epitopes in living tissue and hence demonstrated their power to reveal the in vivo dynamics of sarcomeric protein domains. Together, our toolbox substantiates the multiple advantages of nanobodies to study sarcomere biology. It may inspire the generation of similar toolboxes for other large protein complexes in Drosophila or mammals.


title: Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nano-architecture in flight muscles

abstract: Sarcomeres are the force producing units of all striated muscles. Their nanoarchitecture critically depends on the large titin protein, which in vertebrates spans from the sarcomeric Z-disc to the M-band and hence links actin and myosin filaments stably together. This ensures sarcomeric integrity and determines the length of vertebrate sarcomeres. However, the instructive role of titins for sarcomeric architecture outside of vertebrates is not as well understood. Here, we used a series of nanobodies, the Drosophila titin nanobody toolbox, recognising specific domains of the two Drosophila titin homologs Sallimus and Projectin to determine their precise location in intact flight muscles. By combining nanobodies with DNA-PAINT super- resolution microscopy, we found that, similar to vertebrate titin, Sallimus bridges across the flight muscle I-band, whereas Projectin is located at the beginning of the A- band. Interestingly, the ends of both proteins overlap at the I-band/A-band border, revealing a staggered organisation of the two Drosophila titin homologs. This architecture may help to stably anchor Sallimus at the myosin filament and hence ensure efficient force transduction during flight.


title: Rescue of Escherichia coli auxotrophy by de novo small proteins

abstract: Increasing numbers of small proteins with diverse physiological roles are being identified and characterized in both prokaryotic and eukaryotic systems, but the origins and evolution of these proteins remain unclear. Recent genomic sequence analyses in several organisms suggest that new functions encoded by small open reading frames (sORFs) may emerge de novo from noncoding sequences. However, experimental data demonstrating if and how randomly generated sORFs can confer beneficial effects to cells are limited. Here we show that by up-regulating hisB expression, de novo small proteins ([≤] 50 amino acids in length) selected from random sequence libraries can rescue Escherichia coli cells that lack the conditionally essential SerB enzyme. The recovered small proteins are hydrophobic and confer their rescue effect by binding to the 5 end regulatory region of the his operon mRNA, suggesting that protein binding promotes structural rearrangements of the RNA that allow increased hisB expression. This study adds RNA regulatory elements as another interacting partner for de novo proteins isolated from random sequence libraries, and provides further experimental evidence that small proteins with selective benefits can originate from the expression of nonfunctional sequences.


title: Co-expression of recombinant human collagen α1(III) chain with viral prolyl 4-Hydroxylase in P. pastoris GS115

abstract: Prolyl 4-hydroxylase (P4H) is essential to maintain the stable triple-helix structure and function of human collagen 1([SHcy]) chain (COL3A1). To obtain hydroxylated human COL3A1, the human COL3A1 and the viral P4H A085R were co-expressed in P. pastoris GS115. The sequence of human COL3A1 without N-terminal and C-terminal was selected for expression. Colony PCR analysis and sequencing after transfection showed that the target gene had inserted successfully. Real-time quantitative PCR (RT-qPCR) indicated that human COL3A1 and P4H were expressed at the mRNA levels. SDS-PAGE and Western blotting analysis of supernatant from the recombinant methylotrophic yest culture showed that recombinant human COL3A1 (rhCOL3A1) was secreted into the culture medium with an apparent molecular mass of approximately 130 kDa. It was noted that the rhCOL3A1 expession quantity was higest at 120 h of induction. Furthermore, mass spectrometry analysis demonstrated that the rhCOL3A1 was expressed successfully. His-tagged rhCOL3A1 protein was purified by Ni-affinity column.


title: RADGENNETS: DEEP LEARNING-BASED RADIOGENOMICS MODEL FOR GENE MUTATION PREDICTION IN LUNG CANCER

abstract: AO_SCPLOWBSTRACTC_SCPLOWIn this paper, we present our methodology that can be used for predicting gene mutation in patients with non-small cell lung cancer (NSCLC). There are three major types of gene mutations that a NSCLC patients gene structure can change to: epidermal growth factor receptor (EGFR), Kirsten rat sarcoma virus (KRAS), and Anaplastic lymphoma kinase (ALK). We worked with the clinical and genomics data for each patient as well CT scans. We preprocessed all of the data and then built a novel pipeline to integrate both the image and tabular data. We built a novel pipeline that used a fusion of Convolutional Neural Networks and Dense Neural Networks. Also, using a search approach, we pick an ensemble of deep learning models to classify the separate gene mutations. These models include EfficientNets, SENet, and ResNeXt WSL, among others. Our model achieved a high area under curve (AUC) score of 94% in detecting gene mutation.


title: RadGenNets: Deep Learning-Based Radiogenomics Model For Gene Mutation Prediction In Lung Cancer


title: Rapid microglial phenotype changes modulate neuronal networks and sharp wave-ripple activity in acute slice preparations

abstract: Microglia, the main immune cells of the central nervous system (CNS) have long been known for their remarkable sensitivity to tissue disturbance or injury, but its implications to the interpretation of results from ex vivo models of the CNS have remained largely unclear to date. To this end, we have followed the course of microglial phenotype changes and contribution to neuronal network organisation and functioning in acute brain slices prepared from mice, widely used to study the physiology of the brain from nanoscale events to complex circuits. We found that upon acute slice preparation, microglial cell bodies dislocate and migrate towards the surface of slices, alongside with rapidly progressing morphological changes and altered interactions with neurons. This is accompanied by gradual depolarization and downregulation of P2Y12 receptors, which are instrumental for microglia-neuron communication. Quantitative post-embedding immunofluorescent labelling reveals time-dependent increase in the number of excitatory and inhibitory synapses upon slice preparation in the cerebral cortex, which are markedly influenced by microglia. In line with this, the absence of microglia diminishes the incidence, amplitude and frequency of sharp wave-ripple activity in hippocampal slices. Collectively, our data suggest that microglia are not only inherent modulators of complex neuronal networks, but their specific actions on network reorganisation and functioning must be taken into account when learning lessons from ex vivo models of the CNS.


title: Pneumococcal Evasion of Antibiotics via Metabolic Adaptation During Infection

abstract: Streptococcus pneumoniae is a major human pathogen of global health concern, causing a range of mild to severe infections, including acute otitis media, pneumonia, sepsis, and meningitis. The rapid emergence of antibiotic resistance among S. pneumoniae isolates poses a serious public health problem worldwide. Resistant pneumococcal strains have rendered the mainstay treatment with beta-lactams, fluoroquinolones, and macrolides, ineffective. Antibiotic resistance in S. pneumoniae has spread globally via the emergence of de novo mutations and horizontal transfer of resistance. Fluoroquinolone resistance in S. pneumoniae is an intriguing case because the prevalence of fluoroquinolone resistance does not correlate with increasing usage, as is often the case with other classes of antibiotics. In this study, we demonstrated that deleterious fitness costs constrain the emergence of individual fluoroquinolone resistance mutations in either topoisomerase IV or gyrase A in S. pneumoniae. Generation of double point mutations in the target enzymes in topoisomerase IV and gyrase A conferred high-level fluoroquinolone resistance while restoring fitness comparable to the sensitive wild-type. During an in vivo model of antibiotic resistance evolution, S. pneumoniae was able to circumvent deleterious fitness costs imposed by resistance determinants through development of antibiotic tolerance through metabolic adaptation that reduced the production of reactive oxygen species, an effect that could be recapitulated pharmacologically. The metabolic mutants conferring tolerance resulted in a fitness benefit during infection following antibiotic treatment with fluroquinolones. These data suggest that emergence of fluoroquinolone resistance is tightly constrained in S. pneumoniae by host fitness tradeoffs and that mutational pathways involving metabolic networks to enable tolerance phenotypes may be an important contributor to the evasion of antibiotic mediated killing.


title: Context-dependent sensory modulation underlies Bayesian vocal sequence perception

abstract:
Vocal communication in both songbirds and humans relies on categorical perception of smoothly varying acoustic spaces. Vocal perception can be biased by expectation and context, but the mechanisms of this bias are not well understood. We developed a behavioral task in which songbirds, European starlings, are trained to to classify smoothly varying song syllables in the context of predictive syllable sequences. We find that syllable-sequence predictability biases perceptual categorization following a Bayesian model of probabilistic information integration. We then recorded from populations of neurons in the auditory forebrain while birds actively categorized song syllables, observing large proportions of neurons that track the smoothly varying natural feature space of syllable categories. We observe that predictive information in the syllable sequences dynamically modulates sensory neural representations. These results support a Bayesian model of perception where predictive information acts to dynamically reallocate sensory neural resources, sharpening acuity (i.e. the likelihood) in high-probability regions of stimulus space.

One-Sentence SummaryPredictive information in vocal sequences biases Bayesian categorical perception through rapid sensory reorganization.

Graphical Abstract

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title: What the hippocampus tells the HPA axis: Hippocampal output attenuates acute stress responses via disynaptic inhibition of CRF+ PVN neurons

abstract: The hippocampus exerts inhibitory feedback on the release of glucocorticoids. Because the major hippocampal efferent projections are excitatory, it has been hypothesized that this feedback inhibition is mediated by populations of inhibitory neurons in the hypothalamus or elsewhere. These regions would be excited by hippocampal efferents and project to corticotropin-releasing factor (CRF) cells in the paraventricular nucleus of the hypothalamus (PVN). A direct demonstration of the synaptic responses elicited by hippocampal outputs in PVN cells or upstream GABAergic interneurons has not been provided previously. Here, we used viral vectors to express channelrhodopsin (ChR) and enhanced yellow fluorescent protein (EYFP) in pyramidal cells in the ventral hippocampus (vHip) in mice expressing tdTomato in GABA-or CRF-expressing neurons. We observed dense innervation of the bed nucleus of the stria terminalis (BNST) by labelled vHip axons and sparse labeling within the PVN. Using whole-cell voltage-clamp recording in parasagittal brain slices containing the BNST and PVN, photostimulation of vHip terminals elicited monosynaptic excitatory postsynaptic currents (EPSCs) and disynaptic inhibitory postsynaptic potentials (IPSCs) in both CRF+ and GAD+ cells. The balance between synaptic excitation and inhibition were maintained in CRF+ cells during 20 Hz stimulus trains. Photostimulation of hippocampal afferents to the BNST and PVN in vivo inhibited the rise in blood glucocorticoid levels produced by acute restraint stress. We thus provide functional evidence that hippocampal output to the BNST results in a net inhibition of the hypothalamic-pituitary axis, gaining further mechanistic insights into this process using methods with enhanced spatial and temporal resolution.


title: Hormones do not make the mole-rat: no steroid hormone signatures of subordinate behavioral phenotypes

abstract: In some cooperatively breeding groups, individuals have distinct behavioral characteristics that are often stable and predictable across time. However, in others, like the eusocial naked mole-rat, evidence for behavioral phenotypes is ambiguous. Here, we study whether the naked mole-rat can be divided into discrete phenotypes and if circulating hormone levels underpin these differences. Naked mole-rat colonies consist of a single breeding female and dozens to hundreds of non-reproductive subordinates. The subordinates can potentially be divided into soldiers, who defend the colony; workers, who maintain it; and dispersers, who want to leave it. We established six colonies de novo, tracked them over three years, and assessed the behavior and hormone levels of the subordinates. We found that soldiers tended to be from earlier litters and were higher ranked compared to workers, whereas dispersers were distributed throughout litters and rankings. There was no difference in estradiol, testosterone, or dehydroepiandrosterone (DHEA) levels amongst phenotypes. Progesterone levels were higher in soldiers but this difference appeared to be driven by a few individuals. Principal component analysis demonstrated that soldiers separated into a discrete category relative to workers/dispersers, with the highest ranked loadings being age, weight, and testosterone levels. However, the higher testosterone in soldiers was correlated with large body size instead of strictly behavioral phenotype. Workers and dispersers have more overlap with each other and no hormonal differences. Thus the behavioral variation in subordinate naked mole-rats is likely not driven by circulating steroid hormone levels but rather stems from alternative neural and/or neuroendocrine mechanisms.

category: animal behavior and cognition


title: The recovery of biodiversity and species composition after agricultural abandonment across spatial scales

abstract:
O_LI1. Human impacts have led to dramatic biodiversity change which can be highly scale-dependent across space and time. A primary means to manage these changes is via passive or active ecological restoration. The recovery of biodiversity following the removal of disturbance (passive) is often incomplete. The magnitude of recovery can very much depend on the landscape matrix in which a local (within plots) community or patch (site) is embedded. Our detection of recovery depends on the temporal and spatial scales at which biodiversity is measured.
C_LIO_LI2. We measured the recovery of biodiversity and species composition over 27 years in 17 temperate grasslands with known agricultural land-use histories, comparing them to never-disturbed sites as benchmarks. We specifically measured aspects of diversity at the local plot-scale (-scale, 0.5m2), site-scale ({gamma}-scale, 10m2), as well as the within-site heterogeneity ({beta}-diversity) and among-site variation in species composition (turnover and nestedness).
C_LIO_LI3. At the -scale (within-plot), sites recovering after agricultural abandonment only had 70% of the plant species richness (and [~]30% of the evenness), even up to 80 years post-recovery, compared to never-ploughed sites. Within-site {beta}-diversity (heterogeneity) recovered following agricultural abandonment to around 90% after 80 years. This effect, however, was not enough to lead to recovery at the {gamma}-scale (site-scale) richness in recovering sites was [~]65% of that in remnant never-ploughed sites. The presence of species characteristic of the never disturbed sites increased in the recovering sites through time, suggesting the lack of full recovery was at least partially due to dispersal limitation.
C_LIO_LI4. Synthesis. We found that, during the 80 years after agricultural abandonment, old-fields did not recover to the level of biodiversity in remnant never-ploughed sites at any scale. {beta}-diversity recovered more than -scale or {gamma}-scales, and plant species composition recovered, but not completely, over time. Time to full recovery is predicted to take an extremely long time, and to take different time spans at different spatial scales without intervention. Patterns of ecological recovery in degraded ecosystems across space and long time-scales can inform effective, targeted active restoration interventions and perhaps, help to lead to better outcomes.
C_LI

category: ecology


title: Vaeda computationally annotates doublets in single-cell RNA sequencing data

abstract:
MotivationSingle-cell RNA sequencing (scRNA-seq) continues to expand our knowledge by facilitating the study of transcriptional heterogeneity at the level of single cells. Despite this technologys utility and success in biomedical research, technical artifacts are present in scRNA-seq data. Doublets/multiplets are a type of artifact that occurs when two or more cells are tagged by the same barcode, and therefore they appear as a single cell. Because this introduces non-existent transcriptional profiles, doublets can bias and mislead downstream analysis. To address this limitation computational methods to annotate and remove doublets form scRNA-seq datasets are needed.

ResultsWe introduce vaeda, a new approach for computational annotation of doublets in scRNA-seq data. Vaeda integrates a variational auto-encoder and Positive-Unlabeled learning to produce doublet scores and binary doublet calls. We apply vaeda, along with seven existing doublet annotation methods, to sixteen benchmark datasets and find that vaeda performs competitively in terms of doublet scores and doublet calls. Notably, vaeda outperforms other python-based methods for doublet annotation. All together, vaeda is a robust and competitive method for scRNA-seq doublet annotation and may be of particular interest in the context of python-based workflows.

AvailabilityVaeda is available at https://github.com/kostkalab/vaeda

Contactkostka@pitt.edukostka@pitt.edu


title: Violation of the fluctuation-response relation from a linear model of hair bundle oscillations

abstract: Spontaneous hair-bundle oscillations have been proposed to underlie the ears active process, which amplifies acoustic signals, sharpens frequency selectivity, and broadens the dynamic range. Although this activity is critical for proper hearing, we know very little about its energetics and its nonequilibrium properties. Systems obey fluctuation-response relations, whose violation signals nonequilibrium conditions. Here we demonstrate the violation of the fluctuation-response relation of a linear model for hair bundle oscillations. Combining analytical results with experimental data, we estimate that an energy of at least 146 kBT is dissipated per oscillatory cycle, implying a power output of about 5aW. Our model indicates that this dissipation attains a minimum at a certain characteristic frequency. For high frequencies, we derive a linear scaling behavior of this dissipated energy with the characteristic frequency.

category: biophysics


title: Hijacking of Cellular Functions by SARS-CoV-2. Permeabilization and Polarization of the Host Lipid Membrane by Viroporins.

abstract:
As all viral infections, SARS-CoV-2 acts at multiple levels hijacking fundamental cellular functions and assuring its replication and immune system evasion. In particular, it has been observed that the viral 3 Open Reading Frame (ORF3) codes for a hydrophobic protein which embeds in the cellular membrane, where it acts as an ion viroporin and is related to strong inflammatory response. Here we report equilibrium and enhanced sampling molecular dynamic simulation of the SARS-CoV-2 ORF3 in a model lipid bilayer, showing how the protein permeabilizes the lipid membrane, via the formation of a water channel, which in turn assures ion transport. We report the free energy profile for both K+ and Cl- transfer from the cytosol to the extracellular domain. The important role of ORF3 in the viral cycle, and its highly conservation among coronaviruses, may also make it a target of choice for future antiviral development, further justifying the elucidation of its mechanism at the atomistic level.

TOC GRAPHICS

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title: Arousal neurons that anticipate deviations in blood glucose.

abstract: The brain needs to track body energy state to optimize physiology and behavior. Important information about current and future energy states is contained in minute-to-minute fluctuations in blood glucose. However, it is unclear whether brain glucose sensors are capable of responding to this temporal structure to extract such information. In behaving mammals, hypothalamic hypocretin/orexin neurons (HONs) control arousal and are proposed to sense energy balance, yet recent studies show that HON activity varies rapidly (over seconds) with locomotion. It remains unknown if and how HONs reflect blood glucose fluctuations. Here, for the first time, we co-monitored HON activity and blood glucose in behaving mice, uncovering inhibition of HONs by rising blood glucose. Surprisingly, peak HON signals (population waves) anticipated peak glucose deviations by several minutes. In multivariate analysis of diverse HON activity correlates (locomotion, metabolic variables), the glucose temporal gradient (negative first derivative) emerged as prime predictor of HON population activity on the minute timescales, thereby explaining this anticipatory response. Furthermore, 2-photon imaging of >900 individual HONs revealed parallel communication of absolute blood glucose values and their derivatives in distinct HON subsets. Thus, HONs transmit multiplexed slowly and rapidly changing information, and, in the slow bandwidth, extract temporal features from blood glucose dynamics that suggest a hybrid of anticipatory and proportional logic in brain responses to blood glucose.


title: Kinesin-1 transports morphologically distinct intracellular virions during vaccinia infection

abstract: Intracellular mature virions (IMV) are the first and most abundant infectious form of vaccinia virus to assemble during its replication cycle. IMV can undergo microtubule-based motility, but their directionality and the motor involved in their transport remain unknown. Here, we demonstrate that IMV, like intracellular enveloped virions (IEV), the second form of vaccinia, undergo anterograde transport and recruit kinesin-1. In vitro reconstitution of virion transport reveals that IMV and IEV move toward microtubule plus-ends with respective velocities of 0.66 and 0.56 m/s. Quantitative imaging establishes IMV and IEV recruit an average of 65 and 115 kinesin-1 motor complexes respectively. In the absence of kinesin-1 there is a near-complete loss of in vitro motility and defects in the cellular spread of both virions. Our observations demonstrate kinesin-1 transports two morphologically distinct forms of vaccinia. Reconstitution of vaccinia-based microtubule motility in vitro provides a new model to investigate how motor number and regulation impacts transport of a bona fide kinesin-1 cargo.


title: Meiotic Nuclear Pore Complex Remodeling Provides Key Insights into Nuclear Basket Organization

abstract:
Nuclear pore complexes (NPCs) are large proteinaceous assemblies that mediate nuclear compartmentalization. NPCs undergo largescale structural rearrangements during mitosis in metazoans and some fungi. However, our understanding of NPC remodeling beyond mitosis remains limited. Using time-lapse fluorescence microscopy, we discovered that NPCs undergo two mechanistically-separable remodeling events during budding yeast meiosis whereby parts or all of the nuclear basket transiently dissociate from the NPC core during meiosis I and II, respectively. Meiosis I detachment, observed for Nup60 and Nup2, is driven by Polo kinase-mediated phosphorylation of Nup60 at its interface with the Y-complex. Subsequent reattachment of Nup60-Nup2 to the NPC core is mediated by a lipid-binding amphipathic helix in Nup60. Preventing Nup60-Nup2 reattachment causes misorganization of the entire nuclear basket in gametes. Strikingly, meiotic nuclear basket remodeling also occurs in the distantly related fission yeast, Schizosaccharomyces pombe. Our study reveals a conserved and developmentally programmed aspect of NPC plasticity, providing key mechanistic insights into nuclear basket organization.

SUMMARYKing and Wettstein et al. reveal that nuclear pore complexes undergo two distinct remodeling events during budding yeast meiosis: partial and full nuclear basket detachment. By dissecting the regulation of these events, the study provides mechanistic insights into NPC organization.


title: Covert attention to obstacles biases escapes via the Mauthner cell

abstract: To study the evolutionary origins of object perception, we investigated whether a primitive vertebrate, the larval zebrafish, is sensitive to the presence of obstacles. The zebrafish, which has become a useful model to study brain-wide circuit dynamics, executes fast escape swims when in danger of predation. We posited that collisions with solid objects during escape would be maladaptive to the zebrafish, and therefore the direction of escape swims should be informed by the locations of barriers. To answer this question, we developed a novel closed-loop high-speed imaging rig outfitted with barriers of various qualities. Using this system, we show that when larval zebrafish escape in response to a non-directional vibrational stimulus, they use visual scene information to avoid collisions with obstacles. Our study demonstrates that fish compute absolute distance to obstacles, as distant barriers outside of collision range elicit less bias than nearby collidable barriers that occupy the same visual field. The computation of barrier features is covert, as the fishs reaction to barriers during routine swimming does not predict that they will avoid barriers when escaping. Finally, through two-photon laser ablations, we reveal a novel excitatory input from the visual system to Mauthner cells in the brainstem escape network that is responsible for escape direction bias. We propose that zebrafish construct "object solidity" via an integrative visual computation that is more complex than retinal occupancy alone, suggesting a primitive understanding of object features and possibly the origins of a structured model of the physical world.