IgE-mediated activation of mast cells and basophils contributes to protective immunity against helminths but also causes allergic responses. The development and persistence of IgE responses are poorly understood, which is in part due to the low number of IgE-producing cells. Here, we used next generation sequencing to uncover a striking overlap between the IgE and IgG1 repertoires in helminth-infected or OVA/alum-immunized wild-type BALB/c mice. The memory IgE response after secondary infection induced a strong increase of IgE+ plasma cells in spleen and lymph nodes. In contrast, germinal center B cells did not increase during secondary infection. Unexpectedly, the memory IgE response was lost in mice where the extracellular part of IgG1 had been replaced with IgE sequences. Adoptive transfer studies revealed that IgG1+ B cells were required and sufficient to constitute the memory IgE response in recipient mice. T cell-derived IL-4/IL-13 was required for the memory IgE response but not for expansion of B cells from memory mice. Together, our results reveal a close relationship between the IgE and IgG1 repertoires in vivo and demonstrate that the memory IgE response is mainly conserved at the level of memory IgG1+ B cells. Therefore, targeting the generation and survival of allergen-specific IgG1+ B cells could lead to development of new therapeutic strategies to treat chronic allergic disorders.

Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant-virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses.

Plants produce large amounts of secondary metabolites in their shoots and roots and store them in specialized secretory structures. Although secondary metabolites and their secretory structures are commonly assumed to have a defensive function, evidence that they benefit plant fitness under herbivore attack is scarce, especially below ground. Here, we tested whether latex secondary metabolites produced by the common dandelion (Taraxacum officinale agg.) decrease the performance of its major native insect root herbivore, the larvae of the common cockchafer (Melolontha melolontha), and benefit plant vegetative and reproductive fitness under M. melolontha attack. Across 17 T. officinale genotypes screened by gas and liquid chromatography, latex concentrations of the sesquiterpene lactone taraxinic acid β-D-glucopyranosyl ester (TA-G) were negatively associated with M. melolontha larval growth. Adding purified TA-G to artificial diet at ecologically relevant concentrations reduced larval feeding. Silencing the germacrene A synthase ToGAS1, an enzyme that was identified to catalyze the first committed step of TA-G biosynthesis, resulted in a 90% reduction of TA-G levels and a pronounced increase in M. melolontha feeding. Transgenic, TA-G-deficient lines were preferred by M. melolontha and suffered three times more root biomass reduction than control lines. In a common garden experiment involving over 2,000 T. officinale individuals belonging to 17 different genotypes, high TA-G concentrations were associated with the maintenance of high vegetative and reproductive fitness under M. melolontha attack. Taken together, our study demonstrates that a latex secondary metabolite benefits plants under herbivore attack, a result that provides a mechanistic framework for root herbivore driven natural selection and evolution of plant defenses below ground.

The source of symmetry breaking in vertebrate oocytes is unknown. Animal-vegetal oocyte polarity is established by the Balbiani body (Bb), a conserved structure found in all animals examined that contains an aggregate of specific mRNAs, proteins, and organelles. The Bb specifies the oocyte vegetal pole, which is key to forming the embryonic body axes as well as the germline in most vertebrates. How Bb formation is regulated and how its asymmetric position is established are unknown. Using quantitative image analysis, we trace oocyte symmetry breaking in zebrafish to a nuclear asymmetry at the onset of meiosis called the chromosomal bouquet. The bouquet is a universal feature of meiosis where all telomeres cluster to one pole on the nuclear envelope, facilitating chromosomal pairing and meiotic recombination. We show that Bb precursor components first localize with the centrosome to the cytoplasm adjacent to the telomere cluster of the bouquet. They then aggregate around the centrosome in a specialized nuclear cleft that we identified, assembling the early Bb. We show that the bouquet nuclear events and the cytoplasmic Bb precursor localization are mechanistically coordinated by microtubules. Thus the animal-vegetal axis of the oocyte is aligned to the nuclear axis of the bouquet. We further show that the symmetry breaking events lay upstream to the only known regulator of Bb formation, the Bucky ball protein. Our findings link two universal features of oogenesis, the Bb and the chromosomal bouquet, to oocyte polarization. We propose that a meiotic-vegetal center couples meiosis and oocyte patterning. Our findings reveal a novel mode of cellular polarization in meiotic cells whereby cellular and nuclear polarity are aligned. We further reveal that in zygotene nests, intercellular cytoplasmic bridges remain between oocytes and that the position of the cytoplasmic bridge coincides with the location of the centrosome meiotic-vegetal organizing center. These results suggest that centrosome positioning is set by the last mitotic oogonial division plane. Thus, oocytes are polarized in two steps: first, mitotic divisions preset the centrosome with no obvious polarization yet, then the meiotic-vegetal center forms at zygotene bouquet stages, when symmetry is, in effect, broken.

Leukotriene B4 (LTB4) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB4 and its synthesizing enzymes localize to intracellular multivesicular bodies that, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in a LTB4 receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB4 release. Our findings establish that the exosomal pool of LTB4 acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments.

During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis-free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.

In a process called quorum sensing, bacteria communicate with chemical signal molecules called autoinducers to control collective behaviors. In pathogenic vibrios, including Vibrio cholerae, the accumulation of autoinducers triggers repression of genes responsible for virulence factor production and biofilm formation. The vibrio autoinducer molecules bind to transmembrane receptors of the two-component histidine sensor kinase family. Autoinducer binding inactivates the receptors' kinase activities, leading to dephosphorylation and inhibition of the downstream response regulator LuxO. Here, we report the X-ray structure of LuxO in its unphosphorylated, autoinhibited state. Our structure reveals that LuxO, a bacterial enhancer-binding protein of the AAA+ ATPase superfamily, is inhibited by an unprecedented mechanism in which a linker that connects the catalytic and regulatory receiver domains occupies the ATPase active site. The conformational change that accompanies receiver domain phosphorylation likely disrupts this interaction, providing a mechanistic rationale for LuxO activation. We also determined the crystal structure of the LuxO catalytic domain bound to a broad-spectrum inhibitor. The inhibitor binds in the ATPase active site and recapitulates elements of the natural regulatory mechanism. Remarkably, a single inhibitor molecule may be capable of inhibiting an entire LuxO oligomer.

Planarians display remarkable plasticity in maintenance of their germline, with the ability to develop or dismantle reproductive tissues in response to systemic and environmental cues. Here, we investigated the role of G protein-coupled receptors (GPCRs) in this dynamic germline regulation. By genome-enabled receptor mining, we identified 566 putative planarian GPCRs and classified them into conserved and phylum-specific subfamilies. We performed a functional screen to identify NPYR-1 as the cognate receptor for NPY-8, a neuropeptide required for sexual maturation and germ cell differentiation. Similar to NPY-8, knockdown of this receptor results in loss of differentiated germ cells and sexual maturity. NPYR-1 is expressed in neuroendocrine cells of the central nervous system and can be activated specifically by NPY-8 in cell-based assays. Additionally, we screened the complement of GPCRs with expression enriched in sexually reproducing planarians, and identified an orphan chemoreceptor family member, ophis, that controls differentiation of germline stem cells (GSCs). ophis is expressed in somatic cells of male and female gonads, as well as in accessory reproductive tissues. We have previously shown that somatic gonadal cells are required for male GSC specification and maintenance in planarians. However, ophis is not essential for GSC specification or maintenance and, therefore, defines a secondary role for planarian gonadal niche cells in promoting GSC differentiation. Our studies uncover the complement of planarian GPCRs and reveal previously unappreciated roles for these receptors in systemic and local (i.e., niche) regulation of germ cell development.

The bulk polar movement of the plant signaling molecule auxin through the stem is a long-recognized but poorly understood phenomenon. Here we show that the highly polar, high conductance polar auxin transport stream (PATS) is only part of a multimodal auxin transport network in the stem. The dynamics of auxin movement through stems are inconsistent with a single polar transport regime and instead suggest widespread low conductance, less polar auxin transport in the stem, which we term connective auxin transport (CAT). The bidirectional movement of auxin between the PATS and the surrounding tissues, mediated by CAT, can explain the complex auxin transport kinetics we observe. We show that the auxin efflux carriers PIN3, PIN4, and PIN7 are major contributors to this auxin transport connectivity and that their activity is important for communication between shoot apices in the regulation of shoot branching. We propose that the PATS provides a long-range, consolidated stream of information throughout the plant, while CAT acts locally, allowing tissues to modulate and be modulated by information in the PATS.

Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages) in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR)-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent.

During the first meiotic division, crossovers (COs) between homologous chromosomes ensure their correct segregation. COs are produced by homologous recombination (HR)-mediated repair of programmed DNA double strand breaks (DSBs). As more DSBs are induced than COs, mechanisms are required to establish a regulated number of COs and to repair remaining intermediates as non-crossovers (NCOs). We show that the Caenorhabditis elegans RMI1 homolog-1 (RMH-1) functions during meiosis to promote both CO and NCO HR at appropriate chromosomal sites. RMH-1 accumulates at CO sites, dependent on known pro-CO factors, and acts to promote CO designation and enforce the CO outcome of HR-intermediate resolution. RMH-1 also localizes at NCO sites and functions in parallel with SMC-5 to antagonize excess HR-based connections between chromosomes. Moreover, RMH-1 also has a major role in channeling DSBs into an NCO HR outcome near the centers of chromosomes, thereby ensuring that COs form predominantly at off-center positions.

There is no unified place where genomics researchers can search through all available raw genomic data in a way similar to OMIM for genes or Uniprot for proteins. With the recent increase in the amount of genomic data that is being produced and the ever-growing promises of precision medicine, this is becoming more and more of a problem. DNAdigest is a charity working to promote efficient sharing of human genomic data to improve the outcome of genomic research and diagnostics for the benefit of patients. Repositive, a social enterprise spin-out of DNAdigest, is building an online platform that indexes genomic data stored in repositories and thus enables researchers to search for and access a range of human genomic data sources through a single, easy-to-use interface, free of charge.

Functional residues in proteins tend to be highly conserved over evolutionary time. However, to what extent functional sites impose evolutionary constraints on nearby or even more distant residues is not known. Here, we report pervasive conservation gradients toward catalytic residues in a dataset of 524 distinct enzymes: evolutionary conservation decreases approximately linearly with increasing distance to the nearest catalytic residue in the protein structure. This trend encompasses, on average, 80% of the residues in any enzyme, and it is independent of known structural constraints on protein evolution such as residue packing or solvent accessibility. Further, the trend exists in both monomeric and multimeric enzymes and irrespective of enzyme size and/or location of the active site in the enzyme structure. By contrast, sites in protein-protein interfaces, unlike catalytic residues, are only weakly conserved and induce only minor rate gradients. In aggregate, these observations show that functional sites, and in particular catalytic residues, induce long-range evolutionary constraints in enzymes.

The evolution of heteromorphic sex chromosomes has repeatedly resulted in the evolution of sex chromosome-specific forms of regulation, including sex chromosome dosage compensation in the soma and meiotic sex chromosome inactivation in the germline. In the male germline of Drosophila melanogaster, a novel but poorly understood form of sex chromosome-specific transcriptional regulation occurs that is distinct from canonical sex chromosome dosage compensation or meiotic inactivation. Previous work shows that expression of reporter genes driven by testis-specific promoters is considerably lower-approximately 3-fold or more-for transgenes inserted into X chromosome versus autosome locations. Here we characterize this transcriptional suppression of X-linked genes in the male germline and its evolutionary consequences. Using transgenes and transpositions, we show that most endogenous X-linked genes, not just testis-specific ones, are transcriptionally suppressed several-fold specifically in the Drosophila male germline. In wild-type testes, this sex chromosome-wide transcriptional suppression is generally undetectable, being effectively compensated by the gene-by-gene evolutionary recruitment of strong promoters on the X chromosome. We identify and experimentally validate a promoter element sequence motif that is enriched upstream of the transcription start sites of hundreds of testis-expressed genes; evolutionarily conserved across species; associated with strong gene expression levels in testes; and overrepresented on the X chromosome. These findings show that the expression of X-linked genes in the Drosophila testes reflects a balance between chromosome-wide epigenetic transcriptional suppression and long-term compensatory adaptation by sex-linked genes. Our results have broad implications for the evolution of gene expression in the Drosophila male germline and for genome evolution.

The detailed know-how to implement research protocols frequently remains restricted to the research group that developed the method or technology. This knowledge often exists at a level that is too detailed for inclusion in the methods section of scientific articles. Consequently, methods are not easily reproduced, leading to a loss of time and effort by other researchers. The challenge is to develop a method-centered collaborative platform to connect with fellow researchers and discover state-of-the-art knowledge. Protocols.io is an open-access platform for detailing, sharing, and discussing molecular and computational protocols that can be useful before, during, and after publication of research results.

There is a need for formalised diagrams that both summarise current biological pathway knowledge and support modelling approaches that explain and predict their behaviour. Here, we present a new, freely available modelling framework that includes a biologist-friendly pathway modelling language (mEPN), a simple but sophisticated method to support model parameterisation using available biological information; a stochastic flow algorithm that simulates the dynamics of pathway activity; and a 3-D visualisation engine that aids understanding of the complexities of a system's dynamics. We present example pathway models that illustrate of the power of approach to depict a diverse range of systems.

Recent computational and behavioral studies suggest that motor adaptation results from the update of multiple memories with different timescales. Here, we designed a model-based functional magnetic resonance imaging (fMRI) experiment in which subjects adapted to two opposing visuomotor rotations. A computational model of motor adaptation with multiple memories was fitted to the behavioral data to generate time-varying regressors of brain activity. We identified regional specificity to timescales: in particular, the activity in the inferior parietal region and in the anterior-medial cerebellum was associated with memories for intermediate and long timescales, respectively. A sparse singular value decomposition analysis of variability in specificities to timescales over the brain identified four components, two fast, one middle, and one slow, each associated with different brain networks. Finally, a multivariate decoding analysis showed that activity patterns in the anterior-medial cerebellum progressively represented the two rotations. Our results support the existence of brain regions associated with multiple timescales in adaptation and a role of the cerebellum in storing multiple internal models.

Translational errors occur at high rates, and they influence organism viability and the onset of genetic diseases. To investigate how organisms mitigate the deleterious effects of protein synthesis errors during evolution, a mutant yeast strain was engineered to translate a codon ambiguously (mistranslation). It thereby overloads the protein quality-control pathways and disrupts cellular protein homeostasis. This strain was used to study the capacity of the yeast genome to compensate the deleterious effects of protein mistranslation. Laboratory evolutionary experiments revealed that fitness loss due to mistranslation can rapidly be mitigated. Genomic analysis demonstrated that adaptation was primarily mediated by large-scale chromosomal duplication and deletion events, suggesting that errors during protein synthesis promote the evolution of genome architecture. By altering the dosages of numerous, functionally related proteins simultaneously, these genetic changes introduced large phenotypic leaps that enabled rapid adaptation to mistranslation. Evolution increased the level of tolerance to mistranslation through acceleration of ubiquitin-proteasome-mediated protein degradation and protein synthesis. As a consequence of rapid elimination of erroneous protein products, evolution reduced the extent of toxic protein aggregation in mistranslating cells. However, there was a strong evolutionary trade-off between adaptation to mistranslation and survival upon starvation: the evolved lines showed fitness defects and impaired capacity to degrade mature ribosomes upon nutrient limitation. Moreover, as a response to an enhanced energy demand of accelerated protein turnover, the evolved lines exhibited increased glucose uptake by selective duplication of hexose transporter genes. We conclude that adjustment of proteome homeostasis to mistranslation evolves rapidly, but this adaptation has several side effects on cellular physiology. Our work also indicates that translational fidelity and the ubiquitin-proteasome system are functionally linked to each other and may, therefore, co-evolve in nature.

Evidence has accumulated in recent decades on the drastic impact of climate change on biodiversity. Warming temperatures have induced changes in species physiology, phenology, and have decreased body size. Such modifications can impact population dynamics and could lead to changes in life cycle and demography. More specifically, conceptual frameworks predict that global warming will severely threaten tropical ectotherms while temperate ectotherms should resist or even benefit from higher temperatures. However, experimental studies measuring the impacts of future warming trends on temperate ectotherms' life cycle and population persistence are lacking. Here we investigate the impacts of future climates on a model vertebrate ectotherm species using a large-scale warming experiment. We manipulated climatic conditions in 18 seminatural populations over two years to obtain a present climate treatment and a warm climate treatment matching IPCC predictions for future climate. Warmer temperatures caused a faster body growth, an earlier reproductive onset, and an increased voltinism, leading to a highly accelerated life cycle but also to a decrease in adult survival. A matrix population model predicts that warm climate populations in our experiment should go extinct in around 20 y. Comparing our experimental climatic conditions to conditions encountered by populations across Europe, we suggest that warming climates should threaten a significant number of populations at the southern range of the distribution. Our findings stress the importance of experimental approaches on the entire life cycle to more accurately predict population and species persistence in future climates.

How did evolution generate the extraordinary diversity of vertebrates on land? Zero species are known prior to ~380 million years ago, and more than 30,000 are present today. An expansionist model suggests this was achieved by large and unbounded increases, leading to substantially greater diversity in the present than at any time in the geological past. This model contrasts starkly with empirical support for constrained diversification in marine animals, suggesting different macroevolutionary processes on land and in the sea. We quantify patterns of vertebrate standing diversity on land during the Mesozoic-early Paleogene interval, applying sample-standardization to a global fossil dataset containing 27,260 occurrences of 4,898 non-marine tetrapod species. Our results show a highly stable pattern of Mesozoic tetrapod diversity at regional and local levels, underpinned by a weakly positive, but near-zero, long-term net diversification rate over 190 million years. Species diversity of non-flying terrestrial tetrapods less than doubled over this interval, despite the origins of exceptionally diverse extant groups within mammals, squamates, amphibians, and dinosaurs. Therefore, although speciose groups of modern tetrapods have Mesozoic origins, rates of Mesozoic diversification inferred from the fossil record are slow compared to those inferred from molecular phylogenies. If high speciation rates did occur in the Mesozoic, then they seem to have been balanced by extinctions among older clades. An apparent 4-fold expansion of species richness after the Cretaceous/Paleogene (K/Pg) boundary deserves further examination in light of potential taxonomic biases, but is consistent with the hypothesis that global environmental disturbances such as mass extinction events can rapidly adjust limits to diversity by restructuring ecosystems, and suggests that the gradualistic evolutionary diversification of tetrapods was punctuated by brief but dramatic episodes of radiation.