title: Environmental predictors of electroactive bacterioplankton in small boreal lakes.

abstract: Extracellular electron transfer (EET) by electroactive bacteria in anoxic soils and sediments is an intensively researched subject, but EETs function in planktonic ecology has been less considered. Following the discovery of an unexpectedly high prevalence of EET genes in a bog lakes bacterioplankton, we hypothesized that the redox capacities of dissolved organic matter (DOM) enrich for electroactive bacteria by mediating redox chemistry. We developed the bioinformatics pipeline FEET (Find EET) to identify and summarize EET proteins from metagenomics data. We then applied FEET to several bog and thermokarst lakes and correlated EET protein occurrence values with environmental data to test our predictions. Our results provide evidence that DOM participates in EET by bacterioplankton. We found a similarly high prevalence of EET genes in most of these lakes, where oxidative EET strongly correlated with DOM. Numerous novel clusters of multiheme cytochromes that may enable EET were identified. Taxa previously not considered EET-capable were found to carry EET genes. We conclude that EET and DOM interactions are of major ecological importance to bacterioplankton in small boreal lakes, and that EET, particularly by methylotrophs and phototrophs, should be further studied and incorporated into both conceptual and quantitative methane emission models of melting permafrost.

category: microbiology


title: Antiviral roles of interferon regulatory factor (IRF)-1, 3 and 7 against human coronavirus infection

abstract: Interferon regulatory factors (IRFs) are key elements of antiviral innate responses that regulate transcription of interferons (IFNs) and IFN-stimulated genes (ISGs). As many human coronaviruses are known to be sensitive to IFN, antiviral roles of IRFs are yet to be fully understood. TypeI or II IFN treatment protected MRC5 cells from infection of human coronavirus 229E, but not human coronavirus OC43. Infection of 229E or OC43 efficiently upregulated ISGs, indicating that antiviral transcription is not suppressed during their infection. Antiviral IRFs, IRF1, IRF3 and IRF7, were activated in cells infected with 229E, OC43 or severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2). RNAi knockdown and overexpression of the IRFs demonstrated that IRF1 and IRF3 have antiviral property against OC43 while only IRF3 and IRF7 are effective to restrict 229E infection. Our study demonstrates that IRF3 plays critical roles against infection of human coronavirus 229E and OC43, which may be an anti-human coronavirus therapeutic target.


title: The Envelope Protein of SARS-CoV-2 Inhibits Viral Protein Synthesis and Infectivity of Human Immunodeficiency Virus type 1 (HIV-1)

abstract:
The human coronavirus SARS-CoV-2 encodes for a small 75 amino acid transmembrane protein known as the envelope (E) protein. The E protein forms an ion channel, like the viroporins from human immunodeficiency virus type 1 (HIV-1) (Vpu) and influenza A virus (M2). Here, we analyzed HIV-1 virus infectivity in the presence of four different {beta}-coronavirus E proteins. We observed that the SARS-CoV-2 and SARS-CoV E proteins reduced HIV-1 yields by approximately 100-fold while MERS-CoV or HCoV-OC43 E proteins restricted HIV-1 infectivity to a lesser extent. This was also reflected in the levels of HIV-1 protein synthesis in cells. Mechanistically, we show that that the E protein neither affected reverse transcription nor genome integration. However, SARS-CoV-2 E protein activated the ER-stress pathway associated with the phosphorylation of eIF-2, which is known to attenuate protein synthesis in cells. Finally, we show that these four E proteins and the SARS-CoV-2 N protein did not significantly down-regulate bone marrow stromal cell antigen 2 (BST-2) while the spike (S) proteins of SARS-CoV and SARS-CoV-2, and HIV-1 Vpu efficiently down-regulated cell surface BST-2 expression. The results of this study show for the first time that viroporins from a heterologous virus can suppress HIV-1 infection.

IMPORTANCEThe E protein of coronaviruses is a viroporin that is required for efficient release of infectious virus and for viral pathogenicity. We determined if the E protein from four {beta}-coronaviruses could restrict virus particle infectivity of HIV-1 infection. Our results indicate that the E proteins from SARS-CoV-2 and SARS-CoV potently restricted HIV-1 while those from MERS-CoV and HCoV-OC43 were less restrictive. Substitution of the highly conserved proline in the cytoplasmic domain of SARS-CoV-2 E abrogated the restriction on HIV-1 infection. Mechanistically, the SARS-CoV-2 E protein did not interfere with viral integration or RNA synthesis but rather reduced viral protein synthesis. We show that the E protein-initiated ER stress causing phosphorylation of eIF-2, which is known to attenuate protein synthesis. Companion studies suggest that the E protein also triggers autophagy. These results show for the first time that a viroporin from a coronavirus can restrict infection of another virus.


title: Optimized production and fluorescent labelling of SARS-CoV-2 Virus-Like-Particles to study virus assembly and entry.

abstract: SARS-CoV-2 is an RNA enveloped virus responsible for the COVID-19 pandemia that conducted in 6 million deaths worldwide so far. SARS-CoV-2 particles are mainly composed of the 4 main structural proteins M, N, E and S to form 100nm diameter viral particles. Based on productive assays, we propose an optimal transfected plasmid ratio mimicking the virus RNA ratio allowing SARS-CoV-2 Virus-Like Particle (VLPs) formation composed of the viral structural proteins M, N, E and S. Furthermore, monochrome, dual-color fluorescent or photoconvertible VLPs were produced. Thanks to live fluorescence and super-resolution microscopy, we quantified VLPs size and concentration. It shows a diameter of 110 and 140 nm respectively for MNE-VLPs and MNES-VLPs with a minimum concentration of 10e12 VLP/ml. SARS-CoV-2 VLPs could tolerate the integration of fluorescent N and M tagged proteins without impairing particle assembly. In this condition, we were able to establish incorporation of the mature Spike in fluorescent VLPs. The Spike functionality was then shown by monitoring fluorescent MNES-VLPs docking and endocytosis in human pulmonary cells expressing the receptor hACE2. This work provides new insights on the use of non-fluorescent and fluorescent VLPs to study and visualize the SARS-CoV-2 viral life cycle in a safe environment (BSL-2 instead of BSL-3). Moreover, optimized SARS-CoV-2 VLP production can be further adapted to vaccine design strategies.


title: Engineering a Vaccine Platform using Rotavirus A to Express SARS-CoV-2 Spike Epitopes

abstract:
Human rotavirus (RV) vaccines used worldwide have been developed using live attenuated platforms. The recent development of a reverse genetics system for RVs has delivered the possibility of engineering chimeric viruses expressing heterologous peptides from other virus species to generate polyvalent vaccines. We tested the feasibility of this using two approaches. Firstly, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian SA11 RV strain viral protein (VP) 4. Secondly, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C-terminus of non-structural protein (NSP) 3 of the bovine RF strain RV, with or without an intervening T2A peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RF strain. Except for the RBD mutant, all NSP3 mutants delivered endpoint titres and replication kinetics comparable to that of the WT virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants but only RBM mutant showed cross reactivity with SARS-CoV-2 RBD antibody. The tolerability of large peptide insertions in the NSP3 segment highlights the potential for this approach in the development of vaccine vectors targeting multiple enteric pathogens simultaneously.

IMPORTANCEWe explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an exemplar. Small SARS-CoV-2 peptide insertion (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titre and replication, thus limiting its use as a potential vaccine expression platform. To test RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids. With a T2A-separated 193 amino acid tag on NSP3, there was little effect on the viral rescue efficiency, titre and replication. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. This is the first report describing epitope tagging of VP4, and of a reverse genetics system for the RF strain. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously.


title: The interferon stimulated gene-encoded protein HELZ2 inhibits human LINE-1 retrotransposition and LINE-1 RNA-mediated type I interferon induction

abstract: Some interferon stimulated genes (ISGs) encode proteins that inhibit LINE-1 (L1) retrotransposition. Here, we used immunoprecipitation followed by liquid chromatography-tandem mass spectrometry to identify proteins that associate with the L1 ORF1-encoded protein (ORF1p) in ribonucleoprotein particles. Three ISG proteins that interact with ORF1p inhibit retrotransposition: HECT and RLD domain containing E3 ubiquitin-protein ligase 5 (HERC5); 2-5-oligoadenylate synthetase-like (OASL); and helicase with zinc finger 2 (HELZ2). HERC5 destabilizes ORF1p, but does not affect its cellular localization. OASL impairs ORF1p cytoplasmic foci formation. HELZ2 recognizes sequences and/or structures within the L1 5UTR to reduce L1 RNA, ORF1p, and ORF1p cytoplasmic foci levels. Overexpression of WT or reverse transcriptase-deficient L1s led to a modest induction of IFN- expression, which was abrogated upon HELZ2 overexpression. Notably, IFN- expression was enhanced upon overexpression of an ORF1p RNA binding mutant, suggesting ORF1p binding might protect L1 RNA from "triggering" IFN- induction. Thus, ISG proteins can inhibit retrotransposition by different mechanisms.

category: molecular biology


title: Telomere to telomere sequence of model Aspergillus fumigatus genomes

abstract: The pathogenic fungus Aspergillus fumigatus is a major etiological agent of fungal invasive and chronic diseases affecting tens of millions of individuals worldwide. A high-quality reference genome is a fundamental resource to study its biology, pathogenicity and virulence as well as to discover better and more effective treatments against diseases caused by this fungus. Here, we used PacBio Single Molecule Real-Time (SMRT) and Oxford Nanopore sequencing for de novo genome assembly of two laboratory reference strains of A. fumigatus,CEA10 and A1160. We generated full length chromosome assemblies and a comprehensive telomere to telomere coverage for these two strains including ribosomal repeats and the sequences of centromeres, which we discovered to be composed of long transposon elements.

category: genomics


title: The aggregation potential of Zika virus proteome

abstract:
The ability of human encoded soluble proteins to convert into amyloid fibrils is now recognized as a generic phenomenon in several human illnesses. Typically, such disease causal proteins/peptides consist of aggregation-prone regions (APR) that make them susceptible to misfolding and assemble into highly ordered {beta}-sheet rich fibrils, distinct from their native soluble state. Here, we show that the zika virus (ZIKV) consists of several such aggregation prone hotspots spread across its entire proteome. Using a combination of high-accuracy prediction tools, we identified APRs in both structural and non-structural proteins of ZIKV. Furthermore, we have experimentally validated the bioinformatic results by subjecting the ZIKV proteins and peptides to artificial aggregation inducing environment. Using a combination of dye-based assays (ThT and ANS) and microscopy techniques (HR-TEM and AFM), we further characterized the morphological features of amyloid-like fibrils. We found that Envelope domain III (EDIII) protein, NS1 {beta}-roll peptide, membrane-embedded signal peptide 2K, and cytosolic region of NS4B protein to be highly aggregating in the experimental setup. Our findings also pave the way for an extensive and detailed functional analysis of these predicted APRs in the future to enhance our understanding of the role played by amyloids in the pathogenesis of flavivirus.

Graphical Abstract

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category: biophysics


title: MS1/MMD1 homologs in the moss P. patens are required for male and female gametogenesis and likely for sporogenesis

abstract:
O_LIThe Arabidopsis Plant HomeoDomain (PHD) proteins AtMS1 and AtMMD1 provide chromatin-mediated transcriptional regulation essential for tapetum-dependent pollen formation. Such pollen-based male gametogenesis is a derived trait of seed plants. Male gametogenesis in the common ancestors of land plants is instead likely to have been reminiscent of that in extant bryophytes where flagellated sperms are produced by an elaborate gametophyte generation. Still, also bryophytes possess MS1/MMD1-related PHD proteins.
C_LIO_LIWe addressed the function of two MS1/MMD1-homologs in the bryophyte model moss Physcomitrium patens by the generation and analysis of reporter and loss-of-function lines.
C_LIO_LIThe two genes are together essential for both male and female fertility by providing cell autonomous functions in the gamete-producing inner cells of antheridia and archegonia. They are furthermore expressed in the diploid sporophyte generation suggesting a function during sporogenesis, a process proposed related by descent to pollen formation in angiosperms.
C_LIO_LIWe propose that the moss MS1/MMD1-related regulatory network required for completion of male and female gametogenesis and possibly for sporogenesis, represent a heritage from ancestral land plants.
C_LI

category: plant biology


title: Characteristics of N6-methyladenosine modification during sexual reproduction of Chlamydomonas reinhardtii

abstract: The unicellular green alga Chlamydomonas reinhardtii (referred to as Chlamydomonas throughout) is an ideal model organism which possesses both plant and animal attribute for studying the fundamental processes such as photosynthesis, sexual reproduction and life cycle etc. N6-methyladenosine (m6A) is the most prevalent mRNA modification and plays important roles during sexual reproduction in animals and plants. However, the pattern and function of m6A modification during Chlamydomonas sexual reproduction is still unknown. Here, we performed transcriptome and MeRIP sequencing on the six samples from different stages during sexual reproduction of Chlamydomonas life cycle. The results showed that m6A occurs widely at the main motif of UGKAM (K= U/G, M= A/C) in Chlamydomonas mRNA. Moreover, m6A peak in Chlamydomonas mRNA is mainly enriched in the 3UTR region and negatively correlated with the abundance of the transcripts at each stage. Especially, genes in microtubule-associated pathway showed a significant negative correlation between gene expression level and m6A level, indicating the influences of m6A modification on sexual reproduction and life cycle of Chlamydomonas through regulating microtubule-based movement. In summary, our findings first demonstrate the distributions and the functions of m6A modification in Chlamydomonas and provide new insights into the understandings of m6A modification in the process of sexual reproduction in other plant organisms evolutionarily.


title: Transcriptome analysis reveals robust transcriptional reprogramming during early stages of Cicer-Ascochyta interaction

abstract:
Ascochyta blight (AB) caused by a filamentous fungus Ascochyta rabiei is a major threat to global chickpea production. The mechanisms underlying chickpea response to A. rabiei remain elusive. Here, we investigated the comparative transcriptional dynamics of AB-resistant and susceptible chickpea genotypes upon A. rabiei infection to understand the early host defence response. Our findings revealed that AB-resistant plants underwent rapid and extensive transcriptional reprogramming compared to susceptible host. At early stage (24-hpi), mainly cell wall remodeling and secondary metabolite pathways were highly activated, while DEGs related with signaling components viz. protein kinases, transcription factors, and hormonal pathways show remarkable upsurge at 72-hpi, especially in resistant genotype. Notably, our data suggests imperative role of JA, ET, and ABA signaling in providing immunity against A. rabiei. Furthermore, gene co-expression networks and modules corroborated the importance of cell wall remodeling, signal transduction and phytohormone pathways. The hub genes such as MYB14, PRE6 and MADS-SOC1 discovered in these modules might be the master regulators governing chickpea immunity. Overall, we not only provide novel insights for comprehensive understanding of immune signaling components mediating AB resistance/susceptibility at early Cicer-Ascochyta interactions, but also offer a valuable resource for developing AB-resistant chickpea.

HighlightsComparative transcriptomic and co-expression analysis of AB-resistant and susceptible chickpea genotypes reveals high-amplitude transcriptional dynamics in resistant plants, and also identifies TFs, PKs and phytohormone-crosstalk as core regulators for AB-resistance.


title: CryoET Reveals Organelle Phenotypes in Huntington Disease Patient iPSC-Derived and Mouse Primary Neurons

abstract: Huntingtons Disease (HD) is caused by an expanded CAG repeat in the huntingtin gene, yielding a Huntingtin protein with an expanded polyglutamine tract. Patient-derived induced pluripotent stem cells (iPSCs) can help understand disease; however, defining pathological biomarkers is challenging. Here, we used cryogenic electron tomography to visualize neurites in HD patient iPSC-derived neurons with varying CAG repeats, and primary cortical neurons from BACHD, deltaN17-BACHD, and wild-type mice. In HD models, we discovered mitochondria with enlarged granules and distorted cristae, and thin sheet aggregates in double membrane-bound organelles. We used artificial intelligence to quantify mitochondrial granules, and proteomics to show differential protein content in HD mitochondria. Knockdown of Protein Inhibitor of Activated STAT1 ameliorated aberrant phenotypes in iPSC-neurons and reduced phenotypes in BACHD neurons. We show that integrated ultrastructural and proteomic approaches may uncover early HD phenotypes to accelerate diagnostics and the development of targeted therapeutics for HD.

category: neuroscience


title: T cells induce interferon-responsive oligodendrocytes during white matter aging

abstract: A hallmark of nervous system aging is a decline of white matter volume and function, but the underlying mechanisms leading to white matter pathology are unknown. Here, we found age-related alterations of oligodendrocytes with a reduction of total oligodendrocyte density in the aging murine white matter. Using single-cell RNA sequencing, we identify interferon-responsive oligodendrocytes, which localize in proximity of CD8+ T cells in the aging white matter. Absence of functional lymphocytes decreased oligodendrocyte reactivity and rescued oligodendrocyte loss, while T-cell checkpoint inhibition worsened the aging affect. In summary, we provide evidence that T cells induced interferon-responsive oligodendrocytes are important modifiers of white matter aging.


title: Variation in TAF1 expression in female carrier induced pluripotent stem cells and human brain ontogeny has implications for adult neostriatum vulnerability in X-linked Dystonia Parkinsonism

abstract:
X-linked Dystonia-Parkinsonism (XDP) is an inherited, X-linked, adult-onset movement disorder characterized by degeneration in the neostriatum. No therapeutics alter disease progression. The mechanisms underlying regional differences in degeneration and age of onset are unknown. Developing therapeutics that target XDP-related mechanisms requires a deeper understanding of how XDP-relevant features vary in health and disease. XDP is due, in part, to either a partial loss of TAF1 function and/or a SVA-driven pathological gain of function. A disease-specific SINE-VNTR-Alu (SVA) retrotransposon insertion occurs within intron 32 of TAF1, a subunit of TFIID involved in transcription initiation. While all XDP males are usually clinically affected, females are heterozygous carriers generally not manifesting the full syndrome. As a resource for disease modeling, we characterized eight iPSC lines from XDP female carrier individuals, and identified isogenic lines where one clonal iPSC line expressed the wild-type X, and the two other clonal iPSC lines expressed the XDP haplotype. Furthermore, we characterized XDP-relevant transcript expression variation in humans, and found that SVA-F expression decreases slightly after 30 years of age in the neurotypical human brain and that TAF1 is modestly decreased in the majority of female samples. Uniquely in the caudate nucleus, TAF1 expression is not sexually dymorphic and decreased after 15 years of age. These findings indicate that regional-, age- and sex-specific mechanisms regulate TAF1, highlighting the importance of disease-relevant models and postmortem tissue analysis. We propose that the decreased TAF1 expression in the adult caudate may synergize with the XDP-specific partial loss of TAF1 function in patients, thereby passing a minimum threshold of TAF1 function, and triggering degeneration in the neostriatum.

Significance StatementXDP is an inherited, X-linked, adult-onset movement disorder characterized by degeneration in the neostriatum. No therapeutics alter disease progression. Developing therapeutics requires a deeper understanding of how XDP-relevant features vary in health and disease. XDP is possibly due to a partial loss of TAF1 function. While all XDP males are usually affected, females are heterozygous carriers generally not manifesting the full syndrome. As a resource for disease modeling, we characterized eight stem cell lines from XDP female carrier individuals. Furthermore, we found that, uniquely in the caudate nucleus, TAF1 expression decreases after adolescence in healthy humans. We hypothesize that the decrease of TAF1 after adolescence in human caudate, in general, may underlie the vulnerability of the adult neostriatum in XDP.


title: A Bayesian approach to incorporate structural data into the mapping of genotype to antigenic phenotype of influenza A(H3N2) viruses

abstract: Surface antigens of pathogens are commonly targeted by vaccine-elicited antibodies but antigenic variability, notably in RNA viruses such as influenza, HIV and SARS-CoV-2, pose challenges for control by vaccination. For example, influenza A(H3N2) entered the human population in 1968 causing a pandemic and has since been monitored, along with other seasonal influenza viruses, for the emergence of antigenic drift variants through intensive global surveillance and laboratory characterisation. Statistical models of the relationship between genetic differences among viruses and their antigenic similarity provide useful information to inform vaccine development though accurate identification of causative mutations is complicated by highly correlated genetic signals that arise due to the evolutionary process. Here, using a sparse hierarchical Bayesian analogue of an experimentally validated model for integrating genetic and antigenic data, we identify the genetic changes in influenza A(H3N2) virus that underpin antigenic drift. We show that incorporating protein structural data into variable selection helps resolve ambiguities arising due to correlated signals, with the proportion of variables representing haemagglutinin positions decisively included, or excluded, increased from 59.8% to 72.4%. The accuracy of variable selection judged by proximity to experimentally determined antigenic sites was improved simultaneously. Structure-guided variable selection thus improves confidence in the identification of genetic explanations of antigenic variation and we also show that prioritising the identification of causative mutations is not detrimental to the predictive capability of the analysis. Indeed, incorporating structural information into variable selection resulted in a model that could more accurately predict antigenic assay titres for phenotypically-uncharactrised virus from genetic sequence. Combined, these analyses have the potential to inform choices of reference viruses, the targeting of laboratory assays, and predictions of the evolutionary success of different genotypes, and can therefore be used to inform vaccine selection processes.


title: S51 family peptidases provide resistance to peptidyl-nucleotide antibiotic McC

abstract:
Microcin C-like compounds are natural Trojan horse peptide-nucleotide antibiotics produced by diverse bacteria. The ribosomally-synthesized peptide parts of these antibiotics are responsible for their facilitated transport into susceptible cells. Once inside the cell, the peptide part is degraded, releasing the toxic payload, an isoaspartyl-nucleotide that inhibits aspartyl-tRNA synthetase, an enzyme essential for protein synthesis. Bacteria that produce microcin C-like compounds have evolved multiple ways to avoid self-intoxication. Here, we describe a new strategy through the action of S51 family peptidases, which we name MccG. MccG cleaves the toxic isoaspartyl-nucleotide rendering it inactive. While some MccG homologs are encoded in gene clusters responsible for McC-like compounds biosynthesis, most are encoded by stand-alone genes whose products may provide basal level of resistance to peptide-nucleotide antibiotics in phylogenetically distant bacteria.

SIGNIFICANCEWe identified a natural substrate for a major phylogenetic clade of poorly characterized S51 family proteases from bacteria. We show that these proteins can contribute to basal level of resistance to an important class of natural antibiotics.


title: Detection of Parasites in Microbiomes using Metagenomics

abstract:
Despite a yearly death toll of up to one million people due to parasite-related infections, parasites are still neglected in genomics research. While there is progress in the detection of bacteria and viruses using metagenomics in the context of infectious diseases, there are still challenges in metagenomics-based detection of parasites. Here, we implement a workflow for the detection of parasites from metagenomics data. We employ stringent cut off criteria to limit false positive detections. We analysed a total of 7.120 metagenomics samples of which 359 originated from gut microbiomes of livestock (pigs and chicken) from nine countries, and 6.761 from gut microbiomes of humans (adults and infants) from 25 countries. Five parasite-related genera were detected in livestock, of which Blastocystis sp. was detected in 71% of all pig herds and Eimeria in 83% of all chicken flocks. Distinct gut bacterial taxa were associated with Blastocystis sp. abundance in pigs. Nine parasite-related genera were detected in humans. Blastocystis sp. subtypes ST1, ST2, and ST3 were detected in all countries, and ST3 was most predominant. A higher overall prevalence of Blastocystis sp. was observed in low-income countries as compared to high-income countries, and a higher diversity of Blastocystis subtypes (ST1, ST2, ST3, ST4, ST6, ST7, ST8) was detected in high-income countries as compared to low-income countries. The prevalence of Blastocystis sp. in infant gut microbiome samples was lower as compared to adults. Overall, metagenomics-based analysis may be a promising tool for parasite detection from complex microbiome samples in clinical and veterinary medicine.

Metagenomics could become the preferred method for parasite detection for a wide range of biological samples. Current parasite detection methods often rely on microscopic examination of the sample or using specific PCR. Metagenomics-based analyses may allow for a faster and more convenient way of detecting parasites in humans and animals, as this approach could serve as a one-for-all untargeted approach for pathogen detection, including bacteria, viruses, and parasites.


title: Versican binds collagen via its G3 domain and regulates the organization and mechanics of collagenous matrices

abstract: Type I collagen is the most abundant structural protein in the body and, with other fibrillar collagens, forms the fibrous network of the extracellular matrix (ECM). Another group of ECM polymers, the glycosaminoglycans (GAGs) and GAG-modified proteoglycans, play important roles in regulating collagen behaviors and contribute to the compositional, structural and mechanical complexity of the ECM. While the binding between collagen and small leucine-rich proteoglycans (SLRPs) has been studied in detail, the interactions between collagen and the large bottlebrush proteoglycan versican are not well understood. Here, we report that versican binds collagen directly and regulates collagen structure and mechanics. Versican colocalizes with collagen fibers in vivo and binds to collagen via its C-terminal G3 domain (a non-GAG-modified domain present in all known versican isoforms) in vitro; it promotes the deposition of a highly-aligned collagen-rich matrix by fibroblasts. Versican also shows an unexpected effect on the rheology of collagen gels in vitro, causing decreased stiffness and attenuated shear strain stiffening, and the cleavage of versican in liver results in reduced tissue compression stiffening. Thus, versican is an important collagen binding partner, playing a role in modulating collagen organization and mechanics.

category: cell biology


title: Bone Morphogenetic Protein (BMP) signaling regulates germline stem cell self-renewal in the newly formed  Drosophila  testis stem cell niche

abstract: Bone morphogenetic proteins (BMPs) are a group of multifunctional cytokines and metabologens highly conserved in the transforming growth factor-{beta} (TGF-{beta}) superfamily. BMPs have an established role in controlling cell fate and tissue morphogenesis in a diverse range of organisms and are known to maintain stem cells in adult Drosophila testes. Additional studies in embryonic and larval testes suggest BMP regulates germ cell behavior. However, the roles of BMP signaling in controlling testis stem cell formation have yet to be directly examined. Here, we explore the pattern of BMP activation during embryonic testis niche morphogenesis as well as niche maturation in larval testes. We also assess the impact of altered BMP signaling on these stages of development. Our data suggest that BMP signaling is critical for promoting germ cell identity in primordial germ cells during embryonic niche formation. During niche maturation, we also find that that BMP signaling is both necessary and sufficient for maintenance of a self- renewing germline stem cell (GSC) population, and that newly formed cyst stem cells (CySCs) are a source of BMP activating ligand. As development progresses, however, BMP activation is no longer sufficient to alter GSC self-renewal. Collectively, our work promotes a more thorough understanding of BMP as a key mechanism controlling stem cell development in Drosophila testes that has implications for the development of other organ systems.

category: developmental biology


title: Paternity sharing in insects with female competition for nuptial gifts

abstract: Males contributing costly investments to offspring (i.e., paternal care or nuptial gifts) are expected to have high confidence of paternity and to avoid cuckoldry. While studies of parental care have provided support for this hypothesis, paternity may be more uncertain in systems where males feed each mate. In contrast to many male-care systems, where mating co-occurs with each oviposition giving males control over insemination, in nuptial gift systems (mostly arthropods that feed mates with prey or glandular secretions) mating is separated from egg-laying so that males typically compete more with rivals for fertilizations. Given the large investments these males contribute to offspring, and long refractory periods that limit their ability to gain fitness from frequent copulations, there should be strong selection to avoid cuckoldry, especially complete paternity failure. Further, because females evolve to mate frequently in systems with competition for male-supplied nutrition, males may be 1) unable to avoid sperm competition by mating with virgins and 2) less likely to benefit from behaviours that prevent female remating or depend on last male sperm precedence. Thus, paternity sharing, in which males are not excluded from siring offspring completely, is an expected outcome of sperm competition in these systems. Using wild-caught females in two species (an orthopteran and a dipteran), in which sexually selected females compete for important male nuptial gifts, we examined paternity patterns and compared them to findings in other insects. Using microsatellite analysis of offspring and sperm stores, we found evidence of shared paternity in both study species, and there were very few males failing to sire offspring. Although paternity was not equal among sires, our estimates of paternity bias were similar to other insect species with valuable nuptial gifts and contrasted with patterns in gryllid cricket species where males supply little more than sperm and are frequently excluded from siring offspring. Unlike many insects with strong paternity bias, further support for paternity-sharing is the lack of last-male sperm precedence in either of our study species.

category: evolutionary biology


title: Differences in homologous recombination and maintenance of heteropolyploidy between Haloferax volcanii and Haloferax mediterranei

abstract:
Polyploidy, the phenomenon of having more than one copy of the genome in an organism, is common among haloarchaea. While providing short-term benefits for DNA repair, polyploidy is generally regarded as an  evolutionary trap that by the notion of the Mullers ratchet will inevitably conclude in the species decline or even extinction due to a gradual reduction in fitness. In most reported cases of polyploidy in archaea, the genetic state of the organism is considered as homoploidy i.e. all copies of the genome are identical. Here we demonstrate that while this is indeed the prevalent genetic status in the halophilic archaeon H. volcanii, its close relative H. mediterranei maintains a prolonged heteroploidy state in a non-selective environment once a second allele is introduced. Moreover, a strong genetic linkage was observed between two distant loci in H. mediterranei indicating a low rate of homologous recombination while almost no such linkage was shown in H. volcanii indicating a high rate of recombination in the latter species.

We suggest that H. volcanii escapes Mullers ratchet by means of an effective chromosome-equalizing gene-conversion mechanism facilitated by highly active homologous recombination, whereas H. mediterranei must elude the ratchet via a different, yet to be elucidated mechanism.

category: genetics


title: The role of repetitive DNA in re-patterning of major rDNA clusters in Lepidoptera

abstract: Genes for major ribosomal RNAs (rDNA) are present in multiple copies organized in tandem arrays. Number and position of rDNA loci can change dynamically and their re-patterning is presumably driven by repetitive sequences. We explored a peculiar rDNA organization in several representatives of Lepidoptera with either extremely large or numerous rDNA clusters. We combined molecular cytogenetics with analyses of second and third generation sequencing data to show that rDNA spreads as a transcription unit and reveal association between rDNA and various repeats. Furthermore, we performed comparative long read analyses between the species with derived rDNA distribution and moths with a single rDNA locus, which is considered ancestral. Our results suggest that satellite arrays, rather than mobile elements, facilitate homology-mediated spread of rDNA via either integration of extrachromosomal rDNA circles or ectopic recombination. The latter arguably better explains preferential spread of rDNA into terminal regions of lepidopteran chromosomes as efficiency of ectopic recombination depends on proximity of homologous sequences to telomeres.


title: Single-agent Foxo1 inhibition normalizes glycemia and induces gut β-like cells in streptozotocin-diabetic mice

abstract: Insulin treatment remains the sole effective intervention for Type 1 Diabetes. Here, we investigated the therapeutic potential of converting intestinal epithelial cells to insulin-producing, glucose-responsive {beta}-like cells by targeted inhibition of Foxo1. We have shown that this can be achieved by genetic ablation in gut Neurogenin3 progenitors, adenoviral or shRNA-mediated inhibition in human gut organoids, and chemical inhibition in Akita mice, a model of insulin-deficient diabetes. In the present study, we provide evidence that two novel Foxo1 inhibitors, FBT432 and FBT374 have glucose-lowering and gut {beta}-like cell-inducing properties in mice rendered insulin-deficient by administration of streptozotocin. FBT432 is also highly effective in combination with a Notch inhibitor in this model. The data add to a growing body of evidence suggesting that Foxo1 inhibition be pursued as an alternative treatment to insulin administration in diabetes.

category: pharmacology and toxicology


title: Multicellular Reservoir Computing

abstract: AO_SCPLOWBSTRACTC_SCPLOWThe capacity of cells to process information is currently used to design cell-based tools for ecological, industrial, and biomedical applications such as detecting dangerous chemicals or for bioremediation. In most applications, individual cells are used as the information processing unit. However, single cell engineering is limited by the necessary molecular complexity and the accompanying metabolic burden of synthetic circuits. To overcome these limitations, synthetic biologists have begun engineering multicellular systems that combine cells with designed subfunctions. To further advance information processing in synthetic multicellular systems, we introduce the application of reservoir computing. Reservoir computers (RCs) approximate a temporal signal processing task via a fixed-rule dynamic network (the reservoir) with a regression-based readout. Importantly, RCs eliminate the need of network rewiring, as different tasks can be approximated with the same reservoir. Previous work has already demonstrated the capacity of single cells, as well as populations of neurons, to act as reservoirs. In this work, we extend reservoir computing in multicellular populations with the widespread mechanism of diffusion-based cell-cell signaling. As a proof-of-concept, we simulated a reservoir made of a 3D community of cells communicating via diffusible molecules and used it to approximate two benchmark signal processing tasks, computing median and parity functions from binary input signals. We demonstrate that a diffusion-based multicellular reservoir is a feasible synthetic framework for performing complex temporal computing tasks that provides a computational advantage over single cell reservoirs. We also identified a number of biological properties that can affect the computational performance of these processing systems.

category: systems biology


title: Comparison of rule- and ordinary differential equation-based dynamic model of DARPP-32 signalling network

abstract: AO_SCPLOWBSTRACTC_SCPLOWDynamic modelling has considerably improved our understanding of complex molecular mechanisms. Ordinary differential equations (ODEs) are the most detailed and popular approach to modelling the dynamics of molecular systems. However, their application in signalling networks, characterised by multi-state molecular complexes, can be prohibitive. Contemporary modelling methods, such as rule-based (RB) modelling, have addressed these issues. Although the advantages of RB modelling over ODEs have been presented and discussed in numerous reviews, no direct comparison of the time courses of a molecular system encoded in the two frameworks has been made before. To make such a comparison, a set of reactions that underlie an ODE model by Fernandez et al. [1] was manually encoded in the Kappa language, one of the RB frameworks. A comparison of the models was performed at the level of model specification and results were acquired through model simulations. We found that the Kappa model recapitulated the general dynamics of its ODE counterpart with minor differences. These differences occur whenever molecules have multiple sites binding the same interactor. The notation of such rules requires a complete listing of all possible binding configurations. Furthermore, activation of these molecules in the RB model is slower than in the ODE one but can be corrected by revision of the rate constants used in the relevant rules. We conclude that the RB representation offers a more expressive and flexible syntax that eases access to fine-grain details of the model, facilitating model reuse. In parallel with these analyses, this manuscript reports a refactored model of a DARPP-32 interaction network that can serve as a canvas for the development of a more complex interaction network to study this particular molecular system.


title: 3D-printed decoys are as effective as taxidermied decoys in attracting red-breasted sapsuckers for mist-netting

abstract: Decoys often improve targeted mist-netting efforts by drawing the species of interest to a specific area nearer the net. Traditional decoy constructions include taxidermied carcasses, hand-made wood or clay figures, or professionally made products purchased from companies that provide a limited number of species, sizes, shapes, and markings. 3D-printing allows ornithologists to customize decoys to their own study species specifications using cheap, durable, and replaceable materials. We show that red-breasted sapsuckers (Sphyrapicus ruber ruber) respond with equivocal aggression towards 3D-printed decoys and taxidermied decoys, demonstrating 3D-printed decoys as an effective tool in attracting birds towards a mist net for capture.

category: zoology


title: Deep brain stimulation creates information lesion through membrane depolarization

abstract: Deep brain stimulation (DBS) is a promising neuromodulation therapy that alters neural activity via intracranial electrical stimulation. However, the neurophysiological mechanisms of DBS remain largely unknown, because of the difficulty of obtaining cellular resolution recordings without electrical interference. Here, we performed high-speed membrane voltage fluorescence imaging of individual hippocampal CA1 neurons during DBS in awake mice. We discovered that DBS, delivered at either 40Hz or 140Hz, reliably depolarizes somatic membrane potentials. Further, DBS enhanced spike rates and paced membrane voltage and spike timing at the stimulation frequency, though more prominent at 40Hz than 140Hz. To determine how DBS induced membrane voltage change impacts neurons ability to process inputs, we optogenetically evoked membrane depolarization. We found that neurons become unreliable in responding to optogenetic inputs during DBS, particularly during 140Hz DBS. These results demonstrate that DBS produces powerful membrane depolarization that interferes with neurons ability to process inputs, creating information lesion.


title: Nonlinear EEG signatures of mind wandering during breath focus meditation

abstract: In meditation practices that involve focused attention to a specific object, novice practitioners often experience moments of distraction (i.e., mind wandering). Previous studies have investigated the neural correlates of mind wandering during meditation practice through Electroencephalography (EEG) using linear metrics (e.g., oscillatory power). However, their results are not fully consistent. Since the brain is known to be a chaotic/nonlinear system, it is possible that linear metrics cannot fully capture complex dynamics present in the EEG signal. In this study, we assess whether nonlinear EEG signatures can be used to characterize mind wandering during breath focus meditation in novice practitioners. For that purpose, we adopted an experience sampling paradigm in which 25 participants were iteratively interrupted during meditation practice to report whether they were focusing on the breath or thinking about something else. We compared the complexity of EEG signals during mind wandering and breath focus states using three different algorithms: Higuchis fractal dimension (HFD), Lempel-Ziv complexity (LZC), and Sample entropy (SampEn). Our results showed that EEG complexity was generally reduced during mind wandering relative to breath focus states. We conclude that EEG complexity metrics are appropriate to disentangle mind wandering from breath focus states in novice meditation practitioners, and therefore, they could be used in future EEG neurofeedback and/or neurostimulation protocols to facilitate meditation practice.


title: Adaptation of the yeast gene knockout collection is near-perfectly predicted by fitness and diminishing return epistasis

abstract: Adaptive evolution of clonally dividing cells and microbes is the ultimate cause of cancer and infectious diseases. The possibility of constraining the adaptation of cell populations, by inhibiting proteins that enhance their evolvability has therefore attracted substantial interest. However, our current understanding of how individual genes influence the speed of adaptation is limited, partly because accurately tracking adaptation for many experimental cell populations in parallel is challenging. Here we use a high throughput artificial laboratory evolution (ALE) platform to track the adaptation of >18.000 cell populations corresponding to single gene deletion strains in the haploid yeast deletion collection. We report that the fitness of gene knockout near-perfectly (R2=0.91) predicts their adaptation dynamics under arsenic exposure, leaving virtually no role for dedicated evolvability functions in the corresponding proteins. We tracked the adaptation of another >23.000 yeast gene knockout populations to a diverse range of selection pressures and generalised the almost perfect (R2=0.72 to 0.98) capacity of initial fitness to predict the rate of adaptation. Finally, we reconstruct mutations in the genes FPS1, ASK10, and ARR3, which together account for almost all arsenic adaptation in wildtype cells, in gene deletions covering a broad fitness range. We show that the predictability of arsenic adaptation can be understood almost entirely as a global epistasis phenomenon where excluding arsenic from cells, through these mutations, is more beneficial in cells with low arsenic fitness regardless of what causes the arsenic defects. The lack of genes with a meaningful effect on the adaptation dynamics of clonally reproducing cell populations diminishes the prospects of developing adjuvant drugs aiming to slow antimicrobial and chemotherapy resistance.


title: Benchmarking computational methods for B-cell receptor reconstruction from single-cell RNA-seq data

abstract: Multiple methods have recently been developed to reconstruct full-length B-cell receptors (BCRs) from single-cell RNA-seq (scRNA-seq) data. This need emerged from the expansion of scRNA-seq techniques, the increasing interest in antibody-based drug development and the importance of BCR repertoire changes in cancer and autoimmune disease progression. However, a comprehensive assessment of performance-influencing factors like the sequencing depth, read length or the number of somatic hypermutations (SHMs) as well as guidance regarding the choice of methodology are still lacking. In this work, we evaluated the ability of six available methods to reconstruct full-length BCRs using one simulated and three experimental SMART-seq datasets. In addition, we validated that the BCRs assembled in silico recognize their intended targets when expressed as monoclonal antibodies. We observed that methods like BALDR, BASIC and BRACER showed the best overall performance across the tested datasets and conditions whereas only BASIC demonstrated acceptable results on very short read libraries. Furthermore, the de novo assembly-based methods BRACER and BALDR were the most accurate in reconstructing BCRs harboring different degrees of SHMs in the variable domain, while TRUST4, MiXCR and BASIC were the fastest. Finally, we propose guidelines to select the best method based on the given data characteristics.

category: bioinformatics


title: Leveraging the local genetic structure for trans-ancestry association mapping

abstract: Over the past two decades, genome-wide association studies (GWASs) have successfully advanced our understanding of genetic basis of complex traits. Despite the fruitful discovery of GWASs, most GWAS samples are collected from European populations, and these GWASs are often criticized for their lack of ancestry diversity. Trans-ancestry association mapping (TRAM) offers an exciting opportunity to fill the gap of disparities in genetic studies between non-Europeans and Europeans. Here we propose a statistical method, LOG-TRAM, to leverage the local genetic architecture for TRAM. By using biobank-scale datasets, we showed that LOG-TRAM can greatly improve the statistical power of identifying risk variants in under-represented populations while producing well-calibrated p-values. We applied LOG-TRAM to the GWAS summary statistics of 29 complex traits/diseases from Biobank Japan (BBJ) and UK Biobank (UKBB), and achieved substantial gains in power (the effective sample sizes increased by 49% in average compared to the BBJ GWASs) and effective correction of confounding biases compared to existing methods. Finally, we demonstrated that LOG-TRAM can be successfully applied to identify ancestry-specific loci and the LOG-TRAM output can be further used for construction of more accurate polygenic risk scores (PRSs) in under-represented populations.


title: Key residue on cytoplasmic dynein for asymmetric unbinding and unidirectional movement along microtubule

abstract:
Cytoplasmic dynein 1 is almost exclusively responsible for intracellular transport toward the minus-end of microtubules in animal cells. One of the key factors for the unidirectional movement of dynein is the asymmetry of the unbinding of the motor from the microtubule when an external load is applied; it dissociates more easily from microtubules with minus-end directed loading than with plus-end directed loading. To elucidate the molecular basis for this property, we performed molecular dynamics simulations to identify the key residues responsible for asymmetry, which were then examined experimentally. First, we reproduced asymmetry in the unbinding behavior of dynein using coarse-grained simulations. Second, data analysis together with mutational analysis in silico predicted the specific residues that may be responsible for the asymmetry in unbinding. Third, to examine this prediction, we expressed and purified recombinant dynein with mutations in either of the identified key residues. Consistent with the simulations, one of the mutants did not exhibit asymmetry in the in vitro binding assay. Moreover, the mutant dynein was able to move diffusely along a microtubule but was unable to restrict its movement to the minus-end direction. Thus, our results demonstrate both experimentally and theoretically how the key residue on the microtubule-binding domain generates asymmetry in unbinding, which is a critical mechanism for the unidirectional movement of dynein along a microtubule track.

Significance StatementCytoplasmic dynein moves to the minus end of microtubules. This unidirectional dynein motility provides the driving force for various cellular activities including vesicle transport, organelle positioning and cell division. One of the key factors for dynein to exhibit unidirectional movement is the asymmetry of unbinding of dynein from the microtubule depending on the direction of external load. By combining computational simulations and in vitro experiments, we identified a residue responsible for the asymmetry. A point mutation at the residue indeed abolished unidirectional motility, highlighting the importance of the asymmetric unbinding property in dyneins unidirectional movement.


title: Knockout of TSPO delays and reduces amyloid, Tau, astrocytosis and behavioral dysfunctions in Alzheimer's disease.

abstract: The 18kDa translocator protein (TSPO) is up-regulated in glial cells in neurodegenerative diseases. In Alzheimers disease (AD) animal models, TSPO is first overexpressed in astrocytes and then in microglia. However, the precise role of TSPO in the onset and progression of pathology and symptoms characteristic of the disease remains unknown. Here, we report that in the absence of TSPO in 3xTgAD mice the expected disease onset is significantly delayed and a reduction is seen in the hippocampal load of poorly and highly aggregated forms of Tau (-44% and -82%, respectively) and A{beta}42 (-25% and -95%, respectively), at 9 months of age. In addition, the astrocyte reactivity was decreased in 3xTgAD.TSPO-/- mice with a reduction in the morphologic complexity and the size of astrocytes in the dorso-dorsal hippocampus and the hilus. Functionally, the absence of TSPO ameliorated the cognitive consequences of adeno-associated virus-induced Tau over-expression in the hippocampus. This suggests that TSPO plays an important role in the active disease progression of AD. TSPO-inhibiting drugs thus merit further exploration as to their potential to reduce the rate of neurodegenerative disease progression.

category: pathology


title: Dynamic analysis of sequestration-based feedbacks in cellular and biomolecular circuits

abstract: Nonlinear feedback controllers are ubiquitous features of biological systems at different scales. A key motif arising in these systems is a sequestration-based feedback. As a physiological example of this type of feedback architecture, platelets (specialized cells involved in blood clotting) differentiate from stem cells, and this process is activated by a protein called Thrombopoietin (TPO). Platelets actively sequester and degrade TPO, creating negative feedback whereby any depletion of platelets increases the levels of freely available TPO that upregulates platelet production. We show similar examples of sequestration-based feedback in intracellular biomolecular circuits involved in heat-shock response and microRNA regulation. Our systematic analysis of this feedback motif reveals that platelets induced degradation of TPO is critical in enhancing system robustness to external disturbances. In contrast, reversible sequestration of TPO without degradation results in poor robustness to disturbances. We develop exact analytical results quantifying the limits to which the sensitivity to disturbances can be attenuated by sequestration-based feedback. Next, we consider the stochastic formulation of the circuit that takes into account low-copy number fluctuations in feedback components. Interestingly, our results show that the extent of random fluctuations are enhanced with increasing feedback strength, but can exhibit local maxima and minima across parameter regimes. In summary, our systematic analysis highlights design principles for enhancing the robustness of sequestration-based feedback mechanisms to external disturbances and inherent noise in molecular counts.

category: physiology


title: Stimulus-Response signaling dynamics characterize macrophage polarization states

abstract:
Macrophages show remarkable functional pleiotropy that is dependent on microenvironmental context. Prior studies have characterized how polarizing cytokines alter the transcriptomic and epigenetic landscape. Here we characterized the immune-threat appropriate responses of polarized macrophages by measuring the single-cell signaling dynamics of transcription factor NF{kappa}B. Leveraging a fluorescent protein reporter mouse, primary macrophages were polarized into 6 states and stimulated with 8 different stimuli resulting in a vast dataset. Linear Discriminant Analysis revealed how NF{kappa}B signaling codons compose the immune threat level of stimuli, placing polarization states along a linear continuum between the M1/M2 dichotomy. Machine learning classification revealed losses of stimulus distinguishability with polarization, which reflect a switch from sentinel to more canalized effector functions. However, the stimulus-response dynamics and discrimination patterns did not fit the M1/M2 continuum. Instead, our analysis suggests macrophage functional niches within a multi-dimensional polarization landscape.

HighlightsO_LIPolarization of macrophages affects stimulus-response NF{kappa}B dynamics
C_LIO_LIFor each condition, NF{kappa}B signaling codons quantify the "immune threat" level
C_LIO_LIMachine Learning reveals polarization-induced canalization of stimulus-responses
C_LIO_LINF{kappa}B stimulus-responses may define a landscape of macrophage states
C_LI

eTOC blurbMacrophages are profoundly responsive to their tissue microenvironment, but how that affects their pathogen response functions has not been investigated systematically. Here we studied how their signaling response is affected by six polarizing cytokines. We found each modulates their stimulus-responses highly specifically, producing distinct patterns of stimulus-discrimination. Thereby, these stimulus-response specificities may be used to describe a landscape of functional macrophage states.

Graphical Abstract

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title: Complete sequencing of the mouse pseudoautosomal region, the most rapidly evolving 'chromosome'

abstract: The pseudoautosomal region (PAR) of mammalian sex chromosomes is a small region of sequence identity that allows pairing, crossover, recombination, and proper chromosome segregation during male meiosis. The structure of the mouse PAR is largely unknown. Here, we developed a new assembly method to robustly resolve repetitive sequences and employed highly accurate long-read sequencing data to reveal the entire PAR sequence. The PAR of the widely-used inbred strain C57BL/6J is [~]700 kb, comprising 10 protein- coding genes in a mass of complex repetitive sequences. A large segmental duplication exhibiting copy-number polymorphisms even among C57BL/6J littermates is present. High GC-content exons and short introns are common properties of PAR genes and are the consequence of maintaining gene function, while PAR is rapidly evolving. Elucidating the mouse PAR sequence completes the mouse euchromatic genome sequencing and enables the exploration of the function and evolution of the PAR using modern molecular genetic approaches.


title: DNA Methylation Dynamics and Dysregulation Delineated by High-Throughput Profiling in the Mouse

abstract: We have developed a mouse Infinium DNA methylation array that contains 297,415 probes to capture the diversity of mouse DNA methylation biology. We present a mouse DNA methylation atlas as a rich reference resource of 1,239 DNA samples encompassing distinct tissues, strains, age, sex, and pathologies. We describe applications for comparative epigenomics, genomic imprinting, epigenetic inhibitors, PDX assessment, backcross tracing, and epigenetic clocks. We dissect DNA methylation processes associated with differentiation, aging and tumorigenesis. Notably, we find that tissue-specific methylation signatures localize to binding sites for transcription factors controlling the corresponding tissue development. Age-associated hypermethylation is enriched at regions of Polycomb repression, while hypomethylation is enhanced at regions bound by cohesin complex members. ApcMin/+ polyp-associated hypermethylation affects enhancers regulating intestinal differentiation, while hypomethylation targets AP-1 binding sites. This MM285 mouse array is widely accessible to the research community, and will accelerate future high sample-throughput studies in this important model organism.


title: The dynamics of adaptation to stress from standing genetic variation and de novo mutations

abstract: Adaptation from standing genetic variation is an important process underlying evolution in natural populations but we rarely get the opportunity to observe the dynamics of fitness changes in real time. Here, we used the power of microbial experimental evolution and whole population sequencing to track the phenotypic and genomic changes of genetically diverse yeast populations in environments with different stress levels. We found that populations rapidly and in parallel increased in fitness in stressful environments. The founders genetic diversity was quickly depleted, however, not to the same degree in all populations and environments. Some populations fixed all ancestral variation in < 30 generations while others maintained diversity across hundreds of generations. We also observed parallelism at the gene and pathway level. Specifically, we detected up to seven genes harbouring multiple independent mutations in different populations, and a general enrichment for mutations affecting downstream effectors of the high-osmolarity-glycerol pathway in three out of four environments. Adaptation to the most stressful environment was characterised by the fast evolution of functional haploidy, likely driven by standing genetic variation. Almost 40% of all populations contained aneuploidies (losses or gains of chromosomes) at least once during experimental evolution. Some aneuploidies were maintained for hundreds of generations in parallel in different replicates, suggesting they were adaptive. This work shows that experimental evolution is a great tool to address the interplay between standing variation and the influx of de novo mutations, leading to a better understanding of the demographic and environmental drivers and constraints of a populations capacity to adapt to environmental change.


title: High temperature increases centromere-mediated genome elimination frequency in Arabidopsis deficient in cenH3 or its assembly factor KNL2

abstract: Double haploid production is the most effective way of creating true-breeding lines in a single generation. In Arabidopsis, haploid induction via mutation of the centromere-specific histone H3 (cenH3) has been shown when outcrossed to wild-type. Here we report that a mutant of the cenH3 assembly factor KNL2 can be used as a haploid inducer. We elucidated that short temperature stress of the knl2 mutant increased the efficiency of haploid induction from 1 to 10%. Moreover, we have demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid inducing lines, suggesting that haploid inducing lines in crops can be identified in a naturally occurring or chemically induced mutant population, avoiding the GMO approach at any stage. In addition, we have shown that the cenh3-4 mutant, which does not induce haploids under standard growth conditions, functions as a haploid inducer after exposure to short temperature stress.


title: The Human Motoneuron Expression Signature is Defined by ALS-Related Genes

abstract:
In neurodegenerative diseases of the human spinal cord, such as amyotrophic lateral sclerosis (ALS), motoneurons are particularly vulnerable to degeneration. It is hypothesized that their large size contributes to disease susceptibility, but the link between genetic variants associated with disease and cell-type specific degeneration is not clear. We characterized human spinal cord cells using single-nucleus RNA-sequencing and protein profiling. We found that human motoneurons displayed a unique expression profile characterized by factors involved in cytoskeletal structure, cell size, and degenerative disease (including ALS-associated genes SOD1, NEFH, OPTN, TUBA4A, PRPH, and STMN2) and that protein expression of these genes correlated with larger cell size in tissue. This work suggests a motoneuron-specific signature underlies their selective vulnerability to neurodegeneration.

One-Sentence SummaryHuman spinal motoneurons preferentially express ALS genes, providing an explanation for their selective vulnerability to degeneration.


title: Tree species and genetic diversity increase productivity via functional diversity and trophic feedbacks

abstract: Addressing global biodiversity loss requires an expanded focus on multiple dimensions of biodiversity. While most studies have focused on the consequences of plant interspecific diversity, our mechanistic understanding of how the diversity within a given plant species (genetic diversity) affects plant productivity remains limited. Here, we use a tree species x genetic diversity experiment to disentangle the effects of species diversity and genetic diversity, and how they are related to tree functional diversity and trophic feedbacks. Tree species as well as genetic diversity increased tree productivity via increased tree functional diversity, reduced soil fungal diversity and marginally reduced herbivory. The effect of tree genetic diversity on productivity was partly different between tree species monocultures and mixtures: the functional diversity effect resulting from tree genetic diversity was only found in tree species monocultures, but the trophic feedbacks via herbivory were similar in species monocultures and mixtures. Given the complexity of interactions between tree species and genetic diversity, tree functional diversity and trophic feedbacks on productivity, we suggest that both tree species and genetic diversity should be considered in reforestation.

category: ecology


title: m6A RNA methylation impairs gene expression variability and reproductive thermotolerance in Arabidopsis

abstract: Plants are more susceptible to high temperature stress during reproductive development, which can cause drastic yield loss of fruit and seed crops. Unfortunately, the underlying mechanism remains largely unknown. Here we suggest that m6A RNA methylation level increases in the reproductive tissues of Arabidopsis and negatively regulates gene expression variability. It has been suggested that stochasticity of gene expression can be advantageous to fitness of living organisms under environmental challenges. Indeed, reduced gene expression variability in flowers was associated with compromised transcriptional activation of heat-responsive genes. Importantly, disruption of an RNA demethylase AtALKBH10B led to lower gene expression variability, hypo-responsiveness of heat-activated genes, and strong reduction of plant fertility. Overall, our work proposes a novel mechanism that m6A RNA modification mediates the bet-hedging strategy of plants challenged by heat stress.


title: Altered M2 Cortex - Superior Colliculus visual perception circuit in Huntington's Disease

abstract: The premotor cortical area -M2 cortex in rodents- connection to the striatum is involved in movement and prominently affected in Huntingtons Disease (HD). M2 cortex also projects to the superior colliculus (SC), implicated in oculomotor functions (i.e. saccades) and visual perception. Here, we investigated the contribution of M2 cortex - SC circuit to HD physiopathology in male mice. Using fMRI, we observed that M2 cortex functional connectivity with the SC was the most prominently affected circuit in the symptomatic R6/1 HD mouse model. Structural alterations were also detected by tractography and viral tracing. HD mice showed decreased defensive behavioral responses towards an unexpected visual stimuli, such as a moving robo-beetle, and decreased locomotion upon unexpected flash of light. Additionally, optogenetic M2 cortex - SC stimulation promoted avoidance responses towards the robo-beetle in WT, but not HD mice. Though, DWI measurements in vivo and ex vivo electrophysiological responses to optogenetic and electrical stimuli in the SC suggested preserved brain structure. Finally, GCamp6f fluorescence recordings with fiber photometry indicated that aberrant M2 cortex engagement might be the underlying mechanism of visual perception alterations in HD. Collectively, our findings point to a key role of M2 cortex - SC circuit alterations in HD pathophysiology.


title: Sudden unexpected death in epilepsy is prevented by blocking postictal hypoxia

abstract: Epilepsy is at times a fatal disease. Sudden unexpected death in epilepsy (SUDEP) is the leading cause of mortality in people with intractable epilepsy and is defined by exclusion; non-accidental, non-toxicologic, and non-anatomic causes of death. While SUDEP often follows a bilateral tonic-clonic seizure, the mechanisms that ultimately lead to terminal apnea and then asystole remain elusive and there is a lack preventative treatments. Based on the observation that discrete seizures lead to local vasoconstriction, resulting in hypoperfusion, hypoxia and behavioural disturbances in the forebrain (Farrell et al., 2016), we reasoned that similar mechanisms may play a role in SUDEP when seizures invade the brainstem. Here we tested this neurovascular-based hypothesis of SUDEP in awake non-anesthetized mice by pharmacologically preventing seizure-induced vasoconstriction, with cyclooxygenase-2 or L-type calcium channel antagonists. In both acute and chronic mouse models of SUDEP, ibuprofen and nicardipine extended life. We also examined the potential role of spreading depolarization in the acute model of SUDEP. These data provide a proof of principle for the neurovascular hypothesis of SUDEP and the use of currently available treatments to prevent it.


title: A toolkit of highly selective and sensitive genetically encoded neuropeptide sensors

abstract: Neuropeptides are key signaling molecules in the endocrine and nervous systems that regulate many critical physiological processes, including energy balance, sleep and circadian rhythms, stress, and social behaviors. Understanding the functions of neuropeptides in vivo requires the ability to monitor their dynamics with high specificity, sensitivity, and spatiotemporal resolution; however, this has been hindered by the lack of direct, sensitive and non-invasive tools. Here, we developed a series of GRAB (G protein-coupled receptor activation-based) sensors for detecting somatostatin (SST), cholecystokinin (CCK), corticotropin-releasing factor (CRF), neuropeptide Y (NPY), neurotensin (NTS), and vasoactive intestinal peptide (VIP). These fluorescent sensors utilize the corresponding GPCRs as the neuropeptide-sensing module with the insertion of a circular-permutated GFP as the optical reporter. This design detects the binding of specific neuropeptides at nanomolar concentration with a robust increase in fluorescence. We used these GRAB neuropeptide sensors to measure the spatiotemporal dynamics of endogenous SST release in isolated pancreatic islets and to detect the release of both CCK and CRF in acute brain slices. Moreover, we detect endogenous CRF release induced by stressful experiences in vivo using fiber photometry and 2-photon imaging in mice. Together, these new sensors establish a robust toolkit for studying the release, function, and regulation of neuropeptides under both physiological and pathophysiological conditions.


title: Nuclear receptor corepressor 1 controls regulatory T cell subset differentiation and effector function

abstract: FOXP3+ regulatory T cells (Tregs) are key for immune homeostasis. Tregs are a heterogenous population, however mechanisms regulating their transition from naive to effector Tregs (eTregs) are poorly understood. Here, we reveal a novel role for nuclear receptor corepressor 1 (NCOR1) in effector Tregs (eTregs). NCOR1 represses an effector signature in naive Tregs and NCOR1-deficiency increases the fraction of eTregs at steady-state accompanied with an upregulation of cholesterol biosynthesis pathways. Mechanistically, NCOR1-deficiency in murine and human Tregs results in enhanced expression of MYC, an essential driver of eTreg differentiation, resulting in enrichment of MYC target genes. Disruption of the interaction of liver X receptors (LXRs), crucial regulators of cholesterol biosynthesis, with NCOR1 by an LXR agonist leads to increased MYC expression in in vitro generated WT Tregs. Functionally, NCOR1 deficiency in Tregs compromises their ability to protect mice from severe weight loss and intestinal inflammation in adoptive CD4+ T cell transfer colitis. Our data uncover that an LXR-NCOR1 axis regulates eTreg differentiation, and that NCOR1 restrains MYC expression and eTreg differentiation and positively controls effector functions of Tregs.

category: immunology


title: Identifying transcription factor-bound gene activators and silencers in the chromatin accessible human genome using ATAC-STARR-seq

abstract: Massively parallel reporter assays test the capacity of putative gene regulatory elements to drive transcription on a genome-wide scale. Most gene regulatory activity occurs within accessible chromatin, and recently described methods have combined assays that capture these regions--such as assay for transposase-accessible chromatin using sequencing (ATAC-seq)--with self-transcribing active regulatory region sequencing (STARR-seq) to selectively assay the regulatory potential of accessible DNA (ATAC-STARR-seq). Here, we report a multi-omic approach that quantifies regulatory activity, chromatin accessibility, and transcription factor (TF) occupancy with one assay using ATAC-STARR-seq. Our strategy, including important updates to the ATAC-STARR-seq assay design and workflow, enabled high-resolution testing of [~]50 million unique DNA fragments tiling [~]101,000 accessible chromatin regions in human lymphoblastoid cells. We discovered that 30% of all accessible regions contain an activator, a silencer or both. We demonstrate that activators and silencers represent distinct functional groups that are enriched for unique sets of TF motifs and are marked by specific combinations of histone modifications. Using Tn5 cut-sites retained by the ATAC-STARR library, we performed TF footprinting and stratified these groups by the presence of specific TF footprints that are supported by chromatin immunoprecipitation data. We found that activators and silencers clustered by distinct TF footprint combinations are enriched for distinct gene regulatory pathways, and thus, represent distinct gene regulatory networks of human lymphoblastoid cell function. Altogether, these data highlight the multi-faceted capabilities of ATAC-STARR-seq to comprehensively investigate the regulatory landscape of the human genome all from a single DNA fragment source.


title: Integrated data-driven reannotation of the Kluyveromyces marxianus genome reveals an expanded protein coding repertoire

abstract: Many new biotechnology applications make use of non-traditional yeast species that possess characteristics and traits that are advantageous in specific settings. To a large extent, this is possible because of advances in molecular and genomic technologies that provide opportunities to study and manipulate the genomes of diverse yeasts. There remains, however, a substantial gap between what we know about well-studied models versus emerging industrial yeast species. In this study, we applied a multi-faceted omics analysis to better understand organisation of protein coding gene expression in Kluyveromyces marxianus, a yeast widely used in food and industrial biotechnology. We combined advanced transcriptomic techniques for mapping 5 and 3 ends of RNA transcripts with ribosome profiling to explore the transcriptional and translational landscapes of this yeast. This allowed us to improve the genome annotation and identify over 300 un-annotated or mis-annotated genes. We discovered numerous examples of novel proteoforms due to use of alternative transcription or translation start sites, many genes with translated upstream open reading frames (uORFs) and a novel case of programmed ribosomal frameshifting. Only some of newly discovered features are shared with distant species, for example, being present in Saccharomyces cerevisiae orthologs. The exploration of shared and unique features may provide an insight into the evolution of gene regulation in pre- and post-WGD yeasts. The processed data has been made available on the GWIPS-viz and Trips-Viz browsers, thus providing an accurate data-driven annotation of transcripts and their protein coding regions along with quantitative information on their transcription and translation.


title: Interactions between anthropogenic stressors and recurring perturbations mediate ecosystem resilience or collapse

abstract: Insights into declines in ecosystem resilience, their causes and effects, can inform pre-emptive action to avoid ecosystem collapse and loss of biodiversity, ecosystem services and human well-being. Empirical studies of ecosystem collapse are rare and hampered by ecosystem complexity, non-linear and lagged responses, and interactions across scales. We investigated how an anthropogenic stressor could diminish ecosystem resilience to a recurring perturbation by altering a critical ecosystem driver. We studied groundwater-dependent, peat-accumulating, fire-prone wetlands in southeastern Australia. We hypothesised that underground mining (stressor) reduced resilience of these wetlands to landscape fires (perturbation) by diminishing groundwater, a key ecosystem driver. We monitored soil moisture as an indicator of ecosystem resilience during and after underground mining and, after a landscape fire, we compared the responses of multiple state variables representing ecosystem structure, composition and function in wetlands within the mining footprint to unmined reference wetlands. Soil moisture showed very strong evidence of decline without recovery in mined swamps, but was maintained in reference swamps through eight years. Relative to burnt reference swamps, burnt and mined swamps showed greater loss of peat via substrate combustion, reduced cover, height and biomass of regenerating vegetation, reduced post-fire plant species richness and abundance, altered plant species composition, increased mortality rates of woody plants, reduced post-fire seedling recruitment, and local extinction of a hydrophilc fauna species. Mined swamps therefore showed strong symptoms of post-fire ecosystem collapse, while reference swamps regenerated vigorously. We conclude that an anthropogenic stressor may diminish the resilience of an ecosystem to recurring perturbations, predisposing it to collapse. Avoidance of ecosystem collapse hinges on early diagnosis of mechanisms and preventative risk reduction. It may be possible to delay or ameliorate symptoms of collapse or to restore resilience, but the latter appears unlikely in our study system due to fundamental alteration of a critical ecosystem driver.


title: Quantifying cerebral blood arrival times using hypoxia-mediated arterial BOLD contrast

abstract: Cerebral blood arrival and tissue transit times are sensitive measures of the efficiency of tissue perfusion and can provide clinically meaningful information on collateral blood flow status. We exploit the arterial blood oxygen level dependent (BOLD) signal contrast established by precisely modulating arterial hemoglobin saturation using hypoxic respiratory challenges (dOHb-BOLD) to quantify arterial blood arrival times throughout the brain. A combination of hemodynamic lag with a modified carpet plot analysis yielded lag, onset (blood arrival), mean transit time (MTT) and hypoxic response information, which is indicative of relative total blood volume. Onset times averaged across 12 healthy subjects were 1.1 {+/-} 0.4 and 1.9 {+/-} 0.6 for cortical gray and deep white matter, respectively. The average whole brain MTT was 4.5 {+/-} 0.9 seconds. The dOHb-BOLD response was 1.7 fold higher in grey versus white; in line with known differences in regional blood volume fraction. Our method was also applied in unilateral carotid artery occlusion patient, which revealed prolonged signal onset with normal perfusion in the affected hemisphere. In cases with exhausted reserve capacity or confounding flow effects such as vascular steal, dOHb-BOLD can potentially inform on collateral flow pathways to provide a valuable compliment to clinical vascular reactivity measures.


title: Tract-specific white matter microstructure alterations among young adult APOE ε4 carriers: A replication and extension study

abstract: The parahippocampal cingulum bundle (PHCB) connects regions known to be vulnerable to early Alzheimers disease (AD) pathology, such as posteromedial cortex and medial temporal lobe. While AD-related pathology has been robustly associated with alterations in PHCB microstructure, specifically lower fractional anisotropy (FA) and higher mean diffusivity (MD), emerging evidence indicates that the reverse pattern is evident in younger adults at-risk of AD. In one such study, Hodgetts et al. (2019) reported that healthy young adult carriers of the apolipoprotein-E (APOE) {varepsilon}4 allele - the strongest common genetic risk factor for AD - showed higher FA and lower MD in the PHCB but not the inferior longitudinal fasciculus (ILF). These results are consistent with proposals claiming that heightened neural activity and connectivity have a significant role in posteromedial cortex vulnerability to amyloid-{beta} and tau spread beyond the medial temporal lobe. Given the implications for understanding AD risk, here we sought to replicate Hodgetts et al.s finding in a larger sample (N = 128; 40 APOE {varepsilon}4 carriers, 88 APOE {varepsilon}4 non-carriers) of young adults (age range: 19-33). Extending this work further, we also conducted exploratory analyses using a more advanced measure of microstructure: hindrance modulated orientational anisotropy (HMOA). These analyses included an investigation of hemispheric asymmetry in PHCB and ILF HMOA. Contrary to the original study, we observed no difference in PHCB microstructure between APOE {varepsilon}4 carriers and non-carriers. Bayes factors (BFs) further revealed moderate-to-strong evidence in support of these null findings. APOE {varepsilon}4-related differences in ILF HMOA asymmetry were evident, however, with carriers demonstrating lower leftward asymmetry. Our findings indicate that young adult APOE {varepsilon}4 carriers do not show alterations in PHCB microstructure, as observed by Hodgetts et al., but may show altered asymmetry in ILF microstructure.


title: Rv0233 is not essential for the survival of Mycobacterium tuberculosis in stress conditions

abstract: Mycobacterium tuberculosis is an important global pathogen. We were interested in understanding the role of Rv0233, a proposed subunit of the class IB ribonucleotide reductase. We constructed an in-frame, unmarked deletion strain of M. tuberculosis and characterized its growth and survival under replicating and non-replicating conditions. We confirmed previous studies that Rv0233 is not essential for aerobic growth or survival in the presence of nitrite. We demonstrate that the deletion of Rv0233 does not affect susceptibility to frontline tuberculosis drugs or hydrogen peroxide. The deletion strain survived equally well under nutrient starvation or in hypoxia and was not attenuated for growth in macrophages.

category: microbiology


title: The costs of global protected-area expansion (Target 3 of the post-2020 Global Biodiversity Framework) may fall more heavily on lower-income countries

abstract: One of the biggest stumbling blocks for global environmental agreements is how higher-income and lower-income countries share the costs of implementing them. This problem has become particularly acute as biodiversity and climate ambitions have increased across recent COPs (Conferences of Parties). Here, we estimate the likely distribution of costs for one of the most ambitious proposals: draft Target 3 of the Global Biodiversity Framework (GBF), which would increase coverage of protected and conserved areas (PCAs) to 30% of global land and sea area - more than triple the current value. Since the GBF does not specify where new PCAs would be placed, we use three scenarios of how Target 3 might be implemented, cost those scenarios, and then compare the mean distribution of costs across World Bank income groups. We find that in relative terms, lower-income countries could face considerably larger financial burdens than high-income countries, even though the benefits of conservation are disproportionately enjoyed by high-income countries. Lower-income countries would also face larger increases in the amount of land or sea under conservation, implying higher opportunity and establishment costs. Resolving this potential cost-sharing inequity may be a key requirement to achieve consensus on draft Target 3, and indeed on ambitious environmental proposals more generally.


title: SCExecute: cell barcode-stratified analyses of scRNA-seq data

abstract:
MotivationIn single-cell RNA-sequencing (scRNA-seq) data, stratification of sequencing reads by cellular barcode is necessary to study cell-specific features. However, apart from gene expression, the analyses of cell-specific features are not supported by available tools that are designed for bulk RNA-Seq data.

ResultsWe introduce a tool - SCExecute - which executes a user-provided command on barcode-stratified, extracted on-the-fly, single cell binary alignment map (scBAM) files. SCExecute extracts the cell barcode from aligned, pooled single-cell sequencing data. The user-specified command option executes all the commands defined in the session from monolithic programs and multi-command shell-scripts to complex shell-based pipelines. The execution can be further restricted to barcodes or/and genomic regions of interest. We demonstrate SCExecute with two popular variant callers - GATK and Strelka2 - combined with modules for bam file manipulation and variant filtering, to detect single cell-specific expressed Single Nucleotide Variants (sceSNVs) from droplet scRNA-seq data (10X Genomics Chromium System).

ConclusionSCExecute facilitates custom cell-level analyses on barcoded scRNA-seq data using currently available tools and provides an effective solution for studying low (cellular) frequency transcriptome features.

AvailabilitySCExecute is implemented in Python3 using the PySAM package and distributed for Linux and Python environments from https://github.com/HorvathLab/NGS/tree/master/SCExecute.

category: genomics


title: "Two-story building" of a ctenophore comb plate provides structural and functional integrity for motility of giant multicilia

abstract: Comb plates (CPs) are large compound cilia uniquely seen in a basal metazoan group of ctenophores.1-3 Tens of thousands of cilia are bundled in a CP via structures connecting adjacent cilia, called compartmenting lamella (CL), which are the basis for the structural iridescent color and the coordination of ciliary movement of the CP.4-6 We previously reported the first component of CL, CTENO64, and found that it was convergently acquired in ctenophores and was essential for determination of ciliary orientation.3 However, CTENO64 is localized only in the proximal region of the CL; therefore, the molecular architecture of CL over the entire length has not been elucidated. Here, we identified a second CL component, CTENO189. This ctenophore-specific protein was present in the distal region of the CL, with a localization clearly segregated from CTENO64. Knockdown of the CTENO189 gene with morpholino antisense oligonucleotides resulted in complete loss of CLs in the distal region, but did not affect either the formation of CP or the orientation of each cilium. However, the hexagonal distribution of cilia was disarranged, and the metachronal coordination of CP along a comb row was lost in the CTENO189 morphants. The morphant CP showed asymmetric ciliary-type movement in normal seawater, and in a high-viscosity solution, it could not maintain the normal waveforms, becoming a symmetric flagellar-type. Our findings demonstrate a "two-story building" of CP, comprising the proximal CL, as the building foundation that rigidly fixes the ciliary orientation. The distal CL would reinforce the elastic connection among cilia to overcome the hydrodynamic drag of giant multiciliary plates.


title: Osmotic stress via calmodulin lead to the formation of stress granule in Drosophila S2 cells.

abstract: Cellular stress of S2 cells leads to the formation of stress assemblies by phase separation of cytoplasmic components. We have shown that the cellular stresses of either high increase of the NaCl concentration in the extracellular medium, or a moderate one combined to amino acid starvation, leads to the formation of Sec bodies where components of the endoplasmic Reticulum exit sites (ERES) coalesce. These extracellular stresses lead to both the activation of salt inducible kinase (SIK), and to ER stress triggering the activation of the two downstream kinases IRE1 and PERK. Interestingly, the same stresses also result in the formation of a second stress assembly, the stress granules, which stores specific RNAs and RNA binding proteins. Here we asked whether stress granule formation is governed by the same pathways as Sec bodies. However, we found that the inhibition of SIK, IRE1 and PERK does not affect stress granule formation. Instead, we found that osmotic stress through the addition of either salts (including calcium chloride) or sucrose leads to the formation of stress granules. Interestingly, stress granule formation is partly modulated by calmodulin activation, suggesting the involvement of calcium signaling. Furthermore, as Sec body formation is driven by entirely different pathways, these results show that the same cells under the same stress, form two different stress assemblies by non-overlapping downstream pathway activation, perhaps explaining that they do not coalescence into a single structure.


title: A Coiled-Coil-Based Design Strategy for the Thermostabilization of G-Protein-Coupled Receptors

abstract: The most common methods for generating crystallizable GPCRs are scanning alanine mutagenesis and fusion to crystallization-facilitating partner proteins. The major goal of our work was to create a new GPCR tool that would provide receptor stability and additional soluble surface for crystallization. Towards this aim, we selected the two-stranded antiparallel coiled coil as a domain fold that satisfies both criteria. A selection of antiparallel coiled coils was used for structure-guided substitution of intracellular loop of the {beta}3 adrenergic receptor. Unexpectedly, only the two GPCR variants containing thermostable coiled coils were expressed. We showed that one GPCR chimera is stable upon purification in detergent, retains ligand-binding properties, and can be crystallized. However, the quality of the crystals was not suitable for structure determination. To supply additional surface for promoting crystal contacts, we replaced in a structure-based approach the loop of the antiparallel coiled coil by T4L. Although expression is currently not suitable for structural work, we found that the engineered GPCR is even more stable than the coiled-coil variant. Our approach should be of interest for applications that benefit from stable GPCRs.

category: biophysics


title: Calcineurin-fusion facilitates Cryo-EM Structure Determination of a Family A GPCR

abstract: Advances in singe-particle cryo-electron microscopy (cryo-EM) have made possible to solve the structures of numerous Family A and Family B G protein coupled receptors (GPCRs) in complex with G proteins and arrestins, as well as several Family C GPCRs. Determination of these structures has been facilitated by the presence of large extra-membrane components (such as G protein, arrestin, or Venus flytrap domains) in these complexes that aid in particle alignment during processing of the cryo-EM data. In contrast, determination of the inactive state structure of Family A GPCRs is more challenging due to the relatively small size of the seven transmembrane domain (7TM) and to the surrounding detergent micelle that, in the absence of other features, make particle alignment impossible. Here we describe an alternative protein engineering strategy where the heterodimeric protein calcineurin is fused to a GPCR by three points of attachment, the cytoplasmic ends of TM5, TM6 and TM7. This three-point attachment provides a more rigid link with the GPCR transmembrane domain that facilitates particle alignment during data processing, allowing us to determine the structures of the {beta}2 adrenergic receptor ({beta}2AR) in the apo, antagonist-bound, and agonist-bound states. We expect that this fusion strategy may have broad application in cryo-EM structural determination of other Family A GPCRs.


title: Extracellular vesicle fusion visualized by cryo-EM

abstract: Extracellular vesicles (EVs) transfer bioactive molecules between cells in a process reminiscent of enveloped viruses. EV cargo delivery is thought to occur by protein-mediated and pH-dependent membrane fusion of the EV and the cellular membrane. However, there is a lack of methods to identify the fusion proteins and resolve their mechanism. We developed and benchmarked an in vitro biophysical assay to investigate EV membrane fusion. The assay was standardized by directly comparing EV- and viral-fusion with liposomes. We show that EVs and retroviruses fuse with liposomes mimicking the membrane composition of the late endosome in a pH and protein-dependent manner. Moreover, we directly visualize the stages of membrane fusion using cryo-electron tomography. We find that, unlike most retroviruses, EVs remain fusogenic after acidification and re-neutralization. These results provide novel insights into the EV cargo delivery mechanism and an experimental approach to identify the EV fusion machinery.


title: Nanobubbles are Non-Echogenic for Fundamental-Mode Contrast-Enhanced Ultrasound Imaging

abstract: Microbubbles (1-10 {micro}m diameter) have been used as conventional ultrasound contrast agents (UCAs) for applications in contrast-enhanced ultrasound (CEUS) imaging. Nanobubbles (<1 {micro}m diameter) have recently been proposed as potential extravascular UCAs that can extravasate from the leaky vasculature of tumors or sites of inflammation. However, the echogenicity of nanobubbles for CEUS remains controversial owing to prior studies that have shown very low ultrasound backscatter. We hypothesize that microbubble contamination in nanobubble formulations may explain the discrepancy. To test our hypothesis, we examined the size distributions of lipid-coated nanobubble and microbubble suspensions using multiple sizing techniques, examined their echogenicity in an agar phantom with fundamental-mode CEUS at 7 MHz and 330 kPa peak negative pressure, and interpreted our results with simulations of the modified Rayleigh-Plesset model. We found that nanobubble formulations contained a small contamination of microbubbles. Once the contribution from these microbubbles is removed from the acoustic backscatter, the acoustic contrast of the nanobubbles was shown to be near noise levels. This result indicates that nanobubbles have limited utility as UCAs for CEUS.

category: bioengineering


title: Phase-separated TRB-PRC2 aggregates contribute to Polycomb silencing in plants

abstract:
Local accumulation of Polycomb Repressive Complex 2 (PRC2) is essential to gene silencing. Liquid-liquid phase separation (LLPS) mechanism is emerging as a paradigm to concentrate transcriptional machinery for effective gene regulation. Here, we elucidate that a rice single Myb transcription factor TRBF2 forms phase-separated droplets, which aggregate with PRC2 through direct protein interaction. Furthermore, TRB1, the closest homolog of TRBF2 in Arabidopsis, also forms phase-separated aggregates with PRC2. Mutants of TRBF2 and PRC2 component CLF display similar developmental defects, share common differentially expressed genes, and reduced H3K27me3 chromatin regions. Chromatin immunoprecipitation analysis supports that TRBF2 concentrates PRC2 at target loci to promote H3K27me3 deposition. Therefore, we propose that the aggregation of the plant-specific TRBs with PRC2 by the LLPS mechanism contributes to Polycomb silencing.

One-Sentence SummaryThe phase-separated plant-specific single Myb transcription factor aggregates with PRC2 to facilitate Polycomb silencing.

category: plant biology


title: Histone marks retained during epigenetic reprogramming and their roles essential for fish early development

abstract: Reprograming of epigenetic modifications after fertilization is required for proper embryonic development and cell differentiation. However, histone modifications that escape reprogramming in non-mammalian vertebrates and their potential functional roles are poorly understood. Here, we quantitatively analyzed histone modification dynamics during reprogramming in Japanese Killifish, medaka (Oryzias latipes) embryos, and revealed that H3K27ac, H3K27me3 and H3K9me3 are retained, while H3K4 methylation is completely erased. Furthermore, we experimentally demonstrated the functional roles of such retained modifications at early stages; H3K27ac at promoters is required for proper patterning of H3K4 and H3K27 methylation at zygotic genome activation (ZGA) and specific retention of H3K9me3 at telomeric regions maintains genomic stability during cleavage stage. These results expand the understanding of diversity and conservation of reprogramming in vertebrates and unveil previously uncharacterized functions of histone modifications retained during epigenetic reprogramming.