title: A neuronal code for space in hippocampal coactivity dynamics independent of place fields

abstract: Hippocampus is comprised of [~]20% place cells, discharging in cell-specific locations ("place fields"), standardly interpreted as a dedicated neuronal code for space. However, place cell discharge is temporally unreliable across seconds and days, and spatially multimodal, suggesting an alternative "ensemble cofiring" spatial code with manifold dynamics that does not require reliable spatial tuning. We evaluated these hypotheses using GCaMP6f and miniature microscopes to image mouse CA1 ensemble activity in two environments, across 3 weeks. Both place fields and ensemble coactivity relationships "remap," being distinct between and (weakly) similar within environments. We find that decoding the environment from cell-pair coactivity relationships is effective and improves with experience, even after removing place tuning. Decoding from 1-s ensemble activity is indifferent to firing fields, relying crucially on the most anti-coactive cells because ensemble activity is internally-organized on a low-dimensional manifold of non-linear cofiring relationships that intermittently reregisters to environments according to anti-cofiring subpopulation activity.


title: A single exposure to altered auditory feedback causes observable sensorimotor adaptation in speech

abstract: Sensory errors caused by perturbations to movement-related feedback induce two types of behavioral changes that oppose the perturbation: rapid compensation within a movement, as well as longer-term adaptation of subsequent movements. Although adaptation is hypothesized to occur whenever a sensory error is perceived (including after a single exposure to altered feedback), adaptation of articulatory movements in speech has only been observed after repetitive exposure to auditory perturbations, questioning both current theories of speech sensorimotor adaptation as well as the universality of more general theories of adaptation. Thus, positive evidence for the hypothesized single-exposure or "one-shot" learning would provide critical support for current theories of speech sensorimotor learning and control and align adaptation in speech more closely with other motor domains. We measured one-shot learning in a large dataset in which participants were exposed to intermittent, unpredictable auditory perturbations to their vowel formants (the resonant frequencies of the vocal tract that distinguish between different vowels). On each trial, participants spoke a word out loud while their first formant was shifted up, shifted down, or remained unshifted. We examined whether the perturbation on a given trial affected speech on the subsequent, unperturbed trial. We found that participants adjusted their first formant in the opposite direction of the preceding shift, demonstrating that learning occurs even after a single auditory perturbation as predicted by current theories of sensorimotor adaptation. While adaptation and the preceding compensation responses were correlated, this was largely due to differences across individuals rather than within-participant variation from trial to trial. These findings are more consistent with theories that hypothesize adaptation is driven directly by updates to internal control models than those that suggest adaptation results from incorporation of feedback responses from previous productions.

category: neuroscience


title: Effects of theta transcranial alternating current stimulation (tACS) on exploration and exploitation during uncertain decision-making

abstract:
Exploring ones surroundings may yield unexpected rewards, but is associated with uncertainty and risk. Alternatively, exploitation of certain outcomes is related to low risk, yet potentially better outcomes remain unexamined. As such, risk-taking behavior depends on perceived uncertainty and a trade-off between exploration-exploitation. Previously, it has been suggested that risk-taking may relate to theta activity in the prefrontal cortex. Furthermore, previous studies hinted at a relationship between a right-hemispheric bias in frontal theta asymmetry and risky behavior. In the present double-blind sham-controlled within-subject study, we applied bifrontal transcranial alternating current stimulation (tACS) at the theta frequency (5 Hz) on eighteen healthy volunteers during a gambling task. Two tACS montages with either left-right or posterior-anterior current flow were employed at an intensity of 1 mA. Results showed that, compared to sham, theta tACS increased perceived uncertainty irrespective of current flow direction. Despite this observation, no direct effect of tACS on exploration behavior and general risk-taking was observed. Furthermore, frontal theta asymmetry was more right-hemispherically biased after posterior-anterior tACS, compared to sham. Finally, we used electric field simulation to identify which regions were targeted by the tACS montages as an overlap in regions may explain why the two montages resulted in comparable outcomes. Our findings provide a first step towards understanding the relationship between frontal theta oscillations and different features of risk-taking.

Highlights- Risk taking is related to uncertainty, exploration and exploitation
- Frontal theta tACS was applied to modulate aspects of risk taking
- tACS did increase perceived uncertainty, but not exploration behavior
- tACS induced a right hemispheric shift in frontal theta asymmetry


title: Effect of stimulation time on the expression of human macrophage polarization markers

abstract: Macrophages are highly plastic cells that can polarize into functionally distinct subsets in vivo and in vitro in response to environmental signals. The development of protocols to model macrophage polarization in vitro greatly contributes to our understanding of macrophage biology. Macrophages are divided into two main groups: Pro-inflammatory M1 macrophages (classically activated) and anti-inflammatory M2 macrophages (alternatively activated), based on several key surface markers and the production of inflammatory mediators. However, the expression of these common macrophage polarization markers is greatly affected by the stimulation time used. Unfortunately, there is no consensus yet regarding the optimal stimulation times for particular macrophage polarization markers in in vitro experiments. This situation is problematic, (i) as analysing a particular marker at a suboptimal time point can lead to false-negative results, and (ii) as it clearly impedes the comparison of different studies. Using human monocyte-derived macrophages (MDMs) in vitro, we analysed how the expression of the main polarization markers for M1 (CD64, CD86, CXCL9, CXCL10, HLA-DR, IDO1, IL1{beta}, IL12, TNF), M2a (CD200R, CD206, CCL17, CCL22, IL-10, TGM2), and M2c (CD163, IL-10, TGF{beta}) macrophages changes over time at mRNA and protein levels. Our data establish the most appropriate stimulation time for the analysis of the expression of human macrophage polarization markers in vitro. Providing such a reference guide will likely facilitate the investigation of macrophage polarization and its reproducibility.

category: immunology


title: Selfish bacteria are active throughout the water column of the ocean

abstract: Heterotrophic bacteria use extracellular enzymes to hydrolyze high molecular weight (HMW) organic matter to low molecular weight (LMW) hydrolysis products that can be taken into the cell. These enzymes represent a considerable investment of carbon, nitrogen, and energy, yet the return on this investment is uncertain, since hydrolysis of a HMW substrate outside a cell yields LMW products that can be lost to diffusion and taken up by scavengers that do not produce extracellular enzymes1. However, an additional strategy of HMW organic matter utilization,  selfish uptake2, is used for polysaccharide degradation, and has recently been found to be widespread among bacterial communities in surface ocean waters3. During selfish uptake, polysaccharides are bound at the cell surface, initially hydrolyzed, and transported into the periplasmic space without loss of hydrolysis products2, thereby retaining hydrolysate for the selfish bacteria and reducing availability of LMW substrates to scavenging bacteria. Here we show that selfish bacteria are common not only in the sunlit upper ocean, where polysaccharides are freshly produced by phytoplankton, but also deeper in the oceanic water column, including in bottom waters at depths of more than 5,500 meters. Thus, the return on investment, and therefore also the supply of suitable polysaccharides, must be sufficient to maintain these organisms.

category: microbiology


title: The Alzheimer's gene SORL1 is a key regulator of endosomal recycling in human neurons

abstract:
BackgroundLoss of the Sortilin-related receptor 1 (SORL1) gene seems to act as a causal event for Alzheimers disease (AD). Recent studies have established that loss of SORL1, as well as mutations in autosomal dominant AD genes APP and PSEN1/2, pathogenically converge by swelling early endosomes, ADs cytopathological hallmark. Acting together with the retromer trafficking complex, SORL1 has been shown to regulate the recycling of the amyloid precursor protein (APP) out of the endosome, contributing to endosomal swelling and to APP misprocessing. We hypothesized that SORL1 plays a broader role in neuronal endosomal recycling and used human induced pluripotent stem cell derived neurons (hiPSC-Ns) to test this hypothesis. We examined endosomal recycling of three transmembrane proteins linked to AD pathophysiology: APP, the BDNF receptor Tropomyosin-related kinase B (TRKB), and the glutamate receptor subunit AMPA1 (GLUA1).

MethodsWe used isogenic hiPSCs engineered to have SORL1 depleted or to have enhanced SORL1 expression. We differentiated neurons from these cell lines and mapped the trafficking of APP, TRKB and GLUA1 within the endosomal network using confocal microscopy. We also performed cell surface recycling and lysosomal degradation assays to assess the functionality of the endosomal network in both SORL1 depleted and overexpressing neurons. Finally, we analyzed alterations in gene expression in SORL1 depleted neurons using RNA-sequencing.

ResultsWe find that as with APP, endosomal trafficking of GLUA1 and TRKB is impaired by loss of SORL1. We show that trafficking of all three cargo to late endosomes and lysosomes is affected by manipulating SORL1 expression. We also show that depletion of SORL1 significantly impacts the endosomal recycling pathway for APP and GLUA1 at the level of the recycling endosome and trafficking to the cell surface. This has a functional effect on neuronal activity as shown by multi-electrode array (MEA). Conversely, increased SORL1 expression enhances endosomal recycling for APP and GLUA1. Our unbiased transcriptomic data further support SORL1s role in endosomal recycling. We observe altered expression networks that regulate cell surface trafficking and neurotrophic signaling in SORL1 depleted neurons.

ConclusionCollectively, and together with other recent observations, these findings suggest that SORL1 is a broad regulator of retromer-dependent endosomal recycling in neurons, a conclusion that has both pathogenic and therapeutic implications for Alzheimers disease.

category: cell biology


title: The Alzheimer's gene SORL1 is a key regulator of endosomal traffic and recycling in human neurons


title: MYC hyperactivates WNT signaling in APC/CTNNB1-mutated colorectal cancer cells through miR-92a-dependent repression of DKK3

abstract:
Activation of Wnt signaling is among the earliest events of colon cancer development. It is achieved either via activating mutations in the CTNNB1 gene encoding {beta}-catenin, the key transcription factor in the Wnt pathway, or most commonly by inactivating mutations in APC, a major {beta}-catenin binding partner and negative regulator. However, our analysis of recent Pan Cancer Atlas data revealed that CTNNB1 mutations significantly co-occur with those affecting Wnt receptor complex components (e.g., Frizzled and LRP6), underscoring the importance of additional regulatory events even in the presence of common APC/CTNNB1 mutations. In our effort to identify non-mutational hyperactivating events, we determined that KRAS-transformed murine colonocytes overexpressing direct {beta}-catenin target MYC show significant upregulation of the Wnt signaling pathway and reduced expression of Dickkopf 3 (DKK3), a reported ligand for Wnt co-receptors. We demonstrate that Myc suppresses Dkk3 transcription through one of mir-17-92 cluster microRNAs, miR-92a. We further examined the role of DKK3 by overexpression and knockdown and discovered that DKK3 suppresses Wnt signaling in APC-null murine colonic organoids and human colon cancer cells despite the presence of downstream activating mutations in the Wnt pathway. Conversely, MYC overexpression in the same cell lines resulted in hyperactive Wnt signaling, acquisition of epithelial-to-mesenchymal transition markers, and enhanced migration and invasion and metastasis in syngeneic orthotopic mouse colon cancer model. Our results suggest that the MYC->miR-92a-|DKK3 axis hyperactivates Wnt signaling, forming a feedforward oncogenic loop.

SIGNIFICANCECommon APC and CTNNB1 mutations activate Wnt signaling in colorectal cancers. Here we demonstrate that further potentiation of this pathway involves microRNA-dependent repression of the DKK3 gene by the Myc oncoprotein.

category: cancer biology


title: PEGR: a flexible management platform for reproducible epigenomic and genomic research

abstract: Reproducibility is a significant challenge in (epi)genomic research due to the complexity of experiments composed of traditional biochemistry and informatics. Recent advances have exacerbated this challenge as high-throughput sequencing data is generated at an unprecedented pace. Here we report on our development of a Platform for Epi-Genomic Research (PEGR), a web-based project management platform that tracks and quality controls experiments from conception to publication-ready figures, compatible with multiple assays and bioinformatic pipelines. It supports rigor and reproducibility for biochemists working at the wet bench, while continuing to fully support reproducibility and reliability for bioinformaticians through integration with the Galaxy platform.

category: bioinformatics


title: Single-cell chemical imaging of engineered strains reveals heterogeneity in fatty acid production

abstract: Engineered microbes can produce valuable chemicals, however production strains often require extensive optimization before they can be used at industrial scales. To quantify production, metabolic engineers typically employ mass spectrometry, which offers excellent chemical specificity. However, with this approach samples are derived from bulk cultures, obscuring potential cell-to-cell differences in production, and precluding selection methods that rely on measurements of a single cells phenotype, such as directed evolution. Here, we use stimulated Raman scattering (SRS) based chemical imaging to directly visualize free fatty acids in metabolically engineered Escherichia coli. We uncover substantial heterogeneity in fatty acid production among and within colonies. We then demonstrate longitudinal SRS imaging, allowing fatty acids to be linked with the cells that generated them. Further, using the hyperspectral images, we develop an approach for compositional analysis that determines the chain length and unsaturation of the fatty acids in living cells. Our results demonstrate that cell-to-cell heterogeneity in production of metabolic targets can be substantial and indicate that controlling this to eliminate low producers represents a critical area for future optimization. Further, because SRS imaging is rapid, chemically specific, and can be acquired longitudinally, it is a promising method for metabolic engineers, allowing for direct imaging of biosynthesis at the single-cell level.

category: synthetic biology


title: Probabilistic Evaluation of Null Models for West Nile Virus in the United States

abstract: Null models provide a useful baseline for the development of new models. A variety of options for null models exist. These options have become more sophisticated with the advent of probabilistic modeling approaches. Here, we evaluate 10 different null models for West Nile virus, a primarily mosquito-borne disease introduced to the United States in 1999. The Historical Null was significantly better than all models other than the Negative Binomial. We recommend the use of either of these models as a baseline when developing new models to predict spatial and temporal dynamics of West Nile virus at the county-annual scale. We expect these results to be scale-dependent, and a future direction is to examine performance of null models at finer spatial and temporal scales.

category: ecology


title: Divining Deamidation and Isomerization in Therapeutic Proteins: Effect of Neighboring Residue

abstract: Deamidation of asparagine (ASN) and isomerization of aspartic acid (ASP) residues are among the most commonly observed spontaneous post-translational modifications (PTMs) in proteins. Understanding and predicting a protein sequences propensity for such PTMs can help expedite protein therapeutic discovery and development. In this study, we utilized proton-affinity calculations with semi-empirical quantum mechanics (QM) and {micro}s long equilibrium molecular dynamics (MD) simulations to investigate mechanistic roles of structure and chemical environment in dictating spontaneous degradation of asparagine and aspartic acid residues in 131 clinical-stage therapeutic antibodies. Backbone secondary structure, side-chain rotamer conformation and solvent accessibility were found as three key molecular indicators of ASP isomerization and ASN deamidation. Comparative analysis of backbone dihedral angles along with N-H proton affinity calculations provides a mechanistic explanation for the strong influence of the identity of the n+1 residue on the rate of ASP/ASN degradation. With these findings, we propose a minimalistic physics-based classification model that can be leveraged to predict deamidation and isomerization propensity of therapeutic proteins.

category: biochemistry


title: Gametophyte genome activation occurs at pollen mitosis I in maize

abstract: Flowering plants alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. One consequence of this life cycle is that plants face substantial selection during the haploid phase1-3. Pollen actively transcribes its haploid genome4, providing phenotypic diversity even among pollen grains from a single plant. Currently, the timing that pollen precursors first establish this independence is unclear. Starting with an endowment of transcripts from the diploid parent, when do haploid cells generated by meiosis begin to express genes? Here, we follow the shift to haploid expression in maize pollen using allele-specific RNA-sequencing (RNA-Seq) of single pollen precursors. We observe widespread biallelic expression for 11 days after meiosis, indicating that transcripts synthesized by the diploid sporophyte persist long into the haploid phase. Subsequently, there was a rapid and global conversion to monoallelic expression at pollen mitosis I (PMI), driven by active new transcription from the haploid genome. Genes expressed during the haploid phase showed reduced rates of nonsynonymous relative to synonymous substitutions (dn/ds) if they were expressed after PMI, but not before, consistent with purifying selection acting on the haploid gametophyte. This work establishes the timing with which haploid selection may act in pollen and provides a detailed time-course of gene expression during pollen development.

category: plant biology


title: Modular assembly of dynamic models in systems biology

abstract:
It is widely acknowledged that the construction of large-scale dynamic models in systems biology requires complex modelling problems to be broken up into more manageable pieces. To this end, both modelling and software frameworks are required to enable modular modelling. While there has been consistent progress in the development of software tools to enhance model reusability, there has been a relative lack of consideration for how underlying biophysical principles can be applied to this space. Bond graphs combine the aspects of both modularity and physics-based modelling. In this paper, we argue that bond graphs are compatible with recent developments in modularity and abstraction in systems biology, and are thus a desirable framework for constructing large-scale models. We use two examples to illustrate the utility of bond graphs in this context: a model of a mitogen-activated protein kinase (MAPK) cascade to illustrate the reusability of modules and a model of glycolysis to illustrate the ability to modify the model granularity.

Author summaryThe biochemistry within a cell is complex, being composed of numerous biomolecules and reactions. In order to develop fully detailed mathematical models of cells, smaller submodels need to be constructed and connected together. Software and standards can assist in this endeavour, but challenges remain in ensuring submodels are both consistent with each other and consistent with the fundamental conservation laws of physics.

In this paper, we propose a new approach using bond graphs from engineering. In this approach, connections between models are defined using physical conservation laws. We show that this approach is compatible with current software approaches in the field, and can therefore be readily used to incorporate physical consistency into existing model integration methodologies. We illustrate the utility of this approach in streamlining the development of models for a signalling network (the MAPK cascade) and a metabolic network (the glycolysis pathway).

The advantage of this approach is that models can be developed in a scalable manner while also ensuring consistency with the laws of physics, enhancing the range of data available to train models. This approach can be used to quickly construct detailed and accurate models of cells, facilitating future advances in biotechnology and personalised medicine.

category: systems biology


title: Recording and neural circuit-based manipulation of the maternal oxytocin pulses in mice

abstract: For mammals, successful parturition and breastfeeding are critical to the survival of offspring. Pulsatile release of the hormone oxytocin mediates uterine contraction during parturition and milk ejection during lactation. These oxytocin pulses are generated by unique activity patterns of the central neuroendocrine oxytocin neurons located in the paraventricular and supraoptic hypothalamus. However, the maternal activities of oxytocin neurons remain elusive because most classical electrophysiological studies in anesthetized rats have lacked the genetically defined cell identity of oxytocin neurons. We herein introduce viral genetic approaches in mice to characterize the maternal pulsatile activities of oxytocin neurons by fiber-photometry-based chronic in vivo Ca2+ imaging. We also demonstrate the pharmaco-genetic manipulation of oxytocin pulses during lactation via activating a prominent pre-synaptic structure of oxytocin neurons defined by retrograde trans-synaptic tracing. Collectively, our study opens a new avenue for the neuroscience of maternal neuroendocrine functions.


title: CAR/CXCR5-T cell immunotherapy is safe and potentially efficacious in promoting sustained remission of SIV infection

abstract:
During chronic human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infection prior to AIDS progression, the vast majority of viral replication is concentrated within B cell follicles of secondary lymphoid tissues. We investigated whether infusion of T cells expressing an SIV-specific chimeric antigen receptor (CAR) and the follicular homing receptor, CXCR5, could successfully kill viral-RNA+ cells in targeted lymphoid follicles in SIV-infected rhesus macaques. In this study, CD4 and CD8 T cells from rhesus macaques were genetically modified to express antiviral CAR and CXCR5 moieties (generating CAR/CXCR5-T cells) and autologously infused into a chronically infected animal. At 2 days post-treatment, the CAR/CXCR5-T cells were located primarily in spleen and lymph nodes both inside and outside of lymphoid follicles. Few CAR/CXCR5-T cells were detected in the rectum and lung, and no cells were detected in the bone marrow, liver, brain, or ileum. Within follicles, CAR/CXCR5-T cells were found in direct contact with SIV viral RNA+ cells. We next infused CAR/CXCR5-T cells into ART-suppressed SIV-infected rhesus macaques, in which the animals were released from ART at the time of infusion. These CAR/CXCR5-T cells replicated in vivo within both the extrafollicular and follicular regions of lymph nodes and accumulated within lymphoid follicles. CAR/CXR5-T cell concentrations in follicles peaked during the first week post-infusion but declined to undetectable levels after 2 to 4 weeks. Overall, CAR/CXCR5-T cell-treated animals maintained lower viral loads and follicular viral RNA levels than untreated control animals, and no outstanding adverse reactions were noted. These findings indicate that CAR/CXCR5-T cell treatment is safe and holds promise as a future treatment for the durable remission of HIV.

Author summaryA person infected with human immunodeficiency virus (HIV) has replicating virus concentrated within the follicles of lymphoid tissues. The cells needed to clear the infection, cytotoxic T lymphocytes, have limited access to follicles and, thus, the cytotoxic T lymphocytes are never completely able to clear all of the HIV from the body. In this study, we have produced immunotherapeutic T cells that home to follicles and clear infected cells. These T cells express a viral targeting chimeric antigen receptor (CAR) and a molecule called CXCR5, which leads to homing of the cells to follicles. Upon administration of these CAR T-cells to virus-infected primates, we found that the cells localized to the follicle, replicated, and directly interacted with infected cells. While the cells were not maintained in the animals for more than 4 weeks, most of the treated animals maintained lower levels of virus in the blood and follicles than untreated control animals. This study shows that this immunotherapy has potential as a treatment leading to long-term remission of HIV without the need for antiretroviral drugs.


title: The WOPR family protein Ryp1 is a key regulator of gene expression, development, and virulence in the thermally dimorphic fungal pathogen Coccidioides posadasii

abstract:
Coccidioides spp. are mammalian fungal pathogens endemic to the southwestern US and other desert regions of Mexico, central and South America, with the bulk of US infections occurring in California and Arizona. In the soil, Coccidioides grows in a hyphal form that differentiates into 3-5 micron asexual spores (arthroconidia). When arthroconidia are inhaled by mammals they undergo a unique developmental transition from polar hyphal growth to isotropic expansion with multiple rounds of nuclear division, prior to segmentation, forming large spherules filled with endospores. Very little is understood about the molecular basis of spherule formation. Here we characterize the role of the conserved transcription factor Ryp1 in Coccidioides development. We show that Coccidioides {Delta}ryp1 mutants have altered colony morphology under hypha-promoting conditions and are unable to form mature spherules under spherule-promoting conditions. We analyze the transcriptional profile of wild-type and {Delta}ryp1 mutant cells under hypha- and spherule-promoting conditions, thereby defining a set of hypha- or spherule-enriched transcripts ("morphology-regulated" genes) that are dependent on Ryp1 for their expression. Forty percent of morphology-regulated expression is Ryp1-dependent, indicating that Ryp1 plays a dual role in both hyphal and spherule development. Ryp1-dependent transcripts include key virulence factors such as SOWgp, which encodes the spherule outer wall glycoprotein. Concordant with its role in spherule development, we find that the {Delta}ryp1 mutant is completely avirulent in the mouse model of coccidioidomycosis, indicating that Ryp1-dependent pathways are essential for the ability of Coccidioides to cause disease. Vaccination of C57BL/6 mice with live {Delta}ryp1 spores does not provide any protection from lethal C. posadasii intranasal infection, consistent with our findings that the {Delta}ryp1 mutant fails to make mature spherules and likely does not express key antigens required for effective vaccination. Taken together, this work identifies the first transcription factor that drives mature spherulation and virulence in Coccidioides.

Author SummaryCoccidioides species, C. immitis and C. posadasii, are dimorphic fungal pathogens that commonly infect humans in North, Central, and South America, causing the respiratory fungal disease known as Valley Fever. Coccidioides grows as hyphae in the soil and differentiates into unique structures called spherules in the mammalian host. Spherules expand and internally divide to form endospores, which are released to facilitate dissemination of the pathogen within the host. The mechanisms underlying spherule differentiation remain largely unknown. In this study, we generated knockout mutants ({Delta}ryp1) of the conserved transcription factor Ryp1 in C. posadasii and characterized its role in spherule formation and virulence. We found that Ryp1 is required for the formation of mature spherules and colonization of mouse lungs. Transcriptional profiling of the {Delta}ryp1 mutant and the wild-type strain shows that Ryp1 regulates the expression of a subset of the transcripts that are either upregulated in wild-type spherules or in wild-type hyphae. These findings suggest that Ryp1 has a dual role in regulating morphology and virulence under host conditions as well as regulating genes involved in hyphal growth in the environment.


title: Cell-type specific innate immune responses shape rabies virus tropism

abstract:
Viral tropism, or the specificity of a particular virus to infect a certain cell type, is crucial in determining virus replication, viral spread, and ultimately host survival. Rabies, one of the deadliest known zoonotic diseases, is still causing 60.000 human deaths annually. Upon central nervous system (CNS) entry, neurotropic rabies virus (RABV) preserves the neural network by limiting apoptosis and inflammation. To date, we do not fully understand the factors determining RABV tropism and why glial cells are unable to clear RABV from the infected brain. Here, we compare susceptibilities and innate immune responses of CNS cell types towards infection with virulent dog RABV Tha and less virulent Th2P-4M in vitro, highlighting differences in cellular susceptibility and antiviral responses. Less virulent Th2P-4M bears mutations introduced in viral phosphoprotein (P-protein) and matrix protein (M-protein) thereby hindering viral immune evasion of the host nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B) and Janus kinase (JAK) - signal transducer and activator of transcription protein (STAT) pathways. Our results reveal that human neural stem cell (hNSC)-derived neurons and astrocytes, in contrast to human iPSC-derived microglia, are highly susceptible to Tha and Th2P-4M infection in vitro. Surprisingly, Th2P-4M presents a stronger neurotropism in hNSC-derived CNS cultures compared to Tha suggesting that NF-{kappa}B- and JAK-STAT-mediated antiviral host responses are defining RABV replication and thereby its tropism. Further, we show that astrocyte-like (SVGp12) and microglia-like (HMC3) cells protect neuroblastoma cells (SK-N-SH) from Tha infection in vitro. Transcription profiles and quantification of intracellular protein levels revealed major differences in antiviral immune responses mediated by neurons, astrocytes (IFNB1, CCL5, CXCL10, IL1B, IL6, LIF), and microglia (CCL5, CXCL10, ISG15, MX1, IL6) upon virulent Tha infection. Overall, we provide evidence that RABV tropism depends on its capability to evade cell-type specific immune responses via P- and M-proteins.

Author summaryRabies virus (RABV) neurotropism is widely reported as a unique feature of rabies, and still the exact mechanism underlying RABV susceptibility remains to be elucidated. Several receptors are known to accelerate RABV entry to the cell (1-4) and yet, none of them seems to be essential for successful infection (5,6) questioning a marked cellular tropism. Although RABV is classically reported as strictly neurotropic (7), recent studies report profound infection of glial cells in vivo depending on the viral strain and the infection route used (8,9). Here, we provide evidence that human neural stem cell (hNSC)-derived neurons (hiNeurons) and astrocytes (hiAstrocytes) are highly susceptible towards infection with the virulent field RABV strain Tha and less virulent Th2P-4M. In contrast, human iPSC-derived microglia-like cells (hiMicros) are resistant to viral replication in vitro. Whereas hiNeurons are immunologically quiescent upon Tha infection, fetal astrocytes and hiMicros establish strong antiviral responses. In contrast to Tha, Th2P-4M, which is unable to evade NF-{kappa}B and JAK-STAT pathways (10), shows a more profound neurotropism suggesting that cell-type specific responses shape RABV tropism. Hence, we conclude that viral evasion mechanisms mediated by P- and M-proteins partly determine Tha tropism of human CNS cell types in vitro.


title: Regulatory cross-talk supports resistance to Zn intoxication in Streptococcus

abstract:
Metals such as copper (Cu) and zinc (Zn) are important trace elements that can effect bacterial cell physiology but can also intoxicate bacteria at high concentrations. Discrete genetic systems for management of Cu and Zn efflux have been described in several bacteria pathogens, including streptococci. However, insight into molecular cross-talk between systems for Cu and Zn management in bacteria that drive metal detoxification, is limited. Here, we describe a biologically consequential cross-system effect of metal management in group B Streptococcus (GBS) governed by the Cu-responsive copY regulator in response to Zn. RNAseq analysis of wild-type (WT) and copY-deficient GBS exposed to metal stress revealed unique transcriptional links between the systems for Cu and Zn detoxification. We show that the Cu-sensing functions of CopY extend beyond Cu, and enable CopY to regulate Cu and Zn stress responses to effect genes involved in central cellular processes, including riboflavin synthesis. CopY also contributed to supporting GBS virulence in vivo following infection of mice. Identification of the Zn resistome of GBS using TraDIS revealed a suite of genes essential for GBS growth in metal stress. Several of the genes identified are novel to systems that support bacterial survival in metal stress, and represent a diversity of mechanisms of microbial metal homeostasis during cell stress. Overall, this study reveals a new and important mechanism of cross-system complexity driven by CopY in bacteria to regulate cell management of metal stress and survival.

Author SummaryMetals, such as Cu and Zn, can be used by the mammalian immune system to target bacterial pathogens, and consequently, bacteria have evolved discrete genetic systems that subvert this host-derived antimicrobial response. Systems for Cu and Zn homeostasis are well characterized, including the transcriptional control of sensing and responding to metal stress. Here we have discovered novel features of metal response sytems in Streptococcus that have major implications for pathogenesis and virulence. We show that Streptococcus resists Zn intoxication by utilizing a bona fide Cu regulator, CopY, to maintain cellular metal homeostasis, which enables the bacteria to survive stressful conditions. We identify new genes in Streptococcus that confer resistance to zinc intoxication, including several that have not previously been linked to metal ion homeostasis in any bacterium. The identification of cross-system metal management and new resistance mechanisms enhances our understanding of metal ion homeostasis in bacteria and its effect on pathogenesis.


title: The determinants of African Swine Fever Virus Virulence - the Georgia 2007/1 strain and the host macrophage response

abstract:
African swine fever virus (ASFV) has a major global economic impact. With a case fatality in domestic pigs approaching 100%, it currently presents the largest threat to animal farming. Although genomic differences between attenuated and highly virulent ASFV strains have been identified, the molecular determinants for virulence at the level of gene expression have remained opaque. Here we characterise the transcriptome of ASFV genotype II Georgia 2007/1 (GRG) during infection of the physiologically relevant host cells, porcine macrophages. In this study we applied Cap Analysis Gene Expression sequencing (CAGE-seq) to map the 5 ends of viral mRNAs at 5 and 16 hours post-infection. A bioinformatics analysis of the sequence context surrounding the transcription start sites (TSSs) enabled us to characterise the global early and late promoter landscape of GRG. We compared transcriptome maps of the GRG isolate and the lab-attenuated BA71V strain that highlighted GRG virulent-specific transcripts belonging to multigene families, including two predicted MGF 100 genes I7L and I8L. In parallel, we monitored transcriptome changes in the infected host macrophage cells. Of the 9,384 macrophage genes studied, transcripts for 652 host genes were differentially regulated between 5 and 16 hours-post-infection compared with only 25 between uninfected cells and 5 hours post-infection. NF-kB activated genes and lysosome components like S100 were upregulated, and chemokines such as CCL24, CXCL2, CXCL5 and CXCL8 downregulated.

ImportanceAfrican swine fever virus (ASFV) causes haemorrhagic fever in domestic pigs with case fatality rates approaching 100%, and no approved vaccines or antivirals. The highly-virulent ASFV Georgia 2007/1 strain (GRG) was the first isolated when ASFV spread from Africa to the Caucasus region in 2007. Then spreading through Eastern Europe, and more recently across Asia. We used an RNA-based next generation sequencing technique called CAGE-seq to map the starts of viral genes across the GRG DNA genome. This has allowed us to investigate which viral genes are expressed during early or late stages of infection and how this is controlled, comparing their expression to the non-virulent ASFV-BA71V strain to identify key genes that play a role in virulence. In parallel we investigated how host cells respond to infection, which revealed how the ASFV suppresses components of the host immune response to ultimately win the arms race against its porcine host.


title: African Swine Fever Virus and host response - transcriptome profiling of the Georgia 2007/1 strain and porcine macrophages


title: ADAP1 promotes latent HIV-1 reactivation by selectively tuning a T cell signaling-transcriptional axis

abstract: Immune stimulation fuels cell signaling-transcriptional programs inducing biological responses to eliminate virus-infected cells. Yet, retroviruses that integrate into host cell chromatin, such as HIV-1, co-opt these programs to switch between latent and reactivated states; however, the regulatory mechanisms are still unfolding. Here, we implemented a functional screen leveraging HIV-1s dependence on CD4+ T cell signaling-transcriptional programs and discovered ADAP1 is an undescribed modulator of HIV-1 proviral fate. Specifically, we report ADAP1 (ArfGAP with dual PH domain-containing protein 1), a previously thought neuronal-restricted factor, is an amplifier of select T cell signaling programs. Using complementary biochemical and cellular assays, we demonstrate ADAP1 inducibly interacts with the immune signalosome to directly stimulate KRAS GTPase activity thereby augmenting T cell signaling through targeted activation of the ERK-AP-1 axis. Single cell transcriptomics analysis revealed loss of ADAP1 function blunts gene programs upon T cell stimulation consequently dampening latent HIV-1 reactivation. Our combined experimental approach defines ADAP1 as an unexpected tuner of T cell programs co-opted by HIV-1 for latency escape.


title: Profound structural conservation of chemically cross-linked HIV-1 envelope glycoprotein experimental vaccine antigens

abstract: Chemical cross-linking is used to stabilise protein structure with additional benefits of pathogen and toxin inactivation for vaccine use, but its use is restricted by potential induction of local or global structural distortion. This is of particular importance when the protein in question requires a high degree of structural conservation for the purposes of understanding function, or for inducing a biological outcome such as elicitation of antibodies to conformationally-sensitive epitopes. The HIV-1 envelope glycoprotein (Env) trimer is metastable and shifts between different conformational states, complicating its functional analysis and use as a vaccine antigen. Here we have used the hetero-bifunctional zero-length reagent EDC to cross-link two soluble Env trimers, selected well-folded trimers using an antibody affinity column, and transferred this process to good manufacturing practice (GMP) for clinical trial use. Cross-linking enhanced GMP trimer stability to biophysical and enzyme attack, and had broadly beneficial effects on morphology, antigenicity and immunogenicity. Cryo-EM analysis revealed that cross-linking essentially completely retained overall structure with RMSDs between unmodified and cross-linked Env trimers of 0.4-0.5 [A]. Despite this negligible distortion of global trimer structure we identified individual inter-subunit, intra-subunit and intra-protomer cross-links. Thus, EDC cross-linking maintains protein folding, improves stability, and is readily transferred to GMP, consistent with use of this approach in probing protein structure/function relationships and in the design of vaccines.

category: molecular biology


title: Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production

abstract:
-galactosidase (-GAL) and -N-acetylgalactosaminidase (-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki-diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important -GAL and -NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansonis -GAL and -NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative -GAL/-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for -GAL/-NAGAL substrate cleavage. Both -GAL and -NAGAL enzymatic activities were higher in females compared to males (p<0.05; -NAGAL > -GAL), which was consistent with smp_089290s female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited -NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in -GAL activities were observed in the same extracts. Despite this, decreases in -NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an -NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Pharmacological inhibition of SmNAGAL may lead to the development of a novel anthelmintic class of compounds.

Author summarySchistosomiasis is a parasitic disease caused by infection with blood flukes, which leads to acute and chronic pathology in millions of infected individuals located in deprived tropical and subtropical regions. Elucidating the function of schistosome genes has provided a clearer view on their roles in various molecular pathways, which are critical to successful parasitism. This information is invaluable when progressing novel drug and vaccine candidates. Here, we add to the existing knowledge of the Schistosoma mansoni parasitic glycan processing and modification machinery by functionally characterising a glycosyl hydrolase (S. mansoni -N-acetylgalactosaminidase, SmNAGAL). We demonstrate that this protein is enzymatically active and important in coordinating parasite movement in adult male and female schistosomes. Additionally, we provide evidence that this protein regulates pathways associated with egg production in female schistosomes, which is responsible for inducing pathological reactions. Developing drugs that inhibit SmNAGAL enzymatic activity could provide a novel approach for controlling schistosomiasis.


title: Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction

abstract: Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct functional assessment of SNVs in model organisms. However, the lack of bioinformatic tools aiding target prediction limits the application of base editing in vivo. Here, we provide a framework for adenine and cytosine base editing in medaka (Oryzias latipes) and zebrafish (Danio rerio), ideal for scalable validation studies. We developed an online base editing tool ACEofBASEs (a careful evaluation of base-edits), to facilitate decision-making by streamlining sgRNA design and performing off-target evaluation. We used state-of-the-art adenine (ABE) and cytosine base editors (CBE) in medaka and zebrafish to edit eye pigmentation genes and transgenic GFP function with high efficiencies. Base editing in the genes encoding troponin T and the potassium channel ERG faithfully recreated known cardiac phenotypes. We finally validated missense mutations in novel candidate genes of congenital heart disease (CHD) dapk3, ube2b, usp44, and ptpn11 and present the resulting heart specific phenotypes. This base editing framework applies to a wide range of SNV-susceptible traits accessible in fish, facilitating straight-forward candidate validation and prioritization for detailed mechanistic downstream studies.

category: genetics


title: Bmmp influences wing shape by regulating anterior-posterior and proximal-distal axis development

abstract: Insect wings are subject to strong selective pressure, resulting in the evolution of remarkably diverse wing shapes that largely determine flight capacity. However, the genetic basis and regulatory mechanisms underlying wing shape development are not well understood. The silkworm Bombyx mori micropterous (mp) mutant exhibits shortened wing length and enlarged vein spacings, albeit without changes in total wing area. Thus, the mp mutant comprises a valuable genetic resource for studying wing shape development. In this study, we used molecular mapping to identify the gene responsible for the mp phenotype and designated it Bmmp. Phenotype-causing mutations were identified as indels and single nucleotide polymorphisms in non-coding regions. These mutations resulted in decreased Bmmp mRNA levels and changes in transcript isoform composition. Bmmp null mutants were generated by CRISPR/Cas9 and exhibited significantly smaller wings. By examining the expression of genes critical to wing development in wildtype and Bmmp null mutants, we found that Bmmp exerts its function by coordinately modulating anterior-posterior and proximal-distal axis development. We also studied a Drosophila mp mutant and found that Bmmp is functionally conserved in Drosophila. The Drosophila mp mutant strain exhibits curly wings of reduced size and a complete loss of flight capacity. Our results increase our understanding of the mechanisms underpinning insect wing development and reveal potential targets for pest control.


title: Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation

abstract:
CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying ( homing) therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel gene drive in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations.

Author summaryGene drives have the potential to be applied as novel control strategy of disease-transmitting mosquitoes, by spreading genetic traits that suppress or modify the target population. Many gene drive elements work by recognising and cutting a specific target sequence in the mosquito genome and copying themselves into that target sequence allowing the gene drive to increase in frequency in the population.

Like other mosquito control interventions, efficacy will greatly depend on minimising the development of resistance to the gene drive mechanism - most likely via a change in the target sequence that prevents further cutting. One strategy to reduce resistance is to target sequences that are highly conserved, which implies that changes cannot easily be tolerated. We developed a strategy that simulates high selection pressure, under which resistance is most likely to emerge, and therefore provides a stringent test of its propensity to arise. Unlike previous results with another gene drive, we recovered a resistant allele within a few generations of gene drive exposure and at high frequency. Our results show that conserved sequences can vary hugely in ability to tolerate mutations and highlights the need to functionally validate future candidate gene drive target sites for their robustness to resistance.


title: Genetic loci of the R. anatipestifer serotype discovered by Pan-GWAS and its application for the development of a multiplex PCR serotyping method

abstract:
The disease caused by Riemerella anatipestifer (R. anatipestifer) causes large economic losses to the global duck industry every year. Serotype-related genomic variation (such as in O-antigen and capsular polysaccharide gene clusters) has been widely used for the serotyping in many gram-negative bacteria. To date, there have been few studies focused on genetic basis of serotypes in R. anatipestifer. Here, we used pan-genome-wide association studies (Pan-GWAS) to identify the serotype-specific genetic loci of 38 R. anatipestifers strain. Analyses of the loci of 11 serotypes showed that the loci could be well mapped with the serotypes of the corresponding strains. We constructed the knockout strain for the wzy gene at the locus, and the results showed that the mutant lost the agglutination characteristics to positive antisera. Based on the of Pan-GWAS results, we developed a multiple PCR method to identify serotypes 1, 2, and 11 of R. anatipestifer. Our study provides a precedent for systematically analysing the genetic basis of the R anatipestifer serotypes and establishing a complete serotyping system in the future.

HighlightsO_LIR. anatipestifer serotype-specific locus was identified by Pan-GWAS for the first time.
C_LIO_LIMolecular serotyping multiplex PCR was developed based on O-antigen biosynthesis gene clusters
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category: genomics


title: CIGB-300 synthetic peptide, an antagonist of CK2 kinase activity, as a treatment for Covid-19. A computational biology approach

abstract: Drug repositioning became the first choice for treating Covid-19 patients due to the urgent need to deal with the pandemic. Similarities in the hijacking mechanisms used by SARS-CoV-2 and several type of cancer, suggest the repurposing of cancer drugs to treat Covid-19. CK2 kinase antagonists have been proposed for the treatment of cancer. A recent study in cells infected with SARS-CoV-2 virus found a significant CK2 kinase activity, and the use of a CK2 inhibitor showed antiviral responses. CIGB-300, originally designed as an anticancer peptide, is an antagonist of CK2 kinase activity that binds to CK2 phospho-acceptor sites. Recent preliminary results show an antiviral activity of CIGB-300 versus a surrogate model of coronavirus. Here we present a computational biology study that provides evidences at the molecular level of how CIGB-300 might interfere with SARS-CoV-2 life cycle inside infected human cells. First, from SARS-CoV studies, we infer the potential incidence of CIGB-300 in SARS-CoV-2 interference on immune response. Next, from the analysis of multiple Omics data, we propose the action of CIGB-300 since early stage of viral infections perturbing the virus hijacking of RNA splicing machinery. It was also predicted the interference of CIGB-300 in virus-host interactions responsible for the high infectivity and the particular immune response to SARS-CoV-2 infection. Further, we provide evidences of CIGB-300 attenuation of phenotypes related to muscle, bleeding, coagulation and respiratory disorders.


title: Representational learning of brain responses in executive function and higher-order cognition using deep graph convolutions

abstract:
Brain decoding aims to infer human cognition from recordings of neural activity using modern neuroimaging techniques. Studies so far often concentrated on a limited number of cognitive states and aimed to classifying patterns of brain activity within a local area. This procedure demonstrated a great success on classifying motor and sensory processes but showed limited power over higher cognitive functions. In this work, we investigate a high-order graph convolution model, named ChebNet, to model the segregation and integration organizational principles in neural dynamics, and to decode brain activity across a large number of cognitive domains. By leveraging our prior knowledge on brain organization using a graph-based model, ChebNet graph convolution learns a new representation from task-evoked neural activity, which demonstrates a highly predictive signature of cognitive states and task performance. Our results reveal that between-network integration significantly boosts the decoding of high-order cognition such as visual working memory tasks, while the segregation of localized brain activity is sufficient to classify motor and sensory processes. Using twin and family data from the Human Connectome Project (n = 1,070), we provide evidence that individual variability in the graph representations of working-memory tasks are under genetic control and strongly associated with participants in-scanner behaviors. These findings uncover the essential role of functional integration in brain decoding, especially when decoding high-order cognition other than sensory and motor functions.

TeaserO_LIModelling functional integration through graph convolution is a necessary step towards decoding high-order human cognition.
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Significance statementOver the past two decades, many studies have applied multivariate pattern analysis to decode what task a human participant is performing, based on a scan of her brain. The vast majority of these studies have however concentrated on select regions and a specific domain, because of the computational complexity of handling full brain data in a multivariate model. With the fast progress in the field of deep learning, it is now possible to decode a variety of cognitive domains simultaneously using a full-brain model. By leveraging our prior knowledge on brain organization using a graph-based model, we uncovered different organizational principles in brain decoding for motor execution and high-order cognition by modelling functional integration through graph convolution.


title: Selective ancestral sorting and de novo evolution in the agricultural invasion of Amaranthus tuberculatus

abstract: The relative role of hybridization, de novo evolution, and standing variation in weed adaptation to agricultural environments is largely unknown. In Amaranthus tuberculatus, a widespread North American agricultural weed, adaptation is likely influenced by recent secondary contact and admixture of two previously isolated subspecies. We characterized the extent of adaptation and phenotypic differentiation accompanying the spread of A. tuberculatus into agricultural environments and the contribution of subspecies divergence. We generated phenotypic and whole-genome sequence data from a manipulative common garden experiment, using paired samples from natural and agricultural populations. We found strong latitudinal, longitudinal, and sex differentiation in phenotypes, and subtle differences among agricultural and natural environments that were further resolved with ancestry inference. The transition into agricultural environments has favoured southwestern var. rudis ancestry that leads to higher biomass and environment-specific phenotypes: increased biomass and earlier flowering under reduced water availability, and reduced plasticity in fitness-related traits. We also detected de novo adaptation to agricultural habitats independent of ancestry effects, including marginally higher biomass and later flowering in agricultural populations, and a time to germination home advantage. Therefore, the invasion of A. tuberculatus into agricultural environments has drawn on adaptive variation across multiple timescales--through both preadaptation via the preferential sorting of var. rudis ancestry and de novo local adaptation.

category: evolutionary biology


title: Bayesian Inference of Joint Coalescence Times for Sampled Sequences

abstract: The site frequency spectrum (SFS) is a commonly used statistic to summarize genetic variation in a sample of genomic sequences from a population. Such a genomic sample is associated with an imputed genealogical history with attributes such as branch lengths, coalescence times and the time to the most recent common ancestor (TMRCA) as well as topological and combinatorial properties. We present a Bayesian model for sampling from the joint posterior distribution of coalescence times conditional on the SFS associated with a sample of sequences in the absence of selection. In this model, the combinatorial properties of a genealogy, which is represented as a coalescent tree, are expressed as matrices. This facilitates the calculation of likelihoods and the effective sampling of the entire space of tree structures according to the Equal Rates Markov (or Yule-type) measure. Unlike previous methods, assumptions as to the type of stochastic process that generated the genealogical tree are not required. Novel approaches to defining both uninformative and informative prior distributions are employed. The uncertainty in inference due to the stochastic nature of mutation and the unknown tree structure is expressed by the shape of the posterior distributions. The method is implemented using the general purpose Markov Chain Monte Carlo software PyMC3. From the sampled posterior distribution of coalescence times, one can also infer related quantities such as the number of ancestors of a sample at a given time in the past (ancestral distribution) and the probability of specific relationships between branch lengths (for example, that the most recent branch is longer than all the others). The performance of the method is evaluated against simulated data and is also applied to historic mitochondrial data from the Nuu-Chah-Nulth people of North America. The method can be used to obtain estimates of the TMRCA of the sample. The relationship of these estimates to those given by "Thomsons estimator" is explored.


title: Individual repeatability, species differences, and the influence of socio-ecological factors on neophobia in 10 corvid species

abstract: Behavioural responses to novelty, including fear and subsequent avoidance of novel stimuli, behaviours referred to as neophobia, determine how animals interact with their environment. Neophobia aids in navigating risk and impacts on adaptability and survival. There is variation within and between individuals and species, however, lack of large-scale, comparative studies critically limits investigation of the socio-ecological drivers of neophobia. In this study, we tested responses to novel objects and food (alongside familiar food) versus a baseline (familiar food alone) in 10 corvid species (241 subjects) across 10 labs worldwide. There were species differences in the latency to touch familiar food in the novel object and food conditions relative to the baseline. Three of seven socio-ecological factors influenced object neophobia: 1) use of urban habitat (vs not), 2) territorial pair vs family group sociality and 3) large vs small flock size (whereas range, caching, hunting live animals, and genus did not); while only flock size influenced food neophobia. We found that, overall, individuals were temporally and contextually repeatable (i.e. consistent) in their novelty responses in all conditions, indicating neophobia is a stable behavioural trait. With this study, we have established a network of corvid researchers, demonstrating potential for further collaboration to explore the evolution of cognition in corvids and other bird species. These novel findings enable us, for the first time in corvids, to identify the socio-ecological correlates of neophobia and grant insight into specific elements that drive higher neophobic responses in this avian family group.

category: animal behavior and cognition


title: Cell-specific expression buffering promotes cell survival and cancer robustness

abstract:
Functional buffering that ensures biological robustness is critical for maintaining tissue homeostasis, organismal survival, and evolution of novelty. However, the mechanism underlying functional buffering, particularly in multicellular organisms, remains largely elusive. Here, we developed an inference index (C-score) for Cell-specific Expression- BUffering (CEBU), whereby functional buffering is mediated via expression of buffering genes in specific cells and tissues in humans. By computing C-scores across 684 human cell lines using genome-wide CRISPR screens and transcriptomic RNA-seq, we report that C- score-identified putative buffering gene pairs are enriched for members of the same duplicated gene family, pathway, and protein complex. Furthermore, CEBU is especially prevalent in tissues of low regenerative capacity (e.g., bone and neuronal tissues) and is weakest in highly regenerative blood cells, linking functional buffering to tissue regeneration. Clinically, the buffering capacity enabled by CEBU can help predict patient survival for multiple cancers. Our results reveal CEBU as a buffering mechanism contributing to tissue homeostasis and cancer robustness in humans.

Summary blurbWe unveil a genome-wide functional buffering mechanism, termed Cell-specific Expression Buffering (CEBU), whereby gene expression contributes to functional buffering in specific cell types and tissues. We link CEBU to genetic interactions, tissue homeostasis and cancer robustness.


title: Functional buffering via cell-specific gene expression promotes tissue homeostasis and cancer robustness


title: TCF12 controls oligodendroglial cell proliferation and regulates signaling pathways conserved in gliomas

abstract: Diffuse gliomas are primary brain tumors originating from the transformation of glial cells. In particular, oligodendrocyte precursor cells constitute the major tumor-amplifying population in the gliomagenic process. We previously identified the TCF12 gene, encoding a transcription factor of the E protein family, as being recurrently mutated in oligodendrogliomas. In this study, we sought to understand the function of TCF12 in oligodendroglial cells, the glioma lineage of origin. We first describe TCF12 mRNA and protein expression pattern in oligodendroglial development in the mouse brain. Second, by TCF12 genome wide chromatin profiling in oligodendroglial cells, we show that TCF12 binds active promoters of genes involved in proliferation, translation/ribosomes, and pathways involved in oligodendrocyte development and cancer. Finally, we perform OPC-specific Tcf12 inactivation in vivo and demonstrate by immunofluorescence and transcriptomic analyses that TCF12 is transiently required for OPC proliferation but dispensable for oligodendrocyte differentiation. We further show that Tcf12 inactivation results in deregulation of biological processes that are also altered in oligodendrogliomas. Together, our data suggest that TCF12 directly regulates transcriptional programs in oligodendroglia development that are relevant in a glioma context.


title: Alzheimer's Disease Classification Accuracy is Improved by MRI Harmonization based on Attention-Guided Generative Adversarial Networks

abstract: Alzheimers disease (AD) accounts for 60% of dementia cases worldwide; patients with the disease typically suffer from irreversible memory loss and progressive decline in multiple cognitive domains. With brain imaging techniques such as magnetic resonance imaging (MRI), microscopic brain changes are detectable even before abnormal memory loss is detected clinically. Patterns of brain atrophy can be measured using MRI, which gives us an opportunity to facilitate AD detection using image classification techniques. Even so, MRI scanning protocols and scanners differ across studies. The resulting differences in image contrast and signal to noise make it important to train and test classification models on multiple datasets, and to handle shifts in image characteristics across protocols (also known as domain transfer or domain adaptation). Here, we examined whether adversarial domain adaptation can boost the performance of a Convolutional Neural Network (CNN) model designed to classify AD. To test this, we used an Attention-Guided Generative Adversarial Network (GAN) to harmonize images from three publicly available brain MRI datasets - ADNI, AIBL and OASIS - adjusting for scanner-dependent effects. Our AG-GAN optimized a joint objective function that included attention loss, pixel loss, cycle-consistency loss and adversarial loss; the model was trained bidirectionally in an end-to-end fashion. For AD classification, we adapted the popular 2D AlexNet CNN to handle 3D images. Classification based on harmonized MR images significantly outperformed classification based on the three datasets in non-harmonized form, motivating further work on image harmonization using adversarial techniques.


title: A novel mobile RND-type efflux pump gene cluster, tmexC3D2-toprJ3, confers tigecycline resistance in Pseudomonas alcaligenes

abstract: Tigecycline exhibits promising activity against multidrug-resistant Gram-negative bacteria (MDR-GNB), whose infections are difficult to treat with antimicrobials. However, mobile tigecycline resistance genes, such as tmexCD-toprJ, have emerged in Enterobacterales isolated in China, Vietnam, and possibly other countries in the world. In this study, we investigated tigecycline-nonsusceptible GNB in Japan. Eight tigecycline- and carbapenem-nonsusceptible isolates of Pseudomonas alcaligenes were obtained from sewage water from a medical institution in Japan in 2020. Whole genome analysis of all P. alcaligenes isolates was performed using short-read sequencing, and the isolate KAM426 was further analyzed using long-read sequencing. For important antimicrobial resistance genes, analysis of surrounding structures and comparison with similar sequences in the public genome database were performed. We identified a novel hybrid type of tmexCD-toprJ gene cluster, tmexC3D2-toprJ1b consisting of tmexC3, tmexC2, and toprJ1b, in phylogenetically clonal isolates of P. alcaligenes. The complete genome sequence of KAM426 revealed that this isolate co-harbors tmexC3D2-toprJ1b and two copies of the carbapenemase gene blaIMP-1 on the chromosome. tmexC3D2-toprJ1b in KAM426 was flanked by the IS5/IS1182 family transposase gene, suggesting that the gene cluster was acquired by horizontal gene transfer (HGT). tmexC3D2-toprJ1b seemed to have spread to other Pseudomonas species such as Pseudomonas aeruginosa via HGT mediated by mobile gene elements such as a plasmid. This study identified tmexCD-toprJ-like tigecycline resistance genes in Japan for the first time and suggests that diverse tmexCD-toprJ-like gene clusters, including tmexC3D2-toprJ1b, have spread among MDR-GNB worldwide. Further epidemiological genomic studies in clinical and environmental settings are needed.


title: Emergence of a novel mobile RND-type efflux pump gene cluster, tmexC3D2-toprJ1b, in Pseudomonas species


title: Kveik brewing yeasts demonstrate wide flexibility in beer fermentation temperature and flavour metabolite production and exhibit enhanced trehalose accumulation

abstract: AO_SCPLOWBSTRACTC_SCPLOWTraditional Norwegian Farmhouse ale yeasts, also known as kveik, have captured the attention of the brewing community in recent years. Kveik were recently reported as fast fermenting thermo- and ethanol tolerant yeasts with the capacity to produce a variety of interesting flavour metabolites. They are a genetically distinct group of domesticated beer yeasts of admixed origin with one parent from the "Beer 1" clade and the other unknown. While kveik are known to ferment wort efficiently at warmer temperatures, its range of fermentation temperatures and corresponding flavour metabolites produced, remain uncharacterized. In addition, the characteristics responsible for its increased thermotolerance remain largely unknown. Here we demonstrate variation in kveik strains at a wide range of fermentation temperatures and show not all kveik strains are equal in fermentation performance, flavour metabolite production and stress tolerance. Furthermore, we uncovered an increased capacity of kveik strains to accumulate intracellular trehalose, which likely contributes to its increased thermo- and ethanol tolerances. Taken together our results present a clearer picture of the future opportunities presented by Norwegian kveik yeasts and offer further insight into their applications in brewing.


title: A dual barcoding approach to bacterial strain nomenclature: Genomic taxonomy of Klebsiella pneumoniae strains

abstract:
Sublineages within microbial species can differ widely in their ecology and pathogenicity, and their precise definition is important in basic research and industrial or public health applications. Whereas the classification and naming of prokaryotes is unified at the species level and higher taxonomic ranks, universally accepted definitions of sublineages within species are largely missing, which introduces confusion in population biology and epidemiological surveillance.

Here we propose a broadly applicable genomic classification and nomenclature approach for bacterial strains, using the prominent public health threat Klebsiella pneumoniae as a model. Based on a 629-gene core genome multilocus sequence typing (cgMLST) scheme, we devised a dual barcoding system that combines multilevel single linkage (MLSL) clustering and life identification numbers (LIN). Phylogenetic and clustering analyses of >7,000 genome sequences captured population structure discontinuities, which were used to guide the definition of 10 infra-specific genetic dissimilarity thresholds. The widely used 7-gene multilocus sequence typing (MLST) nomenclature was mapped onto MLSL sublineages (threshold: 190 allelic mismatches) and clonal group (threshold: 43) identifiers for backwards nomenclature compatibility. The taxonomy is publicly accessible through a community-curated platform (https://bigsdb.pasteur.fr/klebsiella), which also enables external users genomic sequences identification.

The proposed strain taxonomy combines two phylogenetically informative barcodes systems that provide full stability (LIN codes) and nomenclatural continuity with previous nomenclature (MLSL). This species-specific dual barcoding strategy for the genomic taxonomy of microbial strains is broadly applicable and should contribute to unify global and cross-sector collaborative knowledge on the emergence and microevolution of bacterial pathogens.


title: A SUMO-dependent regulatory switch connects the piRNA pathway to the heterochromatin machinery in Drosophila

abstract: Nuclear Argonaute proteins, guided by small RNAs, mediate sequence-specific heterochromatin formation. The molecular principles that link Argonaute-small RNA complexes to cellular heterochromatin effectors upon binding to nascent target RNAs are poorly understood. Here, we elucidate the mechanism by which the PIWI interacting RNA (piRNA) pathway connects to the heterochromatin machinery in Drosophila. Piwi-mediated stabilization of the corepressor complex SFiNX on chromatin leads to SUMOylation of its subunit Panoramix. SUMOylation, together with an amphipathic LxxLL motif in Panoramixs intrinsically disordered repressor domain, are necessary and sufficient to recruit small ovary (Sov), a multi-zinc finger protein essential for general heterochromatin formation and viability. Structure-guided mutations that abrogate the Panoramix-Sov interaction or that prevent SUMOylation of Panoramix uncouple Sov from the piRNA pathway, resulting in viable but sterile flies in which Piwi-targeted transposons are derepressed. Thus, by coupling recruitment of a corepressor to nascent transcripts with its SUMOylation, Piwi engages the heterochromatin machinery specifically at transposon loci.


title: Airborne environmental DNA metabarcoding for the monitoring of terrestrial insects - a proof of concept

abstract: Biodiversity is in decline due to human land use, exploitation, and climate change. To be able to counteract this alarming trend it is paramount to closely monitor biodiversity at global scales. Because this is practically impossible with traditional methods, the last decade has seen a strong push for solutions. In aquatic ecosystems the monitoring of species from environmental DNA (eDNA) has emerged as one of the most powerful tools at our disposal but in terrestrial ecosystems the power of eDNA for monitoring has so far been hampered by the local scale of the samples. In this study we report the first attempt to detect insects from airborne eDNA. We compare our results to two traditional insect monitoring projects (1) using light trapping for moth monitoring and (2) transect counts for the monitoring of butterflies and wild bees. While we failed to detect many of the same species monitored with the traditional methods, airborne eDNA metabarcoding revealed DNA from from six classes of Arthropods, and twelve order of Insects - including representatives from all of the four largest orders: Diptera (flies), Lepidoptera (butterflies and moths), Coleoptera (beetles) and Hymenoptera (bees, wasps and ants). We also recovered DNA from nine species of vertebrates, including frogs, birds and mammals as well as from 12 other phyla. We suggest that airborne eDNA has the potential to become a powerful tool for terrestrial biodiversity monitoring, with many impactful applications including the monitoring of pests, invasive or endangered species or disease vectors.


title: Newly identified parasitic nematode beta-tubulin alleles confer resistance to benzimidazoles

abstract:
Infections by parasitic nematodes cause large health and economic burdens worldwide. We use anthelmintic drugs to reduce these infections. However, resistance to anthelmintic drugs is extremely common and increasing worldwide. It is essential to understand the mechanisms of resistance to slow its spread. Recently, four new parasitic nematode beta-tubulin alleles have been identified in benzimidazole (BZ) resistant parasite populations: E198I, E198K, E198T, and E198stop. These alleles have not been tested for the ability to confer resistance or for any effects that they might have on organismal fitness. We introduced these four new alleles into the sensitive C. elegans laboratory-adapted N2 strain and exposed these genome-edited strains to both albendazole and fenbendazole. We found that all four alleles conferred resistance to both BZ drugs. Additionally, we tested for fitness consequences in both control and albendazole conditions over seven generations in competitive fitness assays. We found that none of the edited alleles had deleterious effects on fitness in control conditions and that all four alleles conferred strong and equivalent fitness benefits in BZ drug conditions. Because it is unknown if previously validated alleles confer a dominant or recessive BZ resistance phenotype, we tested the phenotypes caused by five of these alleles and found that none of them conferred a dominant BZ resistance phenotype. Accurate measurements of resistance, fitness effects, and dominance caused by the resistance alleles allow for the generation of better models of population dynamics and facilitate control practices that maximize the efficacy of this critical anthelmintic drug class.

HighlightsO_LIFour newly identified parasitic nematode beta-tubulin alleles confer benzimidazole resistance
C_LIO_LIThe four newly identified alleles do not confer deleterious fitness consequences
C_LIO_LIFive beta-tubulin alleles confer recessive benzimidazole resistance
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Graphical abstract

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title: Contribution of 3D genome topological domains to genetic risk of cancers

abstract:
Genome-wide association studies have identified statistical associations between various diseases, including cancers, and a large number of single-nucleotide polymorphisms (SNPs). However, they provide no direct explanation of the mechanisms underlying the association. Based on the recent discovery that changes in 3-dimensional genome organization may have functional consequences on gene regulation favoring diseases, we investigated systematically the genome-wide distribution of disease-associated SNPs with respect to a specific feature of 3D genome organization: topologically-associating domains (TADs) and their borders.

For each of 449 diseases, we tested whether the associated SNPs are present in TAD borders more often than observed by chance, where chance (i.e. the null model in statistical terms) corresponds to the same number of pointwise loci drawn at random either in the entire genome, or in the entire set of disease-associated SNPs listed in the GWAS catalog. Our analysis shows that a fraction of diseases display such a preferential location of their risk loci. Moreover, cancers are relatively more frequent among these diseases, and this predominance is generally enhanced when considering only intergenic SNPs. The structure of SNP-based diseasome networks confirms that TAD border enrichment in risk loci differ between cancers and non-cancer diseases. Different TAD border enrichments are observed in embryonic stem cells and differentiated cells, which agrees with an evolution along embryogenesis of the 3D genome organization into topological domains.

Our results suggest that, for certain diseases, part of the genetic risk lies in a local genetic variation affecting the genome partitioning in topologically-insulated domains. Investigating this possible contribution to genetic risk is particularly relevant in cancers. This study thus opens a way of interpreting genome-wide association studies, by distinguishing two types of disease-associated SNPs: one with a direct effect on an individual gene, the other acting in interplay with 3D genome organization.

Author summaryGenome-wide association studies comparing patients and healthy subjects have evidenced correlations between diseases and the presence of pointwise genetic variations known as single-nucleotide polymorphisms (SNPs). We exploit and extend this statistical analysis by investigating the location of risk loci, i.e. disease-associated SNPs, with respect to the 3D organization of the genome into spatially-insulated domains, the topologically-associating domains (TADs).

We show that for certain diseases, mostly cancers, their associated risk loci are preferentially located in the borders of these topological domains. The predominance of cancers among these diseases is confirmed and even enhanced when considering only intergenic SNPs. A different enrichment behavior is observed in embryonic stem cells and derived cell lines at an early developmental stage, presumably due to the not fully mature TAD structure in these cells.

Overall, our results show that genome variations in specific TAD borders may increase the risk of developing certain diseases, especially cancers. Our work underlines the importance of considering the genetic risk loci within their 3D genomic context, and suggests a role of 3D genome partitioning into topological domains in the genetic risk which differs between cancers and non-cancer diseases.


title: MicroRNA childhood Cancer Catalog (M3Cs): A Resource for Translational Bioinformatics Toward Health Informatics in Pediatric Cancer.

abstract:
BackgroundMicroRNA childhood Cancer Catalog (M3Cs) is high-quality curated collection of published miRNA research studies on 16 pediatric cancer diseases. M3Cs scope was based on two approaches: data-driven clinical significance and data-driven human pediatric cell line models.

MethodM3Cs development passed through three phases: 1. Literature Mining: It includes external database search and screening. 2. Data processing that includes 3 steps: a-Data Extraction, b-Data Curation & annotation, c-Web Development. 3. Publishing: Shinyapps.io was used as a web interface for the deployment of M3Cs. M3Cs is now available online and can be accessed through https://m3cs.shinyapps.io/M3Cs/.

ResultsFor Data-driven clinical significance Approach, 538 miRNAs from 268 publications were reported in the clinical domain while 7 miRNAs from 5 publications were reported in the clinical & drug domain. For data-driven human pediatric cell line models approach, 538 miRNAs from 1268 publications were reported in cell line domain while 211 miRNAs from 177 publications in cell line & drug domain.

ConclusionM3Cs acted to fill the gap by applying translational bioinformatics (TBI) general pathway to transfer data-driven research toward data-driven clinical care and/or hypothesis generation. Aggregated and well-curated data of M3Cs will enable stakeholders in health care to incorporate miRNA in the clinical policy.


title: AJILE12: Long-term naturalistic human intracranial neural recordings and pose

abstract: Understanding the neural basis of human movement in naturalistic scenarios is critical for expanding neuroscience research beyond constrained laboratory paradigms. Here, we describe our Annotated Joints in Long-term Electrocorticography for 12 human participants (AJILE12) dataset, the largest human neurobehavioral dataset that is publicly available; the dataset was recorded opportunistically during passive clinical epilepsy monitoring. AJILE12 includes synchronized intracranial neural recordings and upper body pose trajectories across 55 semi-continuous days of naturalistic movements, along with relevant metadata, including thousands of wrist movement events and annotated behavioral states. Neural recordings are available at 500 Hz from at least 64 electrodes per participant, for a total of 1280 hours. Pose trajectories at 9 upper-body keypoints were estimated from 118 million video frames. To facilitate data exploration and reuse, we have shared AJILE12 on The DANDI Archive in the Neurodata Without Borders (NWB) data standard and developed a browser-based dashboard.


title: Cognitive process modeling addresses context independence violations in the ABCD Study stop-signal task

abstract: The Adolescent Brain Cognitive Development (ABCD) Study is a longitudinal neuroimaging study of unprecedented scale that is in the process of following over 11,000 youth from middle childhood though age 20. However, a design feature of the studys stop-signal task violates "context independence", an assumption critical to current non-parametric methods for estimating stop-signal reaction time (SSRT), a key measure of inhibitory ability in the study. This has led some experts to call for the task to be changed and for previously collected data to be used with caution. We present a formal cognitive process model, the BEESTS-ABCD model, that provides a mechanistic explanation for the impact of this design feature, describes key behavioral trends in the ABCD data, and allows biases in SSRT estimates resulting from context independence violations to be quantified. We use the model to demonstrate that, although non-parametric SSRT estimates generally preserve the rank ordering of participants SSRT values, failing to account for context independence violations can lead to erroneous inferences in several realistic scenarios. Nonetheless, as the BEESTS-ABCD model can be used to accurately recover estimates of SSRT and other mechanistic parameters of interest from ABCD data, the impact of such violations can be effectively mitigated.


title: Ultrastructural and functional analysis of extra-axonemal structures in trichomonads

abstract: Trichomonas vaginalis and Tritrichomonas foetus are extracellular flagellated parasites that inhabit humans and other mammals, respectively. In addition to motility, flagella act in a variety of biological processes in different cell types; and extra-axonemal structures (EASs) has been described as fibrillar structures that provide mechanical support and act as metabolic, homeostatic and sensory platforms in many organisms. Here, we identified the presence of EASs forming prominent flagellar swellings in T. vaginalis and T. foetus and we observed that their formation was associated with the parasites adhesion on the host cells, fibronectin, and precationized surfaces; and parasite:parasite interaction. A high number of rosettes, clusters of intramembrane particles that has been proposed as sensorial structures, and microvesicles protruding from the membrane were observed in the EASs. The protein VPS32, a member of the ESCRT-III complex crucial for diverse membrane remodeling events, the pinching off and release of microvesicles, was found in the surface as well as in microvesicles protruding from EASs. Moreover, we demonstrated that overexpression of VPS32 protein induce EAS formation and increase parasite motility in semi-solid medium. These results provide valuable data about the role of the flagellar EASs in the cell-to-cell communication and pathogenesis of these extracellular parasites.


title: Nociceptor subtypes are born continuously over DRG development peaking at E10.5 - E11.5.

abstract: Sensory neurogenesis in the dorsal root ganglion (DRG) occurs in two waves of differentiation with larger, myelinated proprioceptive and low-threshold mechanoreceptor (LTMR) neurons differentiating before smaller, unmyelinated (C) nociceptive neurons. This temporal difference was established from early birthdating studies based on DRG soma cell size. However, distinctions in birthdates between molecular subtypes of sensory neurons, particularly nociceptors, is unknown. Here, we assess the birthdate of lumbar DRG neurons in mice using a thymidine analog, EdU, to label developing neurons exiting mitosis combined with co-labeling of known sensory neuron markers. We find that different nociceptor subtypes are born on similar timescales, with continuous births between E9.5 to E13.5, and peak births from E10.5 to E11.5. Notably, we find that thinly myelinated A{delta}-fiber nociceptors and peptidergic C-fibers are born more broadly between E10.5 and E11.5 than previously thought and that non-peptidergic C-fibers and C-LTMRs are born with a peak birth date of E11.5. Moreover, we find that the percentages of nociceptor subtypes born at a particular timepoint are the same for any given nociceptor cell type marker, indicating that intrinsic or extrinsic influences on cell type diversity are occurring similarly across developmental time. Overall, the patterns of birth still fit within the classical "two wave" description, as touch and proprioceptive fibers are born primarily at E10.5, but suggest that nociceptors have a slightly broader wave of birthdates with different nociceptor subtypes continually differentiating throughout sensory neurogenesis irrespective of myelination.

category: developmental biology


title: Interactive effects of herbivory and the level and fluctuations of nutrient availability on dominance of alien plants in synthetic native communities

abstract:
Numerous studies have highlighted the role of nutrient availability and fluctuations therein for invasion success of alien plants. Others also highlighted the role of herbivores in invasion success. However, how herbivory and the level and fluctuations in nutrient availability interact in driving alien plant invasion into resident communities remains largely unexplored.

We grew eight invasive alien species as target species in pot{square}mesocosms with five different synthetic native communities in a three-factorial design with two levels of nutrient availability (low vs high), two levels of nutrient fluctuations (constant vs pulsed) and two levels of herbivory (with vs without).

The relative biomass production of the alien target plants decreased in response to an increase in nutrient availability, and increased in response to the presence of herbivores. Furthermore, herbivory could interact with changes in nutrient availability and nutrient fluctuations to affect the dominance of the alien target species (a marginally significant interaction; 90% CIs: [0.125, 2.712]).

Our multispecies experiment indicates that herbivory could mediate the interactive effect of nutrient enrichment and variability in nutrient supply on invasion of alien plants into native communities. Therefore, we recommend that studies testing the fluctuating resources hypothesis should also consider interactive effect of other trophic levels.


title: Soil fauna may buffer the negative effects of drought on alien plant invasion

abstract: Although many studies have tested the direct effects of drought on alien plant invasion, less is known about how drought affects alien plant invasion indirectly via other groups of organisms such as soil fauna. To test for such indirect effects, we grew single plant of nine naturalized alien target species in pot-mesocosms with a native community of five native grassland species under four combinations of two drought (well-watered vs drought) and two soil-fauna-inoculation (with vs without) treatments. We found that drought decreased the absolute and the relative biomass production of the alien plants, and thus reduced their competitive advantage in the native community. Drought decreased the abundance of soil fauna, particularly the soil mites, but did not affect abundance and richness of soil herbivores. Soil-fauna inoculation increased the biomass of the native plant community and thereby decreased the relative biomass production of the alien species. The increased invasion resistance due to soil fauna, however, tended (p = 0.09) to be stronger for plants growing under well-watered conditions than under drought. Our multispecies experiment thus shows that soil fauna might help native resident communities to resist alien plant invasions, but that this effect might be diminished by drought.


title: Novel modification by L/F-tRNA-protein transferase (LFTR) generates a Leu/N-degron ligand in Escherichia coli.

abstract: The N-degron pathways are a set of proteolytic systems that relate the half-life of a protein to its N-terminal (Nt) residue. In Escherchia coli the principal N-degron pathway is known as the Leu/N-degron pathway of which an Nt Leu is a key feature of the degron. Although the physiological role of the Leu/N-degron pathway is currently unclear, many of the components of the pathway are well defined. Proteins degraded by this pathway contain an Nt degradation signal (N-degron) composed of an Nt primary destabilizing (Nd1) residue (Leu, Phe, Trp or Tyr) and an unstructured region which generally contains a hydrophobic element. Most N-degrons are generated from a pro-N-degron, either by endoproteolytic cleavage, or by enzymatic attachment of a Nd1 residue (Leu or Phe) to the N-terminus of a protein (or protein fragment) by the enzyme Leu/Phe tRNA protein transferase (LFTR) in a non-ribosomal manner. Regardless of the mode of generation, all Leu/N-degrons are recognized by ClpS and delivered to the ClpAP protease for degradation. To date, only two physiological Leu/N-degron bearing substrates have been verified, one of which (PATase) is modified by LFTR. In this study, we have examined the substrate proteome of LFTR during stationary phase. From this analysis, we have identified several additional physiological Leu/N-degron ligands, including AldB, which is modified by a previously undescribed activity of LFTR. Importantly, the novel specificity of LFTR was confirmed in vitro, using a range of model proteins. Our data shows that processing of the Nt-Met of AldB generates a novel substrate for LFTR. Importantly, the LFTR-dependent modification of T2-AldB is essential for its turnover by ClpAPS, in vitro. To further examine the acceptor specificity of LFTR, we performed a systematic analysis using a series of peptide arrays. These data reveal that the identity of the second residue modulates substrate conjugation with positively charged residues being favored and negatively charged and aromatic residues being disfavored. Collectively, these findings extend our understanding of LFTR specificity and the Leu/N-degron pathway in E. coli.


title: Engineering Viral Vectors for Acoustically Targeted Gene Delivery

abstract: Targeted gene delivery to the brain is a critical tool for neuroscience research and has significant potential to treat human disease. However, the site-specific delivery of common gene vectors such as adeno-associated viruses (AAVs) is typically performed via invasive injections, limiting their scope of research and clinical applications. Alternatively, focused ultrasound blood-brain-barrier opening (FUS-BBBO), performed noninvasively, enables the site-specific entry of AAVs into the brain from systemic circulation. However, when used in conjunction with natural AAV serotypes, this approach has limited transduction efficiency, requires ultrasound parameters close to tissue damage limits, and results in undesirable transduction of peripheral organs. Here, we use high throughput in vivo selection to engineer new AAV vectors specifically designed for local neuronal transduction at the site of FUS-BBBO. The resulting vectors substantially enhance ultrasound-targeted gene delivery and neuronal tropism while reducing peripheral transduction, providing a more than ten-fold improvement in targeting specificity. In addition to enhancing the only known approach to noninvasively target gene delivery to specific brain regions, these results establish the ability of AAV vectors to be evolved for specific physical delivery mechanisms.

category: bioengineering