title: GJA1-20k, an internally translated isoform of Connexin 43, is an actin capping protein

abstract: Previously, we identified that GJA1-20k, an internally translated isoform of Connexin 43, mediates an actin-dependent protective form of mitochondrial fission (Shimura, Nuebel et al. 2021). We found that when GJA1-20k is present, bands of actin surround mitochondria at locations enriched with GJA1-20k, inducing mitochondrial fission which generates less oxygen free radicals, protecting hearts subjected to ischemia-reperfusion injury. Here, we report that GJA1-20k is a direct actin binding protein and thereby identify the mechanism by which GJA1-20k is able to recruit and stabilize actin filaments around the mitochondria. Surprisingly, GJA1-20k functions as a canonical actin capping protein, producing both truncated actin puncta and stabilized actin filaments. GJA1-20k contains an RPEL-like actin binding motif, and we confirm with both computational modeling and biochemistry, that this domain is crucial for actin capping. The actin capping functionality of GJA1-20k adds GJA1-20k to the family of proteins that regulate actin dynamics. As a stress responsive protein, GJA1-20k can help explain cytoskeletal dependent responses to cellular stress, from delivery of channels to affecting mitochondrial size and function.


title: Simultaneous inference of parental admixture proportions and admixture times from unphased local ancestry calls

abstract: Population genetic analyses of local ancestry tracts routinely assume that the ancestral admixture process is identical for both parents of an individual, an assumption that may be invalid when considering recent admixture. Here we present Parental Admixture Proportion Inference (PAPI), a Bayesian tool for inferring the admixture proportions and admixture times for each parent of a single admixed individual. PAPI analyzes unphased local ancestry tracts and has two components models: a binomial model that exploits the informativeness of homozygous ancestry regions to infer parental admixture proportions, and a hidden Markov model (HMM) that infers admixture times from tract lengths. Crucially, the HMM employs an approximation to the pedigree crossover dynamics that accounts for unobserved within-ancestry recombination, enabling inference of parental admixture times. We compared the accuracy of PAPIs admixture proportion estimates with those of ANCESTOR in simulated admixed individuals and found that PAPI outperforms ANCESTOR by an average of 46% in a representative set of simulation scenarios, with PAPIs estimates deviating from the ground truth by 0.047 on average. Moreover, PAPIs admixture time estimates were strongly correlated with the ground truth in these simulations (R = 0.76), but have an average downward bias of 1.01 generations that is partly attributable to inaccuracies in local ancestry inference. As an illustration of its utility, we ran PAPI on real African Americans from the PAGE study (N = 5, 786) and found strong evidence of assortative mating by ancestry proportion: couples ancestry proportions are closer to each other than expected by chance (P < 10-6), and are highly correlated (R = 0.87). We anticipate that PAPI will be useful in studying the population dynamics of admixture and will also be of interest to individuals seeking to learn about their personal genealogies.

category: genetics


title: The genomic basis of host and vector specificity in non-pathogenic trypanosomatids

abstract:
The ability of trypanosome parasites to survive and sustain infections is dependent on diverse and intricate immune evasion mechanisms. Pathogenic trypanosomes often have broad host niches that preclude identification of host specific adaptations. In contrast, some non-pathogenic species of the genus Trypanosoma have highly specific hosts and vectors. Trypanosoma theileri, a non-pathogenic parasite of bovines, has a predicted surface protein architecture that likely aids survival in its mammalian host, distinct from the dominant variant surface glycoprotein coat of pathogenic African trypanosomes. In both species, their surface proteins are encoded by genes which account for [~]10% of their genome. A non-pathogenic parasite of sheep, Trypanosoma melophagium, is transmitted by the sheep ked and is closely related to T. theileri. To explore host and vector specificity between these closely related species, we sequenced the T. melophagium genome and transcriptome and an annotated draft genome was assembled. T. melophagium was compared to 43 kinetoplastid genomes, including T. theileri. T. melophagium and T. theileri have an AT biased genome, the greatest bias of publicly available trypanosomatids. This trend may result from selection acting to decrease the genome nucleotide cost. The T. melophagium genome is 6.3Mb smaller than T. theileri and large families of proteins, characteristic of the predicted surface of T. theileri, were found to be absent or greatly reduced in T. melophagium. Instead, T. melophagium has modestly expanded protein families associated with the avoidance of complement-mediated lysis. The genome of T. melophagium contains core genes required for development, glycolysis, RNA interference, and meiotic exchange, each being shared with T. theileri. Comparisons between T. melophagium and T. theileri provide insight into the specific adaptations of these related trypanosomatids to their distinct mammalian hosts and arthropod vectors.

Author summaryNon-pathogenic trypanosomes can have narrow host niches, with closely related trypanosome species expanding into distinct mammalian host and insect vectors. T. theileri, a non-pathogenic trypanosome of bovines, is predicted to have an intricate cell surface which allows it to evade the immune response of its mammalian host. In contrast, T. melophagium is closely related to T. theileri but infects sheep and is transmitted by the sheep ked rather than tabanid flies that transmit T. theileri. Here, we sequence and assemble the T. melophagium genome to identify the genomic basis of host and vector specificity in these non-pathogenic trypanosomes. We confirm the two species are closely related, however, T. melophagium has a smaller genome than T. theileri. Most of the discrepancy in genome size is due to an expansion of putative cell surface genes in T. theileri. The differential investment in cell surface proteins could be due to a focus on adaptation to the mammalian host in T. theileri and the insect host in T. melophagium.

Data summaryThe genomes, transcriptomes and proteomes used in this study were accessed from the TriTrypDB repository or NCBI. T. theileri genome sequencing data was downloaded from NCBI SRA (SRR13482812). T. melophagium data generated during this study is available from the NCBI BioProject PRJNA786535.

RepositoriesT. melophagium DNA and RNA sequencing data, along with the draft genome assembly and its annotation, can be found under the NCBI BioProject PRJNA786535.

category: genomics


title: Insights into I-motif stabilization by high resolution primer extension assays: Its strengths and limitations

abstract: Cytosine-rich DNA can fold into four-stranded intercalated structures, i-motif (iM), in acidic pH and require hemi-protonated C:C+ base pairs to form. However, its formation and stability rely on many other factors that are not yet fully understood. In here, we combined biochemical and biophysical approaches to determine the factors influencing iM stability in a wide range of experimental conditions. By using high resolution primer extension assays, circular dichroism and absorption spectroscopies, we demonstrate that the stability of three different biologically relevant iMs are not dependent on molecular crowding agents. Instead, some of the crowding agents affected overall DNA synthesis. We also tested a range of small molecules to determine their effect on iM stabilization at physiological temperature, and demonstrated that the G-quadruplex-specific molecule, CX-5461, is also a promising candidate for selective iM stabilization. This work provides important insights into the requirements needed for different assays to accurately study iM stabilization, which will serve as important tools for understanding iMs biological roles and their potential as therapeutic targets.

category: biochemistry


title: Discovery of novel compounds as potent activators of Sirt3

abstract: Among the sirtuin enzymes, Sirt3 is one of the most important deacetylases as it regulates acetylation levels in mitochondria, which are linked to the metabolism of multiple organs and therefore involved in many types of age-related human diseases such as cancer, heart diseases and metabolic diseases. Given the dearth of direct activators of Sirt3, the identification of new modulators could be a key step in the development of new therapeutics. Here we report the discovery of Sirt3 modulators, including activators, through the use of DNA encoded library technology (DEL). The most enriched compounds after DEL selection against SIRT3 were evaluated according to their activity and affinity. Our best activator seems at least as potent as Honokiol (HKL) while the docking studies suggest that our modulators interact with Sirt3 at an atypical site. Our results establish the attractiveness of the DEL technology in identifying novel and potent Sirt3 activators and, therefore, in associated therapeutic applications.


title: In vitro characterization of the full-length human dynein-1 cargo adaptor BicD2

abstract: The cargo adaptors are crucial in coupling motor proteins with their respective cargos and regulatory proteins. BicD2 is one of the most prominent examples within the cargo adaptor family. BicD2 is able to recruit the microtubule motor dynein to RNA, viral particles and nuclei. The BicD2-mediated interaction between the nucleus and dynein is implicated in mitosis as well as interkinetic nuclear migration (INM) in radial glial progenitor cells, and neuron precursor migration during embryonic neocortex development. In vitro studies involving full-length cargo adaptors are difficult to perform due to the hydrophobic character, low-expression levels, and intrinsic flexibility of cargo adaptors. Here we report the recombinant production of full-length human BicD2 and confirm its biochemical activity by interaction studies with RanBP2 and cytoplasmic dynein-1. We also describe pH-dependent conformational changes of BicD2 using cryoEM, template-free structure predictions, and biophysical tools. Our results will help defining the biochemical parameters for the invitro reconstitution of higher order BicD2 protein complexes.


title: Intracellular copper storage and delivery in a bacterium

abstract: A family of cytosolic copper (Cu) storage proteins (the Csps) are widespread in bacteria. The Csps can bind large quantities of Cu(I) via their Cys-lined four-helix bundles, and the majority are cytosolic (Csp3s). This is inconsistent with the current dogma that bacteria, unlike eukaryotes, have evolved not to maintain intracellular pools of Cu due to its potential toxicity. Sporulation in Bacillus subtilis has been used to investigate if a Csp3 can store Cu(I) in the cytosol for a target enzyme. The activity of the Cu-requiring endospore multi-Cu oxidase BsCotA (a laccase) increases under Cu-replete conditions in wild type B. subtilis, but not in the strain lacking BsCsp3. Cuprous ions readily transfer from BsCsp3, but not from the cytosolic copper metallochaperone BsCopZ, to BsCotA in vitro producing active enzyme. Both BsCsp3 and BsCotA are upregulated during late sporulation. The hypothesis we propose is that BsCsp3 acquires and stores Cu(I) in the cytosol for BsCotA.


title: Three million images and morphological profiles of cells treated with matched chemical and genetic perturbations

abstract: We present a new, carefully designed and well-annotated dataset of images and image-based profiles of cells that have been treated with chemical compounds and genetic perturbations. Each gene that is perturbed is a known target of at least two compounds in the dataset. The dataset can thus serve as a benchmark to evaluate methods for predicting similarities between compounds and between genes and compounds, measuring the effect size of a perturbation, and more generally, learning effective representations for measuring cellular state from microscopy images. Advancements in these applications can accelerate the development of new medicines.

category: bioinformatics


title: Cryo-EM structure of human somatostatin receptor 2 complex with its agonist somatostatin delineates the ligand binding specificity

abstract: Somatostatin is a peptide hormone regulating endocrine systems through binding to G-protein-coupled somatostatin receptors. somatostatin receptor 2 (SSTR2) is one of the human somatostatin receptors and highly implicated in cancers and neurological disorders. Here, we report the high resolution cryo-EM structure of full-length human SSTR2 bound to the agonist somatostatin (SST-14) complex with inhibitory G (Gi) proteins. Our structure shows that seven transmembrane helices form a deep pocket for ligand binding and that the highly conserved Trp-Lys motif of SST-14 positions at the bottom of the pocket. Furthermore, our sequence analysis combined with AlphaFold modeled structures of other SSTR isoforms provide how SSTR family proteins specifically interact with their cognate ligands. This work provides the first glimpse into the molecular recognition of somatostatin receptor and crucial resource to develop therapeutics targeting somatostatin receptors.

category: biophysics


title: Neomorphic DNA-binding enables tumor-specific therapeutic gene expression in fusion-addicted childhood sarcoma

abstract:
Chimeric fusion transcription factors are oncogenic hallmarks of several devastating cancer types including pediatric sarcomas, such as Ewing sarcoma (EwS) and alveolar rhabdomyosarcoma (ARMS). Despite their exquisite specificity, these driver oncogenes have been considered largely undruggable due to their lack of enzymatic activity.

Here, we show in the EwS model that - capitalizing on neomorphic DNA-binding preferences - the addiction to the respective fusion transcription factor EWSR1-FLI1 can be leveraged to express therapeutic genes.

We genetically engineered a de novo enhancer-based, synthetic and highly potent expression cassette that can elicit EWSR1-FLI1-dependent expression of a therapeutic payload as evidenced by episomal and CRISPR-edited genomic reporter assays. Combining in silico screens and immunohistochemistry, we identified GPR64 as a highly specific cell surface antigen for targeted transduction strategies in EwS. Functional experiments demonstrated that anti-GPR64-pseudotyped lentivirus harboring our expression cassette can specifically transduce EwS cells to promote the expression of viral thymidine kinase sensitizing EwS for treatment to the otherwise relatively non-toxic (Val)ganciclovir and leading to strong anti-tumorigenic, but no adverse effects in vivo. Further, we prove that similar vector designs can be applied in PAX3-FOXO1-driven ARMS, and to express immunomodulatory cytokines, such as IL-15 and XCL1, in tumor types typically considered to be immunologically  cold.

Collectively, these results generated in pediatric sarcomas indicate that exploiting, rather than suppressing, the neomorphic functions of chimeric transcription factors may open inroads to innovative and personalized therapies, and that our highly versatile approach may be translatable to other cancers addicted to oncogenic transcription factors with unique DNA-binding properties.

category: cancer biology


title: Mutant KRAS-associated proteome is mainly controlled by exogenous factors

abstract: KRAS signaling has been extensively studied, yet the clarification between KRAS-autonomous and non-autonomous mechanisms are still less explored. Understanding how KRAS signaling and effects are affected by exogenous stimuli can provide valuable insights not only to understand resistance mechanisms that justify pathway inhibition failure, but also to uncover novel therapeutic targets for mutant KRAS patients. Hence, aiming at understanding KRAS-autonomous versus non autonomous mechanisms, we studied the response of two mutant KRAS colorectal cancer cell lines (HCT116 and LS174T) - control and KRAS silenced- to TGF{beta}1-activated fibroblasts secretome. By performing a total proteome analysis, we observed that TGF{beta}1-activated fibroblast-secreted factors triggered cell line-specific proteome alterations and that mutant KRAS governs approximately 1/3 of those alterations. Moreover, the analysis of the impact of exogenous factors on the modulation of KRAS proteome revealed that, in both cell lines, more than 2/3 of the KRAS-associated proteome is controlled in a KRAS-non-autonomous manner and dependent on the exogenous factors. This work highlights the context-dependency of KRAS-associated signaling and reinforces the importance of establishing more integrative models resembling the complexity of the tumor microenvironment to study KRAS-associated signals.


title: Limited consequences for loss of RNA-directed DNA methylation in Setaria viridis domains rearranged methyltransferase (DRM) mutants

abstract:
The Domains Rearranged Methyltransferases (DRMs) are crucial for RNA-directed DNA methylation (RdDM) in plant species. Setaria viridis is a model monocot species with a relatively compact genome that has limited transposable element content. CRISPR-based genome editing approaches were used to create loss-of-function alleles for the two putative functional DRM genes in S. viridis to probe the role of RdDM. The analysis of drm1ab double mutant plants revealed limited morphological consequences for the loss of RdDM. Whole-genome methylation profiling provided evidence for wide-spread loss of methylation in CHH sequence contexts, particularly in regions with high CHH methylation in wild-type plants. Evidence was also found for locus-specific loss of CG and CHG methylation, even in some regions that lack CHH methylation. Transcriptome profiling identified a limited number of genes with altered expression in the drm1ab mutants. The majority of genes with elevated CHH methylation directly surrounding the transcription start site or in nearby promoter regions do not have altered expression in the drm1ab mutant even when this methylation is lost, suggesting limited regulation of gene expression by RdDM. Detailed analysis of the expression of transposable elements identified several transposons that are transcriptionally activated in drm1ab mutants. These transposons likely require active RdDM for maintenance of transcriptional repression.

Significance statementMethylation profiling of Setaria viridis plants that lack functional Domains Rearranged Methyltransferase genes reveal widespread loss of DNA methylation in the CHH sequence context. Transcriptome analysis reveals a small set of genes and transposons that are silenced by RNA-directed DNA methylation.

category: plant biology


title: The role of collegiality in academic review, promotion, and tenure

abstract: Review, promotion, and tenure (RPT) processes at universities typically assess candidates along three dimensions: research, teaching, and service. In recent years, some have argued for the inclusion of a controversial fourth criterion: collegiality. While collegiality plays a role in the morale and effectiveness of academic departments, it is amorphic and difficult to assess, and could be misused to stifle dissent or enforce homogeneity. Despite this, some institutions have opted to include this additional element in their RPT documents and processes, but it is unknown the extent of this practice and how it varies across institution type and disciplinary units. This study is based on two sets of data: survey data collected as part of a project that explored the publishing decisions of faculty and how these related to perceived importance in RPT processes, and 864 RPT documents collected from 129 universities from the United States and Canada. We analysed these RPT documents to determine the degree to which collegiality and related terms are mentioned, if they are defined, and if and how they may be assessed during the RPT process. Results show that when collegiality and related terms appear in these documents they are most often just briefly mentioned. It is less common for collegiality and related terms to be defined or assessed in RPT documents. Although the terms are mentioned across all types of institutions, there is a statistically significant difference in how prevalent they are at each. Collegiality is more commonly mentioned in the documents of doctoral research-focused universities (60%), than of masters universities and colleges (31%) or baccalaureate colleges (15%). Results from the accompanying survey of faculty also support this finding: individuals from R-Types were more likely to perceive collegiality to be a factor in their RPT processes. We conclude that collegiality likely plays an important role in RPT processes, whether it is explicitly acknowledged in policies and guidelines or not, and point to several strategies in how it might be best incorporated in the assessment of academic careers.

category: scientific communication and education


title: Nucleoplasmic Lamin C Rapidly Accumulates at Sites of Nuclear Envelope Rupture with BAF and cGAS

abstract:
In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that lamin C but not the other lamin isoforms rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The immunoglobulin-like fold domain and the NLS were required for the recruitment from the nucleoplasm to the rupture sites with the Barrier-to-autointegration factor (BAF). The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF and cGAS concertedly accumulate at sites of NE rupture for repair.

SummaryKono et al. show the rapid recruitment of nucleoplasmic lamin C to sites of nuclear envelope rupture with Barrier-to-autointegration factor. Lamin A/C is also involved in nuclear DNA sensing with cytoplasmic cGAS at the ruptured sites.

category: cell biology


title: Full Spectrum Flow Cytometry for High-Dimensional Immunophenotyping of Murine Innate Lymphoid Cells

abstract:
This 25-parameter, 22-colour full spectrum flow cytometry panel was designed and optimised for the comprehensive enumeration and functional characterisation of innate lymphoid cell (ILC) subsets in mouse tissues (Table 1). The panel presented here allows the discrimination of ILC progenitors (ILCP), ILC1, ILC2, NCR+ ILC3, NCR- ILC3, CCR6+ ILC3 and mature natural killer (NK) cell populations. Further characterisation of ILC and NK cell functional profiles in response to stimulation is provided by the inclusion of subset-specific cytokine markers, and proliferation markers. Development and optimisation of this panel was performed on freshly isolated cells from adult BALB/c lungs and small intestine lamina propria, and ex vivo stimulation with phorbol 12-myrisate 13-acetate, ionomycin, brefeldin A and pro-ILC activating cytokines.

O_TBL View this table:
org.highwire.dtl.DTLVardef@8df9d7org.highwire.dtl.DTLVardef@10dc5d4org.highwire.dtl.DTLVardef@136f44dorg.highwire.dtl.DTLVardef@e10165org.highwire.dtl.DTLVardef@22a0cd_HPS_FORMAT_FIGEXP  M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONSummary Table

C_TABLECAPTION C_TBL


title: Full Spectrum Flow Cytometry for High-Dimensional Immunophenotyping of Mouse Innate Lymphoid Cells


title: Conserved meiotic mechanisms in the cnidarian Clytia hemisphaerica revealed by Spo11 knockout

abstract: During meiosis, each duplicated chromosome pairs and recombines with its unique homolog to ensure the shuffling of genetic information across generations. Functional studies in classical model organisms have revealed a surprising diversity in the chronology and interdependency of the earliest meiotic steps such as chromosome movements, pairing, association via Synaptonemal Complex formation (synapsis), recombination and the formation of chiasmata. A key player is Spo11, an evolutionarily conserved topoisomerase-related transesterase that initiates meiotic recombination via the catalysis of programmed DNA double stranded breaks (DSBs). While DSBs are required for pairing and synapsis in budding yeast and mouse, alternative pathways are employed during female meiosis of the fruit fly and nematode Caenorhabditis elegans. Here, to provide a comparative perspective on meiotic regulation from a distinct animal clade, we chart gametogenesis in Clytia hemisphaerica jellyfish and examine the role of Spo11 using CRISPR-Cas9 mutants, generated clonally from F0 polyp colonies. Spo11 mutant females fail to assemble synaptonemal complexes and chiasmata, such that homologous chromosome pairs disperse during oocyte growth. Subsequent meiotic divisions are abnormal but produce viable progeny. Clytia thus shares an ancient eukaryotic dependence of synapsis and chromosome segregation on Spo11-generated DSBs. It provides a valuable additional experimental model for dissecting meiotic mechanisms during animal gametogenesis, and for building a comparative framework for distinguishing evolutionarily conserved versus flexible features of meiosis.

category: developmental biology


title: Cardiomyocyte differentiation from iPS cells is delayed following knockout of Bcl-2

abstract: Anti-apoptotic B-cell lymphoma 2 (Bcl-2) regulates a wide array of cellular functions involved in cell death, cell survival decisions and autophagy. Bcl-2 acts by both direct interaction with different components of the pathways involved and by intervening in intracellular Ca2+ signalling. The function of Bcl-2 is in turn regulated by post-translational modifications including phosphorylation at different sites by various protein kinases. Besides functions in cell death and apoptosis, Bcl-2 regulates cell differentiation processes, including of cardiomyocytes, although its mechanism of action in this process is not fully elucidated. To further address the role of Bcl-2 in cardiomyocyte differentiation, we investigated the effect of its genetic knockout by CRISPR/Cas9 on the differentiation and functional maturation trajectory of human induced pluripotent stem cells (hiPSC) to cardiomyocytes. Our results indicate that differentiation of hiPSC to cardiomyocytes is delayed by Bcl-2 KO. This effect was associated with smaller areas of spontaneously beating cells. At the cell population level this exhibited in reduced expression and activity of the cardiomyocyte Ca2+ toolkit. However, when restricting the comparison to the active areas exclusively Bcl-2 KO did not show to significantly alter the functionality of the differentiated cardiomyocytes. Finally, Bcl-2 KO reduced c-Myc expression early in the cardiac differentiation process which may account at least in part for the here observed delay in cardiac differentiation resulting reduced efficiency of the differentiation process. These data are supportive of a pivotal role for Bcl-2 in cardiomyocyte differentiation and maturation.


title: Developmental maturation of the hematopoietic system controlled by a Lin28b-let-7-PRC1 axis

abstract: Hematopoiesis changes over life to meet the demands of maturation and aging. Here, we find that the definitive hematopoietic stem and progenitor cell (HSPC) compartment is remodeled from gestation into adulthood, a process regulated by the heterochronic Lin28b/let-7 axis. Native fetal and neonatal HSPCs distribute with a pro-lymphoid/erythroid bias with a shift toward myeloid output in adulthood. By mining transcriptomic data comparing juvenile and adult HSPCs and reconstructing coordinately activated gene regulatory networks, we uncover the Polycomb repressor complex 1 (PRC1) component Cbx2 as an effector of Lin28b/let-7s control of hematopoietic maturation. We find that juvenile Cbx2-/- hematopoietic tissues show impairment of B-lymphopoiesis and a precocious adult-like myeloid bias and that Cbx2/PRC1 regulates developmental timing of expression of key hematopoietic transcription factors. These findings define a novel mechanism of epigenetic regulation of HSPC output as a function of age with potential impact on age-biased pediatric and adult blood disorders.


title: Nanometer-Resolution Long-term Tracking of Single Cargos Reveals Dynein Motor Mechanisms

abstract: Cytoplasmic dynein is essential for intracellular transport, but because of its complexity, we still do not fully understand how this 1.5 megadalton protein works. Here, we used novel optical probes that enable single-particle tracking (SPT) of individual cargos transported by dynein motors in live neurons over 900 m. Analyses using the Fluctuation Theorem (FT) showed that the number of dynein molecules switches between 1-5 motors during the transport. Clearly resolved single-molecular steps revealed that the dwell times between individual steps were accurately described by an enzymatic cycle dominated by two equal and thermally-activated rate constants. Based on these data, we propose a new molecular model whereby each step requires the hydrolysis of 2 ATPs. The model is consistent with extensive structural, single-molecule and biochemical measurements.


title: Integrin signaling is critical for myeloid-mediated support of T-cell acute lymphoblastic leukemia

abstract:
We previously found that T-cell acute lymphoblastic leukemia (T-ALL) requires support from tumor-associated myeloid cells, which activate IGF1R signaling in the leukemic blasts. However, IGF1 is not sufficient to sustain T-ALL survival in vitro, implicating additional myeloid-mediated signals in T-ALL progression. Here, we find that T-ALL cells require close contact with myeloid cells to survive. Transcriptional profiling and in vitro assays demonstrate that integrin-mediated cell adhesion and activation of the downstream FAK/PYK2 kinases are required for myeloid-mediated support of T-ALL cells and promote IGF1R activation. Consistent with these findings, inhibition of integrins or FAK/PYK2 signaling diminishes leukemia burden in multiple organs and confers a survival advantage in a mouse model of T-ALL. Inhibiting integrin-mediated cell adhesion or FAK/PYK2 also diminishes survival of primary patient T-ALL cells co-cultured with myeloid cells. Furthermore, elevated integrin pathway gene signatures correlate significantly with myeloid enrichment and an inferior prognosis in pediatric T-ALL patients.

Statement of significanceAlthough tumor-associated myeloid cells provide critical support for T-ALL, our understanding of the underlying mechanisms remains limited. This study reveals that integrin-mediated adhesion and signaling are key mechanisms by which myeloid cells promote survival and progression of T-ALL blasts in the leukemic microenvironment.


title: The neural dynamics associated with computational complexity

abstract: Many everyday tasks require people to solve computationally complex problems. However, little is known about the effects of computational hardness on the neural processes associated with solving such problems. Here, we draw on computational complexity theory to address this issue. We performed an experiment in which participants solved several instances of the 0-1 knapsack problem, a combinatorial optimization problem, while undergoing ultra-high field (7T) functional magnetic resonance imaging (fMRI). Instances varied in two task-independent measures of intrinsic computational hardness: complexity and proof hardness. We characterise a network of brain regions whose activation was correlated with both measures but in distinct ways, including the anterior insula, dorsal anterior cingulate cortex and the intra-parietal sulcus/angular gyrus. Activation and connectivity changed dynamically as a function of complexity and proof hardness, in line with theoretical computational requirements. Overall, our results suggest that computational complexity theory provides a suitable framework to study the effects of computational hardness on the neural processes associated with solving complex cognitive tasks.

category: neuroscience


title: Transcriptional and cell type profiles of cortical brain regions showing ultradian cortisol rhythm dependent responses to emotional face stimulation

abstract: The characteristic endogenous circadian rhythm of plasma glucocorticoid concentrations is made up from an underlying ultradian pulsatile secretory pattern. Recent evidence has indicated that this ultradian cortisol pulsatility is crucial for normal emotional response in man. In this study, we investigate the anatomical transcriptional and cell type signature of brain regions sensitive to a loss of ultradian rhythmicity in the context of emotional processing. We combine human cell type and transcriptomic atlas data of high spatial resolution with functional magnetic resonance imaging (fMRI) data. We show that the loss of cortisol ultradian rhythm alters emotional processing response in cortical brain areas that are characterized by transcriptional and cellular profiles of GABAergic function. We find that two previously identified key components of rapid non- genomic GC signaling - the ANXA1 gene and retrograde endocannabinoid signaling - show top differential expression and the most significant enrichment. Our results further indicate that specific cell types, including a specific NPY- expressing GABAergic neuronal cell type, and specific G protein signaling cascades underly the cerebral effects of a loss of ultradian cortisol rhythm. Our results provide a biological mechanistic underpinning of our fMRI findings, indicating specific cell types and cascades as a target for manipulation in future experimental studies.


title: Gata2, Nkx2-2 and Skor2 form a transcription factor network regulating development of amidbrain GABAergic neuron subtype with characteristics of REM sleep regulatory neurons

abstract: The midbrain reticular formation is a mosaic of diverse GABAergic and glutamatergic neurons that have been associated with a variety of functions, including the regulation of sleep. However the molecular characteristics and development of the midbrain reticular formation neurons are poorly understood. As the transcription factor Gata2 is required for the development of all GABAergic neurons derived from the embryonic mouse midbrain, we hypothesized that the genes expressed downstream of Gata2 could contribute to the diversification of GABAergic neuron subtypes in this brain region. Here, we show that Gata2 is indeed required for the expression of several lineage-specific transcription factors in post-mitotic midbrain GABAergic neuron precursors. These include a homeodomain transcription factor Nkx2-2 and a SKI family transcriptional repressor Skor2, which are co-expressed in a restricted group of GABAergic precursors in the midbrain reticular formation. Both Gata2, and Nkx2-2 function is required for the expression of Skor2 in GABAergic precursors. In the adult mouse as well as rat midbrain, the Nkx2-2 and Skor2 expressing GABAergic neurons locate at the boundary of the ventrolateral periaqueductal gray and the midbrain reticular formation, an area shown to contain REM-off neurons regulating REM sleep. In addition to the characteristic localization, the Skor2 positive cells increase their activity upon REM sleep inhibition, send projections to a pontine region associated with sleep control and are responsive to orexins, consistent with the known properties of the midbrain REM-off neurons.


title: Function and constraint in enhancers with multiple evolutionary origins

abstract:
MotivationThousands of human gene regulatory enhancers are composed of sequences with multiple evolutionary origins. These evolutionarily "complex" enhancers consist of older "core" sequences and younger "derived" sequences. However, the functional relationship between the sequences of different evolutionary origins within complex enhancers is poorly understood.

ResultsWe evaluated the function, selective pressures, and sequence variation across core and derived components of human complex enhancers. We find that both components are older than expected from the genomic background, and cores are enriched for derived sequences of similar evolutionary ages. Both components show strong evidence of biochemical activity in massively parallel report assays (MPRAs). However, core and derived sequences have distinct transcription factor (TF) binding preferences that are largely stable across evolutionary origins. Given these signatures of function, both core and derived sequences have substantial evidence of purifying selection. Nonetheless, derived sequences exhibit weaker purifying selection than adjacent cores. Derived sequences also tolerate more common genetic variation and are enriched compared to cores for eQTL associated with gene expression variability in human populations.

ConclusionsBoth core and derived sequences have strong evidence of gene regulatory function, but derived sequences have distinct constraint profiles, TF binding preferences, and tolerance to variation compared with cores. We propose that the step-wise integration of younger derived and older core sequences has generated regulatory substrates with robust activity and the potential for functional variation. Our analyses demonstrate that synthesizing study of enhancer evolution and function can aid interpretation of regulatory sequence activity and functional variation across human populations.

Significance StatementThousands of human gene regulatory enhancers are mosaics of sequences from multiple evolutionary origins, yet how these different segments contribute to enhancer function is poorly understood. By dissecting their regulatory functions, transcription factor binding, constraint, and human genetic variation, we show that both older "core" and younger "derived" sequences in complex enhancers have strong evidence of gene regulatory function, but derived sequences are more likely to harbor genetic variants that influence function. Together, our results support a model in which the integration of sequences of different origins generates regulatory substrates with robust activity and the potential for functional variation.

category: evolutionary biology


title: CRISPR-LRS for mapping transgenes in the mouse genome

abstract: Microinjected transgenes, including bacterial artificial chromosomes (BACs), insert randomly in the mouse genome. Traditional methods of mapping a transgene are challenging, thus complicating breeding strategies and the accurate interpretation of phenotypes, particularly when a transgene disrupts critical coding or noncoding sequences. Here, we introduce CRISPR-Cas9 long-read sequencing (CRISPR-LRS) to ascertain transgene integration locus and estimated copy number. This method revealed integration loci for both a BAC and Cre-driver line, and estimated the copy numbers for two other BAC mouse lines. CRISPR-LRS offers an easy approach to establish robust breeding practices and accurate phenotyping of most any transgenic mouse line.


title: Red blood cells protein profile is modified in breast cancer patients

abstract: Metastasis is the primary cause of death for most breast cancer patients who succumb to the disease. During the haematogenous dissemination, circulating tumor cells interact with different blood components. Thus, there are micro-environmental and systemic processes contributing to cancer regulation. We have published that Red Blood Cells (RBCs) that accompany circulating tumor cells have prognostic value in metastatic breast cancer patients. Although the principal known role of RBCs is gas transport, it has been recently assigned additional functions as regulatory cells on circulation. Hence, to explore their potential contribution to tumor progression, we characterized the proteomic composition of RBCs from 53 breast cancer patients, compared with 33 healthy donors. RBCs from breast cancer patients showed a different proteomic profile compared to healthy donors. The differential proteins were mainly related to extracellular components, proteasome, and metabolism. Besides, LAMP2 emerge as a new RBCs marker with diagnostic and prognostic potential for metastatic patients. Seemingly, RBCs are acquiring modifications in their proteomic composition that probably represents the systemic cancer disease, conditioned by the tumor microenvironment.


title: Apical and basal auxin sources pattern shoot branching in a moss

abstract: Shoot branching mechanisms where branches arise in association with leaves - referred to as lateral or axillary branching - evolved by convergence in the sporophyte of vascular plants and the gametophyte of bryophytes, and accompanied independent events of plant architectural diversification1. Previously, we showed that three hormonal cues, including auxin, have been recruited independently to co-ordinate branch patterning in flowering plant leafy shoots and moss gametophores (Coudert, Palubicki et al., 2015)2-4. Moreover, auxin-mediated apical dominance, which relies on local auxin production, has been proposed as a unifying molecular regulatory mechanism of branch development across land plants5. Whilst our previous work in the moss Physcomitrium patens has gathered indirect evidence supporting the notion that auxin synthesized in gametophore apices regulates branch formation at a distance2, direct genetic evidence for a role of auxin biosynthesis in gametophore branching control is still lacking. Here, we show that gametophore apex decapitation promotes branch emergence through massive and rapid transcriptional reprogramming of auxin-responsive genes and altering auxin biosynthesis gene activity. Specifically, we identify a subset of P. patens TRYPTOPHAN AMINO-TRANSFERASE (TAR) and YUCCA FLAVIN MONOOXYGENASE-LIKE (YUC) auxin biosynthesis genes expressed in apical and basal regions of the gametophore, and show that they are essential for branch initiation and outgrowth control. Our results demonstrate that local auxin biosynthesis coordinates branch patterning in moss and thus constitutes a shared and ancient feature of shoot architecture control in land plants.


title: CRISPR/Cas9 knock-in strategy to evaluate phospho-regulation of SAMHD1

abstract:
Sterile  motif (SAM) and HD domain-containing protein 1 (SAMHD1) is a potent restriction factor for immunodeficiency virus 1 (HIV-1), active in myeloid and resting CD4+ T cells. As a dNTP triphosphate triphosphohydrolase (dNTPase), SAMHD1 is proposed to limit cellular dNTP levels correlating with inhibition of HIV-1 reverse transcription. The anti-viral activity of SAMHD1 is regulated by dephosphorylation of the residue T592. However, the impact of T592 phosphorylation on dNTPase activity is still under debate. Whether additional cellular functions of SAMHD1 impact anti-viral restriction is also not completely understood.

We use BlaER1 cells as a novel human macrophage transdifferentiation model combined with CRISPR/Cas9 knock-in (KI) to study SAMHD1 mutations in a physiological context. Transdifferentiated BlaER1 cells, resembling primary human macrophages, harbor active dephosphorylated SAMHD1 that blocks HIV-1 reporter virus infection. Co-delivery of Vpx or CRISPR/Cas9-mediated SAMHD1 knock-out relieves the block to HIV-1. Using CRISPR/Cas9-mediated homologous recombination, we introduced specific mutations into the genomic SAMHD1 locus. Homozygous T592E mutation, but not T592A, leads to loss of HIV-1 restriction, confirming the role of T592 dephosphorylation in the regulation of anti-viral activity. However, T592E KI cells retain wild type dNTP levels, suggesting the antiviral state might not only rely on dNTP depletion.

In conclusion, the role of the T592 phospho-site for anti-viral restriction was confirmed in an endogenous physiological context. Importantly, loss of restriction in T592E mutant cells does not correlate with increased dNTP levels, indicating that the regulation of anti-viral and dNTPase activity of SAMHD1 might be uncoupled.

ImportanceSterile  motif (SAM) and HD domain-containing protein 1 (SAMHD1) is a potent anti-viral restriction factor, active against a broad range of DNA viruses and retroviruses. In myeloid and resting CD4+ T cells, SAMHD1 blocks reverse transcription of immunodeficiency virus 1 (HIV-1), not only inhibiting viral replication in these cell types, but also limiting the availability of reverse transcription products for innate sensing of HIV-1. Manipulating SAMHD1 activity could be an attractive approach to improve HIV-1 therapy or vaccination strategies. Anti-viral activity is strictly dependent on dephosphorylation of SAMHD1 residue T592, however the mechanistic consequence of T592 phosphorylation is still unclear. Here, we use BlaER1 cells as an alternative myeloid cell model in combination with CRISPR/Cas9-mediated KI to study the influence of SAMHD1 T592 phosphorylation on anti-viral restriction and control of cellular dNTP levels in an endogenous context. By using this novel approach, we were able to genetically uncouple SAMHD1s anti-viral and dNTPase activity with regard to regulation by T592 phosphorylation. This suggests that SAMHD1 dNTPase activity may not exclusively be responsible for the anti-lentiviral activity of SAMHD1 in myeloid cells. In addition, our toolkit may inspire further genetic analysis and investigation of SAMHD1-mediated restriction, as wells as its cellular function and regulation, leading to a deeper understanding of SAMHD1 and HIV-1 biology.

category: microbiology


title: Reliable cell retention of mammalian suspension cells in microfluidic cultivation chambers

abstract: We present a new microfluidic trapping concept to retain randomly moving suspension cells inside a cultivation chamber. In comparison to previously published complex multilayer structures, we achieve cell retention by a thin PDMS barrier, which can be easily integrated into various PDMS-based cultivation devices. Cell loss during cultivation is effectively prevented while diffusive media supply is still ensured.

category: bioengineering


title: Light-driven biological actuators to probe the rheology of 3D microtissues

abstract: The mechanical properties of biological tissues are key to the regulation of their physical integrity and function. Although the application of external loading or biochemical treatments allows to estimate these properties globally, it remains problematic to assess how such external stimuli compare with internal, cell-generated contractions. Here we engineered 3D microtissues composed of optogenetically-modified fibroblasts encapsulated within collagen. Using light to control the activity of RhoA, a major regulator of cellular contractility, we induced local mechanical perturbation within 3D fibrous microtissues, while tracking in real time microtissue stress and strain. We thus investigated the dynamic regulation of light-induced, local contractions and their spatio-temporal propagation in microtissues. By comparing the evolution of stresses and strains upon stimulation, we demonstrated the potential of our technique for quantifying tissue elasticity and viscosity, before examining the possibility of using light to map local anisotropies in mechanically heterogeneous microtissues. Altogether, our results open an avenue to non-destructively chart the rheology of 3D tissues in real time, using their own constituting cells as internal actuators.


title: Opposite roles of Rad5 in DNA damage tolerance: playing in both error-free and mutagenic lesion bypass

abstract:
DNA Damage Tolerance (DDT) functions to bypass replication-blocking lesions and is divided into two distinct pathways: error-prone Translesion Synthesis (TLS) and error-free Damage Avoidance (DA). Rad5 is an important player in these processes. Indeed, Saccharomyces cerevisiae Rad5 is a large multifunctional protein that contains three well defined domains: a RING domain that promotes PCNA polyubiquitination and a ssDNA-dependent ATPase/helicase domain, that are both conserved in Rad5 human ortholog HLTF. Yeast Rad5 also contains a Rev1-binding domain.

In this study we used domain-specific mutants to address the contribution of each of the Rad5 functions to lesion tolerance. Using an assay based on the insertion of a single lesion into a defined locus in the genome of a living yeast cell, we demonstrate that Rad5 plays opposite roles in lesion tolerance: i) Rad5 favors error-free lesion bypass by activating template switching through polyubiquitination of PCNA; ii) Rad5 is also required for TLS by recruiting the TLS polymerase Rev1. We also show that the helicase activity does not play any role in lesion tolerance.

category: molecular biology


title: A novel computational method for head-to-tail peptide cyclization: application to urotensin II

abstract: Peptides have recently re-gained interest as therapeutic candidates but their development remains confronted with several limitations including low bioavailability. Backbone head-to-tail cyclization is one effective strategy of peptide-based drug design to stabilize the conformation of bioactive peptides while preserving peptide properties in terms of low toxicity, binding affinity, target selectivity and preventing enzymatic degradation. However, very little is known about the sequence-structure relationship requirements of designing linkers for peptide cyclization in a rational manner. Recently, we have shown that large scale data-mining of available protein structures can lead to the precise identification of protein loop conformations, even from remote structural classes. Here, we transpose this approach to head-to-tail peptide cyclization. Firstly we show that given a linker sequence and the conformation of the linear peptide, it is possible to accurately predict the cyclized peptide conformation improving by over 1 [A] over pre-existing protocols. Secondly, and more importantly, we show that is is possible to elaborate on the information inferred from protein structures to propose effective candidate linker sequences constrained by length and amino acid composition, providing the first framework for the rational peptide head-to-tail cyclization. As functional validation, we apply it to the design of a head-to-tail cyclized derivative of urotensin II, an 11-residue long peptide which exerts a broad array of biologic activities, making its cognate receptor a valuable and innovative therapeutic or diagnostic target. We propose a three amino acid candidate linker, leading to the first synthesized 14-residue long cyclic UII analogue with excellent retention of in vitro activity.


title: Lack of evidence for functional luteinising hormone chorionic gonadotropin receptors in non-pregnant human endometrial stromal cells

abstract: The human luteinising hormone chorionic gonadotropin receptor (LHCGR) is a G-protein coupled receptor activated by both human chorionic gonadotropin (hCG) and luteinizing hormone (LH), two structurally related gonadotropins with essential roles in ovulation and maintenance of the corpus luteum. LHCGR expression predominates in ovarian tissues where it elicits functional responses through cyclic adenosine mononucleotide (cAMP), Ca2+ and extracellular signal-regulated kinase (ERK) signalling. LHCGR has also been localized to the human endometrium, with purported roles in decidualization and implantation. However, these observations are contentious. In this investigation, transcripts encoding LHCGR were undetectable in bulk RNA sequencing datasets from whole cycling endometrial tissue and cultured human endometrial stromal cells (EnSC). However, analysis of single-cell RNA sequencing data revealed cell-to-cell transcriptional heterogeneity and identified a small subpopulation of stromal cells with discernible LHCGR transcripts. In HEK-293 cells expressing recombinant LHCGR, both hCG and LH elicited robust cAMP, Ca2+ and ERK signals that were absent in wild type HEK-293 cells. However, none of these responses were recapitulated in primary EnSC cultures. In addition, proliferation, viability and decidual transformation of EnSC were refractory to both hCG and LH, irrespective of treatment to induce differentiation. Although we challenge the assertion that LHCGR is expressed at a functionally active level in the human endometrium, the discovery of a discrete subpopulation of EnSC that express LHCGR transcripts may plausibly account for the conflicting evidence in the literature.

category: pharmacology and toxicology


title: Machine learning guided batched design of a bacterial Ribosome Binding Site

abstract: Optimisation of gene expression levels is an essential part of the organism design process. Fine control of this process can be achieved through engineering transcription and translation control elements, including the ribosome binding site (RBS). Unfortunately, design of specific genetic parts can still be challenging due to lack of reliable design methods. To address this problem, we have created a machine learning guided Design-Build-Test-Learn (DBTL) cycle for the experimental design of bacterial RBSs to show how small genetic parts can be reliably designed using relatively small, high-quality data sets. We used Gaussian Process Regression for the Learn phase of cycle and the Upper Confidence Bound multi-armed bandit algorithm for the Design of genetic variants to be tested in vivo. We have integrated these machine learning algorithms with laboratory automation and high-throughput processes for reliable data generation. Notably, by Testing a total of 450 RBS variants in four DBTL cycles, we experimentally validated RBSs with high translation initiation rates equalling or exceeding our benchmark RBS by up to 34%. Overall, our results show that machine learning is a powerful tool for designing RBSs, and they pave the way towards more complicated genetic devices.

category: synthetic biology


title: Analytical time-dependent distributions for gene expression models with complex promoter switching mechanisms

abstract: Classical gene expression models assume exponential switching time distributions between the active and inactive promoter states. However, recent experiments have shown that many genes in mammalian cells may produce non-exponential switching time distributions, implying the existence of multiple promoter states and molecular memory in the promoter switching dynamics. Here we analytically solve a gene expression model with random bursting and complex promoter switching, and derive the time-dependent distributions of the mRNA and protein copy numbers, generalizing the steady-state solution obtained in [SIAM J. Appl. Math. 72, 789-818 (2012)] and [SIAM J. Appl. Math. 79, 1007-1029 (2019)]. Using multiscale simplification techniques, we find that molecular memory has no influence on the time-dependent distribution when promoter switching is very fast or very slow, while it significantly affects the distribution when promoter switching is neither too fast nor too slow. By analyzing the dynamical phase diagram of the system, we also find that molecular memory in the inactive gene state weakens the transient and stationary bimodality of the copy number distribution, while molecular memory in the active gene state enhances such bimodality.

category: systems biology


title: An efficient multi-scale CNN model with intrinsic feature integration for motor imagery EEG subject classification in brain-machine interfaces

abstract:
ObjectiveElectroencephalogram (EEG) based motor imagery (MI) classification is an important aspect in brain-machine interfaces (BMIs) which bridges between neural system and computer devices decoding brain signals into recognizable machine commands. However, the MI classification task is challenging due to inherent complex properties, inter-subject variability, and low signal-to-noise ratio (SNR) of EEG signals. To overcome the above-mentioned issues, the current work proposes an efficient multi-scale convolutional neural network (MS-CNN).

ApproachIn the framework, discriminant user-specific features have been extracted and integrated to improve the accuracy and performance of the CNN classifier. Additionally, different data augmentation methods have been implemented to further improve the accuracy and robustness of the model.

Main resultsThe model achieves an average classification accuracy of 93.74% and Cohens kappa-coefficient of 0.92 on the BCI competition IV2b dataset outperforming several baseline and current state-of-the-art EEG-based MI classification models.

SignificanceThe proposed algorithm effectively addresses the shortcoming of existing CNN-based EEG-MI classification models and significantly improves the classification accuracy.


title: A CNN model with feature integration for MI EEG subject classification in BMI


title: Differential serotonergic modulation of principal neurons and interneurons in the anterior piriform cortex

abstract: Originating from the brainstem raphe nuclei, serotonin is an important neuromodulator involved in a variety of physiological and pathological functions. Specific optogenetic stimulation of serotonergic neurons results in the divisive suppression of spontaneous, but not sensory evoked activity in the majority of neurons in the primary olfactory cortex and an increase in firing in a minority of neurons. To reveal the mechanisms involved in this dual serotonergic control of cortical activity we used a combination of in vitro electrophysiological recordings from identified neurons in the primary olfactory cortex, optogenetics and pharmacology and found that serotonin suppressed the activity of principal neurons, but excited local interneurons. The results have important implications in sensory information processing and other functions of the olfactory cortex and related brain areas.


title: Injury suppresses Ras cell competitive advantage through enhanced wild-type cell proliferation

abstract:
Healthy skin is a tapestry of wild-type and mutant clones. Although injury can cooperate with Ras mutations to promote tumorigenesis, the consequences in genetically mosaic skin are unknown. Here, we show that wild-type cells prevent oncogenic Ras-induced aberrant growth after injury. Although HrasG12V/+ and KrasG12D/+ cells outcompete wild-type cells in uninjured, mosaic tissue, their competitive advantage is suppressed after injury due to a selective increase in wild-type cell proliferation. EGFR inhibition abolishes the competitive advantage of wild-type cells after injury of HrasG12V/+-mosaic skin. Global loss of the cell cycle inhibitor p21 increases wild-type cell proliferation even without injury, suppressing the competitive advantage of HrasG12V/+ cells. Thus, injury plays an unanticipated role in switching the competitive balance between oncogenic and wild-type cells in genetically mosaic skin.

One sentence SummaryInjury-repair selectively induces wild-type cell proliferation to suppress oncogenic growth in Ras-mosaic skin epithelium.


title: Characterization of alveolar epithelial lineage heterogeneity during the late pseudoglandular stage of mouse lung development

abstract:
The specification, characterization, and fate of alveolar type 1 and type 2 (AT1 and 2) progenitors during embryonic lung development remains mostly elusive. In this paper, we build upon our previously published work on the regulation of airway epithelial progenitors by fibroblast growth factor receptor 2b (Fgfr2b) signalling during early (E12.5) and mid (E14.5) pseudoglandular lung development. Here, we looked at the regulation by Fgfr2b signalling on alveolar progenitors during late pseudoglandular/early canalicular (E14.5-E16.5) development. Using a dominant negative mouse model to conditionally inhibit Fgfr2b ligands at E16.5, we used gene array analyses to characterize a set of potential direct targets of Fgfr2b signalling. By mining published single-cell RNA sequence (scRNAseq) datasets, we showed that these Fgfr2b signature genes narrow on a discreet subset of AT2 cells at E17.5 and in adult lungs. Furthermore, we demonstrated that Fgfr2b signalling is lost in AT2 cells in their transition to AT1 cells during repair after injury.

We also used CreERT2-based mouse models to conditionally knock-out the Fgfr2b gene in AT2 and in AT1 progenitors, as well as lineage label these cells. We found, using immunofluorescence, that in wildtype controls AT1 progenitors labeled at E14.5-E15.5 contribute a significant proportion to AT2 cells at E18.5; while AT2 progenitors labeled at the same time contribute significantly to the AT1 lineage. We show, using immunofluorescence and FACS-based analysis, that knocking out of Fgfr2b at E14.5-E15.5 in AT2 progenitors leads to an increase in lineage-labeled AT1 cells at E18.5; while the reverse is true in AT1 progenitors. Furthermore, we demonstrate that increased Fgfr signalling in AT2 progenitors reduces their contribution to the AT1 pool.

Taken together, our results suggest that a significant proportion of AT2 and AT1 progenitors are cross-lineage committed during late pseudoglandular development, and that lineage commitment is regulated in part by Fgfr2b signalling. We have characterized a set of direct Fgfr2b targets at E16.5 which are likely involved in alveolar lineage formation. These signature genes concentrate on a subpopulation of AT2 cells later in development, and are downregulated in AT2 cells transitioning to the AT1 lineage during repair after injury in adults.

Our findings highlight the extensive heterogeneity of alveolar cells by elucidating the role of Fgfr2b signalling in these cells during early alveolar lineage formation, as well as during repair after injury.


title: Humanization of wildlife gut microbiota in urban environments

abstract: Urbanization is rapidly altering Earths environments, demanding investigations of the impacts on resident wildlife. Here, we show that urban populations of coyotes (Canis latrans) and crested anole lizards (Anolis cristatellus) acquire gut microbiota constituents found in humans, including the gut bacterial lineages most significantly associated with urbanization in humans (e.g., Bacteroides). Comparisons of urban and rural wildlife and human populations revealed significant convergence of the gut microbiota among urban host populations. Remarkably, all microbial lineages found in humans that were overrepresented in urban wildlife relative to rural wildlife were also overrepresented in urban humans relative to rural humans. These results indicate parallel effects of urbanization on human and wildlife gut microbiota and suggest spillover of bacteria from humans into wildlife in cities.

category: ecology


title: Climatic niche conservatism in a clade of disease vectors (Diptera: Phlebotominae)

abstract: Sandflies of the family Psychodidae show notable diversity in both disease vector status and climatic niche. Some species (in the subfamily Phlebotominae) transmit Leishmania parasites, responsible for the disease leishmaniasis. Other Psychodidae species do not. Psychodid species ranges can be solely tropical, confined to the temperate zones, or span both. Studying the relationship between the evolution of disease vector status and that of climatic niche affords an understanding not only of the climate conditions associated with the presence and species richness of Leishmania vectors, but also allows the study of the extent to which psychodid flies climatic niches are conserved, in a context with implications for global human health. We obtained observation site data, and associated climate data, for 223 psychodid species to understand which aspects of climate most closely predict distribution. Temperature and seasonality are strong determinants of species occurrence within the clade. We built a mitochondrial DNA phylogeny of Psychodidae, and found a positive relationship between pairwise genetic distance and climate niche differentiation, which indicates strong niche conservatism. This result is also supported by strong phylogenetic signals of metrics of climate differentiation. Finally, we used ancestral trait reconstruction to infer the tropicality (i.e., proportion of latitudinal range in the tropics minus the proportion of the latitudinal range in temperate areas) of ancestral species, and counted transitions to and from tropicality states, finding that tropical and temperate species respectively produced almost entirely tropical and temperate descendant species, a result consistent for vector and non-vector species. Taken together, these results imply that while vectors of Leishmania can survive in a variety of climates, their climate niches are strongly predicted by phylogeny.


title: Relaxed risk of predation drives parallel evolution of stickleback behaviour

abstract: The occurrence of similar phenotypes in multiple independent populations (viz. parallel evolution) is a testimony of evolution by natural selection. Parallel evolution implies that populations share a common phenotypic response to a common selection pressure associated with habitat similarity. Examples of parallel evolution at the genetic and phenotypic levels are fairly common, but the driving selective agents often remain elusive. Similarly, the role of phenotypic plasticity in facilitating early stages of parallel evolution is unclear. We investigated whether the relaxation of predation pressure associated with the colonization of freshwater ponds by nine-spined sticklebacks (Pungitius pungitius) likely explains the divergence in complex behaviours between marine and pond populations, and whether this divergence is parallel. Using laboratory-raised individuals exposed to different levels of perceived predation risk, we calculated vectors of phenotypic divergence for four behavioural traits between habitats and predation risk treatments. We found a significant correlation between the directions of evolutionary divergence and phenotypic plasticity, suggesting that habitat divergence in behaviour is aligned with the response to relaxation of predation pressure. Finally, we show that this alignment is found across multiple pairs of populations, and that the relaxation of predation pressure has likely driven parallel evolution of behaviour in this species.


title: Applied phenomics and genomics for improving barley yellow dwarf resistance in winter wheat

abstract: Barley yellow dwarf (BYD) is one of the major viral diseases of cereals. Phenotyping BYD in wheat is extremely challenging due to similarities to other biotic and abiotic stresses. Breeding for resistance is additionally challenging as the wheat primary germplasm pool lacks genetic resistance, with most of the few resistance genes named to date originating from a wild relative species. The objectives of this study were to, i) evaluate the use of high-throughput phenotyping (HTP) from unmanned aerial systems to improve BYD assessment and selection, ii) identify genomic regions associated with BYD resistance, and iii) evaluate genomic prediction models ability to predict BYD resistance. Up to 107 wheat lines were phenotyped during each of five field seasons under both insecticide treated and untreated plots. Across all seasons, BYD severity was lower with the insecticide treatment and plant height (PTHTM) and grain yield (GY) showed increased values relative to untreated entries. Only 9.2% of the lines were positive for the presence of the translocated segment carrying resistance gene Bdv2 on chromosome 7DL. Despite the low frequency, this region was identified through association mapping. Furthermore, we mapped a potentially novel genomic region for resistance on chromosome 5AS. Given the variable heritability of the trait (0.211 - 0.806), we obtained relatively good predictive ability for BYD severity ranging between 0.06 - 0.26. Including Bdv2 on the predictive model had a large effect for predicting BYD but almost no effect for PTHTM and GY. This study was the first attempt to characterize BYD using field-HTP and apply GS to predict the disease severity. These methods have the potential to improve BYD characterization and identifying new sources of resistance will be crucial for delivering BYD resistant germplasm.

category: genetics


title: Alternative splicing analysis benchmark with DICAST

abstract: Alternative splicing is a major contributor to transcriptome and proteome diversity in health and disease. A plethora of tools have been developed for studying alternative splicing in RNA-seq data. Previous benchmarks focused on isoform quantification and mapping. They neglected event detection tools, which arguably provide the most detailed insights into the alternative splicing process. DICAST offers a modular and extensible framework for the analysis of alternative splicing integrating 11 splice-aware mapping and eight event detection tools. We benchmark all tools extensively on simulated as well as whole blood RNA-seq data. STAR and HISAT2 demonstrated the best balance between performance and run time. The performance of event detection tools varies widely with no tool outperforming all others. DICAST allows researchers to employ a consensus approach to consider the most successful tools jointly for robust event detection. Furthermore, we propose the first reporting standard to unify existing formats and to guide future tool development.

category: bioinformatics


title: Phosphate transporter PHT1;1 as a key determinant of phosphorus acquisition in Arabidopsis natural accessions

abstract:
To understand the genetic basis in governing phosphorus (P) acquisition, we performed genome-wide association studies (GWAS) on a diversity panel of Arabidopsis thaliana by two primary determinants of P acquisition, phosphate (Pi)-uptake activity and PHOSPHATE TRANSPORTER 1 (PHT1) protein abundance. Association mapping revealed a shared significant peak on chromosome 5 (Chr5) where the PHT1;1/2/3 genes reside, suggesting a strong correlation between the regulation of Pi-uptake activity and PHT1 protein abundance. Genes encoding transcription factors, kinases, and a metalloprotease associated with both traits were also identified. Conditional GWAS followed by statistical analysis of genotype-dependent expression of PHT1;1 and transcription activity assays revealed an epistatic interaction between PHT1;1 and MYB DOMAIN PROTEIN 52 (MYB52) on Chr1. Analyses of F1 hybrids generated by crossing two subgroups of natural accessions carrying specific SNPs associated with PHT1;1 and MYB52 further revealed the strong effects of potential variants on PHT1;1 expression and Pi uptake activity. Notably, the soil P contents in A. thaliana habitats were found to coincide with PHT1;1 haplotype, underscoring how fine-tuning of the activity of P acquisition by natural variants allows plants to adapt to their environments. This study sheds light on the complex regulation of P acquisition and offers a framework to systematically assess the effectiveness of GWAS approaches in the study of quantitative traits.

One sentence summaryStepwise GWAS analyses reveal insights into the genetic basis in regulating phosphorus acquisition and associations between the phosphate transporter PHT1;1 haplotype and Arabidopsis habitats.


title: HLJ1 amplifies endotoxin-induced sepsis severity by promoting IL-12 heterodimerization in macrophages

abstract: Heat shock protein (HSP) 40 has emerged as a key actor in both innate and adaptive immunity, whereas the role of HLJ1, a molecular chaperone in HSP40 family, in modulating endotoxin-induced sepsis severity is still unclear. Here, we use single-cell RNA sequencing to characterize mouse liver nonparenchymal cell populations under LPS (lipopolysaccharide) stimulation, and show that HLJ1 deletion affected IFN-{gamma}-related gene signatures in distinct immune cell clusters. HLJ1 deficiency also leads to reduced serum levels of IL-12 in LPS-treated mice, contributing to dampened production of IFN-{gamma} in natural killer cells but not CD4+ or CD8+ T cells, and subsequently to improved survival rate. Adoptive transfer of HLJ1-deleted macrophages into LPS-treated mice results in reduced IL-12 and IFN-{gamma} levels and protects the mice from IFN-{gamma}-dependent mortality. In the context of molecular mechanisms, HLJ1 is an LPS-inducible protein in macrophages and converts misfolded IL-12p35 homodimers to monomers, which maintains bioactive IL-12p70 heterodimerization and secretion. This study suggests HLJ1 causes IFN-{gamma}-dependent septic lethality by promoting IL-12 heterodimerization, and targeting HLJ1 has therapeutic potential in inflammatory diseases involving activating IL-12/IFN-{gamma} axis.

category: immunology


title: Spatial scales of competition and a growth-motility tradeoff interact to determine bacterial coexistence

abstract: The coexistence of competing species is a long-lasting puzzle in evolutionary ecology research. Despite abundant experimental evidence showing that the opportunity for coexistence decreases as niche overlap increases between species, bacterial species and strains competing for the same resources are commonly found across diverse spatially heterogeneous habitats. We thus hypothesized that the spatial scale of competition may play a key role in determining bacterial coexistence, and interact with other mechanisms that promote coexistence, including a growth-motility tradeoff. To test this hypothesis, we let two Pseudomonas putida strains compete at local and regional scales by inoculating them either in a mixed droplet or in separate droplets in the same Petri dish, respectively. We also created conditions that allow the bacterial strains to disperse across abiotic or fungal hyphae networks. We found that competition at the local scale led to competitive exclusion while regional competition promoted coexistence. When competing in the presence of dispersal networks, the growth-motility tradeoff promoted coexistence only when the strains were inoculated in separate droplets. Our results provide a mechanism by which existing laboratory data suggesting competitive exclusion at a local scale is reconciled with the widespread coexistence of competing bacterial strains in complex natural environments with dispersal.


title: Plant peptidoglycan precursor biosynthesis: Conservation between moss chloroplasts and Gram negative bacteria

abstract:
An accumulation of evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesised around the chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to land plants at least as evolved as pteridophyte ferns, but the biosynthetic pathway has not been demonstrated. We employed mass spectrometry and enzymology in a twofold approach to characterize the synthesis of peptidoglycan in chloroplasts of the moss Physcomitrium (Physcomitrella) patens. To drive the accumulation of peptidoglycan pathway intermediates, P.patens was cultured with the antibiotics phosphomycin, D-cycloserine and carbenicillin, which inhibit key peptidoglycan pathway proteins in bacteria. Mass spectrometry of the TCA-extracted moss metabolome revealed elevated levels of five of the predicted intermediates from UDP-GlcNAc through to the UDP-MurNAc-D,L-diaminopimelate (DAP)-pentapeptide.

Most Gram negative bacteria, including cyanobacteria, incorporate meso-diaminopimelate (D,L-DAP) into the third residue of the stem peptide of peptidoglycan, as opposed to L-lysine, typical of most Gram positive bacteria. To establish the specificity of D,L-DAP incorporation into the P.patens precursors, we analysed the recombinant protein, UDP-MurNAc-tripeptide ligase (MurE), from both P.patens and the cyanobacterium Anabaena sp. strain PCC 7120. Both ligases incorporated D,L-DAP in almost complete preference to L-Lys, consistent with the mass spectrophotometric data, with catalytic efficiencies similar to previously documented Gram negative bacterial MurE ligases. We discuss how these data accord with the conservation of active site residues common to DL-DAP-incorporating bacterial MurE ligases and of the probability of a horizontal gene transfer event within the plant peptidoglycan pathway.


title: A novel form of macropinocytosis mediates ultra-rapid transfer of pathological alpha- synuclein to lysosomes

abstract: The nervous system spread of alpha-synuclein fibrils leads to Parkinsons disease (PD) and other synucleinopathies, yet the mechanisms underlying internalization and cell-to-cell transfer are enigmatic. Here we use confocal and superresolution microscopy, subcellular fractionation and electron microscopy (EM) of immunogold labelled alpha-synuclein pre-formed fibrils (PFF) to demonstrate that this toxic protein species enters cells using a novel form of ultra-rapid macropinocytosis with transfer to lysosomes in as little as 2 minutes, an unprecedented cell biological kinetic for lysosomal targeting. PFF uptake circumvents classical endosomal pathways and is independent of clathrin. Immunogold-labelled PFF are seen at the highly curved inward edge of membrane ruffles, in newly formed macropinosomes, and in lysosomes. While many of the fibrils remain in lysosomes that continue to take up PFF for hours, a portion are transferred to neighboring naive cells on the external face of vesicles, likely exosomes. These data indicate that PFF uses a novel internalization mechanism as a component of cell-to-cell propagation.


title: A novel form of macropinocytosis mediates ultra-rapid transfer of pathological alpha-synuclein to lysosomes


title: Generation of rat lungs by blastocyst complementation in Fgfr2b-deficient mouse model

abstract: Regenerative medicine is a tool to compensate for the shortage of lungs for transplantation, but it remains difficult to construct a lung in vitro due to the complex three-dimensional structures and multiple cell types required. A blastocyst complementation method using interspecies chimeric animals has been attracting attention as a way to create complex organs in animals, but successful lung formation has not yet been achieved. Here, we applied a "reverse-blastocyst complementation method" to clarify the conditions required to form lungs in an Fgfr2b-deficient mouse model. We then successfully formed a rat-derived lung in the mouse model without generating a mouse line by applying a tetraploid-based organ-complementation method. Importantly, rat lung epithelial cells retained their developmental timing even in the mouse body. This result provides useful insights regarding the need to overcome the barrier of species-specific developmental timing in order to generate functional lungs in interspecies chimeras.


title: Tiling mechanisms of the compound eye through geometrical tessellation

abstract: Tilling patterns are observed in many biological structures. Hexagonal tilling, commonly observed in the compound eyes of wild-type Drosophila, is dominant in nature; this dominance can probably be attributed to physical restrictions such as structural robustness, minimal boundary length, and space filling efficiency. Surprisingly, tetragonal tiling patterns are also observed in some Drosophila small eye mutants and aquatic crustaceans. Herein, geometrical tessellation is shown to determine the ommatidial tiling patterns. In small eye mutants, the hexagonal pattern is transformed into a tetragonal pattern as the relative positions of neighboring ommatidia are stretched along the dorsal-ventral axis. Hence, the regular distribution of ommatidia and their uniform growth collectively play an essential role in the establishment of tetragonal and hexagonal tiling patterns in compound eyes.


title: A western representative of an eastern clade: phylogeographic history of the gypsophilous plant Nepeta hispanica

abstract: The preference of certain plant species for gypsum soils leads to disjunct population structures that are thought to generate island-like dynamics potentially influencing biogeographic patterns at multiple evolutionary scales. Here, we study the evolutionary and biogeographic history of Nepeta hispanica, a western Mediterranean plant associated with gypsum soils and displaying a patchy distribution with populations very distant from each other. Three approaches were used: (a) interspecific phylogenetic analyses based on nuclear DNA sequences of the ITS region to unveil the relationships and times of divergence between N. hispanica and its closest relatives; ((b)phylogeographic analyses using plastid DNA regions trnS-trnG and psbJ-petA to evaluate the degree of genetic isolation between populations of N. hispanica, their relationships and their genetic diversity; and (c) ecological niche modelling to evaluate historical distributional changes. Results reveal that N. hispanica belongs to an eastern Mediterranean and Asian clade diversified in arid environments since the Miocene-Pliocene. This species represents the only extant lineage of this clade that colonized the western Mediterranean, probably through the northern Mediterranean coast (southern Europe). Present Iberian populations display a high plastid genetic diversity and, even if geographically distant from each other, they are highly connected according to the distribution of plastid haplotypes and lineages. This can be explained by a scenario involving a complex history of back-and-forth colonisation events, facilitated by a relative stability of suitable conditions for the species across the Iberian Peninsula throughout the Quaternary.


title: Serine 1283 in extracellular matrix glycoprotein Reelin is crucial for Reelin's function in brain development

abstract: Deficiency in the extracellular matrix glycoprotein Reelin severely affects migration of neurons during development. The function of serine at position 1283 in Reelin has remained uncertain. To explore its relevance we generated rlnA/A mice that carry alanine instead of serine at position 1283, thereby disrupting the putative casein kinase 2 (CK2) phosphorylation site S1283DGD. Mutated mice displayed reeler-like locomotor behavior, abnormal brain anatomy and decrease of Reelin RNA and protein levels during development and in adulthood. Since serine 1283 was previously proposed to mediate proteolysis of adhesion molecules, we investigated proteolysis of cell adhesion molecule L1 and found it normal in rlnA/A mice. Neuronal migration in the embryonic rlnA/A cerebral cortex was impaired, but rescued by in utero electroporation of the Reelin fragment N-R6 containing the putative CK2 phosphorylation site. In rlnA/A mice migration of cerebellar granule cells in vitro was promoted by application of wild-type but not by mutated Reelin. In cerebellar neuron cultures, Reelin expression was decreased upon inhibition of ecto-phosphorylation by CK2. Biochemically purified wild-type, but not mutated Reelin was found phosphorylated. Altogether, the results indicate that ecto-phosphorylation at serine 1283 rather than proteolytic processing of adhesion molecules by Reelin plays an important role in Reelin functions.


title: Where we perceive before we look: The distribution of presaccadic attention assessed with dynamic 1/f noise

abstract: Already before the onset of a saccadic eye movement, we preferentially process visual information at the upcoming eye fixation. This  presaccadic shift of attention is typically assessed via localized test items, which potentially bias the attention measurement. Here we show how presaccadic attention shapes perception from saccade origin to target when no scene-structuring items are presented. Participants made saccades into a 1/f ("pink") noise field, in which we embedded a brief orientation signal at various locations shortly before saccade onset. Local orientation discrimination performance served as a proxy for the allocation of attention. Results demonstrate that (1) saccades are preceded by shifts of attention to their goal location even if they are directed into an unstructured visual field, but the spread of attention, compared to target-directed saccades, is broad; (2) the presaccadic attention shift is accompanied by considerable attentional costs at the presaccadic eye fixation; (3) objects markedly shape the distribution of presaccadic attention - demonstrating the relevance of an item-free approach for measuring attentional dynamics across the visual field.


title: Synaptic plasticity of inhibitory synapses onto medial olivocochlear efferent neurons

abstract:
The descending auditory system modulates the ascending system at every level. The final descending, or efferent stage, is comprised of lateral olivocochlear (LOC) and medial olivocochlear (MOC) neurons. MOC somata in the ventral brainstem project axons to the cochlea to synapse onto outer hair cells (OHC), inhibiting OHC-mediated cochlear amplification. MOC suppression of OHC function is implicated in cochlear gain control with changing sound intensity, detection of salient stimuli, attention, and protection against acoustic trauma. Thus, sound excites MOC neurons to provide negative feedback of the cochlea. Sound also inhibits MOC neurons via medial nucleus of the trapezoid body (MNTB) neurons. However, MNTB-MOC synapses exhibit short-term depression, suggesting reduced MNTB-MOC inhibition during sustained stimuli. Further, due to high rates of both baseline and sound-evoked activity in MNTB neurons in vivo, MNTB-MOC synapses may be tonically depressed. To probe this, we characterized short-term plasticity of MNTB-MOC synapses in mouse brain slices. We mimicked in vivo-like temperature and extracellular calcium conditions, and in vivo-like activity patterns of fast synaptic activation rates, sustained activation, and prior tonic activity. Synaptic depression was sensitive to extracellular calcium concentration and temperature. During rapid MNTB axon stimulation, post-synaptic currents (PSCs) in MOC neurons summated but with concurrent depression, resulting in smaller, sustained currents, suggesting tonic inhibition of MOC neurons during rapid circuit activity. Low levels of baseline MNTB activity did not significantly reduce responses to subsequent rapid activity that mimics sound stimulation, indicating that, in vivo, MNTB inhibition of MOC neurons persists despite tonic synaptic depression.

Key points summaryO_LIInhibitory synapses from MNTB onto MOC neurons exhibit short-term plasticity that is sensitive to calcium and temperature, with enhanced synaptic depression occurring at higher calcium concentrations and at room temperature
C_LIO_LIHigh rates of background synaptic activity that mimic the upper limits of spontaneous MNTB activity cause tonic synaptic depression of MNTB-MOC synapses that limits further synaptic inhibition
C_LIO_LIHigh rates of activity at MNTB-MOC synapses cause synaptic summation concurrent depression to yield a response with an initial large amplitude that decays to a tonic inhibition
C_LI


title: Neuronal apoptosis drives remodeling states of microglia and shifts in survival pathway dependence

abstract: Microglia serve critical remodeling roles that shape the developing nervous system, responding to the changing neural environment with phagocytosis or soluble factor secretion. Recent single-cell sequencing (scRNAseq) studies have revealed the context-dependent diversity in microglial properties and gene expression, but the cues promoting this diversity are not well defined. Here, we ask how interactions with apoptotic neurons shape microglial state, including lysosomal and lipid metabolism gene expression and independence from Colony-stimulating factor 1 receptor (CSF1R) for survival. Using early postnatal mouse retina, a CNS region undergoing significant developmental remodeling, we performed scRNAseq on microglia from mice that are wild-type, lack neuronal apoptosis (Bax KO), or are treated with CSF1R inhibitor (PLX3397). We find that interactions with apoptotic neurons drives multiple microglial remodeling states, subsets of which are resistant to CSF1R inhibition. We find that TAM receptor Mer and complement receptor 3 are required for clearance of apoptotic neurons, but that Mer does not drive expression of remodeling genes. We show TAM receptor Axl is negligible for phagocytosis or remodeling gene expression but is consequential for microglial survival in the absence of CSF1R signaling. Thus, interactions with apoptotic neurons shift microglia towards distinct remodeling states and through Axl, alters microglial dependence on survival pathway, CSF1R.


title: IKKalpha-Mediated Non-canonical NF-kappaB Signaling is Required to Support Murine Gammaherpesvirus 68 Latency In Vivo

abstract:
Non-canonical NF-kappaB signaling is activated in B cells via TNF receptor superfamily members CD40, Lymphotoxin beta-R, and BAFF-R. The non-canonical pathway is required at multiple stages of B-cell maturation and differentiation, including the germinal center reaction. However, the role of this pathway in gammaherpesvirus latency is not well understood. Murine gammaherpesvirus 68 (MHV68) is a genetically tractable system used to define pathogenic determinants. Mice lacking the BAFF-R exhibit defects in splenic follicle formation and are greatly reduced for MHV68 latency. We report a novel approach to disrupt non-canonical NF-kappaB signaling exclusively in cells infected with MHV68. We engineered a recombinant virus that expresses a dominant negative form of IKKalpha, named IKK-SA, with S176A and S180A mutations that prevent phosphorylation by NIK. We controlled for the transgene insertion by introducing two all-frame stop codons into the IKK-SA gene. The IKK-SA mutant but not the IKK-SA.STOP control virus impaired LTbetaR-mediated activation of NF-kappaB p52 upon fibroblast infection. IKK-SA expression did not impact replication in primary fibroblasts or in the lungs of mice following intranasal inoculation. However, the IKK-SA mutant was severely defective in colonization of the spleen and in the establishment of latency compared to the IKK-SA.STOP control and WT MHV68 at 16 dpi. Reactivation was undetectable in splenocytes infected with the IKK-SA mutant, but reactivation in peritoneal cells was not impacted by IKK-SA. Taken together, the non-canonical NF-kappaB signaling pathway is essential for the establishment of latency in the secondary lymphoid organs of mice infected with the murine gammaherpesvirus pathogen MHV68.

IMPORTANCEThe latency programs of the human gammaherpesviruses EBV and KSHV are associated with B cell lymphomas. It is critical to understand the signaling pathways that are used by gammaherpesviruses to establish and maintain latency in primary B cells. We used a novel approach to block non-canonical NF-kappaB signaling only in the infected cells of mice. We generated a recombinant virus that expresses a dominant negative mutant of IKKalpha that is non-responsive to upstream activation. Latency was reduced in a route- and cell type-dependent manner in mice infected with this recombinant virus. These findings identify a significant role for the non-canonical NF-kappaB signaling pathway that might provide a novel target to prevent latent infection of B cells with oncogenic gammaherpesviruses.


title: Genome characteristic of Bordetella parapertussis isolated from Iran

abstract: Pertussis also known as whooping cough is a respiratory infection in humans particularly in infants and usually caused by Bordetella pertussis. However, Bordetella parapertussis can also cause a similar clinical syndrome. During 2012 to 2015, from nasal swabs sent from different provinces to the pertussis reference laboratory of Pasture Institute of Iran for pertussis confirmation, seven B. parapertussis isolates were identified by bacterial culture, biochemical tests, and the presence of IS1001 insertion in the genome by real-time PCR. Furthermore, the expression of pertactin (Prn) as one the major virulence factor for bacterial adhesion was investigated using western blot. Moreover, the genomic characteristic of one recently collected isolate, IRBP134, from a seven-month infant was investigated using Illumina NextSeq sequencing protocol. The results revealed the genome with G+C content 65% and genome size 4.7 Mbp. A total of 81 single nucleotide polymorphisms (SNPs) and 13 short insertion and deletions were found in the genome compared to the B. parapertussis 12822 as a reference genome showing ongoing evolutionary changes in our isolate. A phylogeny relationship of IRBP134 was also investigated using global B. parapertussis available genomes.


title: Unravelling microalgal-bacterial interactions in aquatic ecosystems through 16S co-occurrence networks

abstract: Microalgae and bacteria have a wide spectrum of associations in aquatic environments. Since their interactions can directly influence global carbon and nutrient cycling, understanding these associations help us evaluate their influence on ecosystem productivity. Algal biodiversity is large, and bacterial associations have been characterised for a small fraction of them. While experiments based on algal-bacterial co-culturing are commonly used to infer interactions, deciphering all associations present in nature through such methods is impractical and approaches based on co-occurrence network analysis can help infer associations. In this study, we used microbial co-occurrence networks built from Earth microbiome project 16S metabarcoding data to detect microalgal-bacterial associations in aquatic environments. We analysed marine and freshwater environments to understand what groups of bacteria are tightly co-occurring with different algal groups in both aquatic environments, to see patterns of interactions, and to evaluate the overall use of co-occurrence networks to infer meaningful algal-bacterial interactions. In line with expectations from co-culturing work, our results show that the phyla Proteobacteria and Bacteroidetes are the major bacterial associates of microalgae and the co-occurring bacteria may be specific to the algal host. From the independent analysis of environments, we also show that sample origin may be an important determinant of these interactions. By unravelling previously established microalgal-bacterial links as well as identifying a range of previously unknown interactions, we show that co-occurrence network analysis is a promising hypothesis-generating framework to study microalgal-bacterial interactions that can guide future research into the functional nature of interactions.


title: Genomic signatures of isolation, hybridization, and selection during speciation of island finches

abstract:
Sister species occurring sympatrically on islands are rare and offer unique opportunities to understand how speciation can proceed in the face of gene flow. The Sao Tome grosbeak is a massive-billed,  giant finch endemic to the island of Sao Tome in the Gulf of Guinea, where it has diverged from its co-occurring sister species the Principe seedeater, an average-sized finch that also inhabits two neighbouring islands. Here, we show that the grosbeak carries a large number of unique alleles different from all three Principe seedeater populations, but also shares many alleles with the sympatric Sao Tome population of the seedeater, a genomic signature signifying divergence in isolation as well as subsequent introgressive hybridization. Furthermore, genomic segments that remain unique to the grosbeak are situated close to genes, including genes that determine bill morphology, suggesting the preservation of adaptive variation through natural selection during divergence with gene flow. This study reveals a complex speciation process whereby genetic drift, introgression, and selection during periods of isolation and secondary contact all have shaped the diverging genomes of these sympatric island endemic finches.

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title: Linked-Read Sequencing of Eight Falcons Reveals a Unique Genomic Architecture in Flux

abstract:
Falcons are diverse birds of cultural and economic importance. They have undergone major lineage-specific chromosomal rearrangements, resulting in greatly-reduced chromosome counts relative to other birds. Here, we use 10X Genomics linked reads to provide new high-contiguity genomes for two gyrfalcons, a saker falcon, a lanner falcon, three subspecies of peregrine falcons, and the common kestrel. Assisted by a transcriptome sequenced from 22 gyrfalcon tissues, we annotate these genomes for a variety of genomic features, estimate historical demography, and then investigate genomic equilibrium in the context of falcon-specific chromosomal rearrangements. We find that falcon genomes are not in AT-GC equilibrium with a bias in mutations towards higher AT content; this bias is predominantly driven by--but not dependent on--hypermutability of CpG sites. Small indels and large structural variants were also biased towards insertions rather than deletions. Patterns of disequilibrium were linked to chromosomal rearrangements: falcons have lost GC content in regions that have fused to larger chromosomes from microchromosomes and gained GC content in regions of macrochromosomes that have translocated to microchromosomes. Inserted bases have accumulated on regions ancestrally belonging to microchromosomes, consistent with insertion-biased gene conversion. We also find an excess of interspersed repeats on regions of microchromosomes that have fused to macrochromosomes. Our results reveal that falcon genomes are in a state of flux. They further suggest that many of the key differences between microchromosomes and macrochromosomes are driven by differences in chromosome size, and indicate a clear role for recombination and biased-gene-conversion in determining genomic equilibrium.

SignificanceFalcons are a particularly diverse and widespread genus of birds of particular cultural and economic importance. Falcons have also undergone recent large-scale chromosomal rearrangements to arrive at atypically low chromosome counts relative to other birds. We produced eight new high-quality falcon genomes to support general research on falcons, and we analyze these genomes to assess how chromosome loss has influenced specific aspects of genomic architecture.

category: genomics