title: Scalable Species Tree Inference with External Constraints

abstract:
Species tree inference under the multi-species coalescent (MSC) model is a basic step in biological discovery. Despite the developments in recent years of methods that are proven statistically consistent and that have high accuracy, large datasets create computational challenges. Although there is generally some information available about the species trees that could be used to speed up the estimation, only one method-ASTRAL-J, a recent development in the ASTRAL family of methods-is able to use this information. Here we describe two new methods, NJst-J and FASTRAL-J, that can estimate the species tree given partial knowledge of the species tree in the form of a non-binary unrooted constraint tree.. We show that both NJst-J and FASTRAL-J are much faster than ASTRAL-J and we prove that all three methods are statistically consistent under the multi-species coalescent model subject to this constraint. Our extensive simulation study shows that both FASTRAL-J and NJst-J provide advantages over ASTRAL-J: both are faster (and NJst-J is particularly fast), and FASTRAL-J is generally at least as accurate as ASTRAL-J. An analysis of the Avian Phylogenomics project dataset with 48 species and 14,446 genes presents additional evidence of the value of FASTRAL-J over ASTRAL-J (and both over ASTRAL), with dramatic reductions in running time (20 hours for default ASTRAL, and minutes or seconds for ASTRAL-J and FASTRAL-J, respectively).

AvailabilityFASTRAL-J and NJst-J are available in open source form at https://github.com/RuneBlaze/FASTRAL-constrained and https://github.com/RuneBlaze/NJst-constrained. Locations of the datasets used in this study and detailed commands needed to reproduce the study are provided in the supplementary materials at http://tandy.cs.illinois.edu/baqiao-suppl.pdf.


title: Hybridization and introgression are prevalent in Southern European Erysimum (Brassicaceae) species

abstract:
Background and AimsHybridization is a common and important force in plant evolution. One of its outcomes is introgression - the transfer of small genomic regions from one taxon to another by hybridization and repeated backcrossing. This process is believed to be common in glacial refugia, where range expansions and contractions can lead to cycles of sympatry and isolation, creating conditions for extensive hybridization and introgression. Polyploidization is another genome-wide process with a major influence on plant evolution. Both hybridization and polyploidization can have complex effects on plant evolution. However, these effects are often difficult to understand in recently evolved species complexes.

MethodsWe combined flow cytometry, transcriptomic and genomic analyses, and pollen-tube growth assays to investigate the consequences of polyploidization, hybridization, and introgression on the recent evolution of several Erysimum (Brassicaceae) species from the South of the Iberian Peninsula, a well-known glacial refugium. This species complex differentiated in the last 2Myr, and its evolution has been hypothesized to be determined mainly by polyploidization, interspecific hybridization, and introgression.

Key ResultsOur results support a scenario of widespread hybridization involving both extant and "ghost" taxa. Several taxa studied here, most notably those with purple corollas, are polyploids, likely of allopolyploid origin. Moreover, hybridization in this group might be an ongoing phenomenon, as prezygotic barriers appeared weak in many cases.

ConclusionsThe evolution of Erysimum spp. has been determined by hybridization to a large extent. The adaptive value of such genomic exchanges remains unclear, but our results indicate the importance of hybridization for plant diversification across evolutionary scales.

category: evolutionary biology


title: The Anaphase Promoting Complex/ cyclosome co-activator, Cdh1, is a novel target of human Papillomavirus 16 E7 oncoprotein in cervical oncogenesis

abstract: The transforming properties of the high risk human papillomavirus E7 oncoprotein are indispensable for driving the virus life cycle and pathogenesis. Besides inactivation of retinoblastoma (Rb) family of tumor suppressors as part of its oncogenic endeavors, E7-mediated perturbations of eminent cell cycle regulators, checkpoint proteins and proto-oncogenes are considered to be the tricks of its transformative traits. However, many such critical interactions are still unknown. In the present study, we have identified the anaphase promoting complex/ cyclosome (APC/C) co-activator, Cdh1, as a novel interacting partner and a degradation target of E7. We found that HPV16 E7-induced inactivation of Cdh1 promoted abnormal accumulation of multiple Cdh1 substrates. Such a mode of deregulation possibly contributes to HPV-mediated cervical oncogenesis. Our mapping studies recognized the carboxyl-terminal zinc finger motif of E7 to associate with Cdh1 and interfere with the timely degradation of FoxM1, a bona fide Cdh1 substrate and a potent oncogene. Importantly, the E7 mutant with impaired interaction with Cdh1 exhibited defects in its ability for overriding typical cell cycle transition and oncogenic transformation, thereby validating the functional and pathological significance of the E7-Cdh1 axis during cervical carcinoma progression. Altogether, the findings from our study discover a unique nexus between E7 and APC/C-Cdh1, thereby adding to our understanding of the mechanism of E7-induced carcinogenesis and provide a promising target for the management of cervical carcinoma.

category: biochemistry


title: Injectable Nanoporous Microgels Generate Vascularized Constructs and Support Bone Regeneration in Critical-sized Defects

abstract: Large and aberrant bone fractures require ossification and concomitant vascularization for proper healing. Evidence indicates that osteogenesis and vessel growth are coupled in bone fractures. Although the synergistic role of endothelial cells has been recognized, vascularizing large bone grafts remains a challenge and has apprehended the clinical translation of engineered bone constructs. Here, we describe a facile method to fabricate vascularized constructs using chitosan and gelatin-based microgels that promote osteogenesis of human mesenchymal stromal cells (MSC) while supporting endothelial sprouting and network formation. The microgels are enzymatically degradable and had a high hydration rate with a volume swelling ratio of ~560% and a polymer density of ~430 mg/cm3, which is comparable to that of native skeletal tissues. AFM indentation of the surface showed an average Youngs modulus of 189 kPa falling in a range that is conducive to both osteogenesis and vasculogenesis. The osteogenic microgel containing chitosan, gelatin, and hydroxyapatite, mimicking the bone matrix, supported robust attachment, proliferation, and differentiation of MSC. On the other hand, the vasculogenic microgels containing only gelatin, enriched endothelial phenotype and enabled vascular networks formation when embedded in 3D matrices. Combining the two types of microgels created a hybrid construct that sustained the functions of both osteogenic and vasculogenic microgels and enhanced one another in some instances. Using a murine model, we also show that the osteogenic microgels regenerate bone in a critical-sized defect with ~65% defect closure by week 12. These multifunctional microgels can be administered minimally invasively and can conformally fill large bone defects. This work lays the foundation to establish principles of designing multiphasic scaffolds with tissue-specific biophysical and biochemical properties for regenerating vascularized and interfacial tissues.

category: bioengineering


title: Label-free prediction of Cell Painting from brightfield images

abstract: Cell Painting is a high-content image-based assay which can reveal rich cellular morphology and is applied in drug discovery to predict bioactivity, assess toxicity and understand diverse mechanisms of action of chemical and genetic perturbations. In this study, we investigate label-free Cell Painting by predicting the five fluorescent Cell Painting channels from paired brightfield z-stacks using deep learning models. We train and validate the models with a dataset representing 1000s of pan-assay interference compounds sampled from 17 unique batches. The model predictions are evaluated using a test set from two additional batches, treated with compounds comprised from a publicly available phenotypic set. In addition to pixel-level evaluation, we process the label-free Cell Painting images with a segmentation-based feature-extraction pipeline to understand whether the generated images are useful in downstream analysis. The mean Pearson correlation coefficient (PCC) of the images across all five channels is 0.84. Without actually incorporating these features into the model training we achieved a mean correlation of 0.45 from the features extracted from the images. Additionally we identified 30 features which correlated greater than 0.8 to the ground truth. Toxicity analysis on the label-free Cell Painting resulted a sensitivity of 62.5% and specificity of 99.3% on images from unseen batches. Additionally, we provide a breakdown of the feature profiles by channel and feature type to understand the potential and limitation of the approach in morphological profiling. Our findings demonstrate that label-free Cell Painting has potential above the improved visualization of cellular components, and it can be used for downstream analysis. The findings also suggest that label-free Cell Painting could allow for repurposing the imaging channels for other non-generic fluorescent stains of more targeted biological interest, thus increasing the information content of the assay.

category: bioinformatics


title: USPNet: unbiased organism-agnostic signal peptidepredictor with deep protein language model

abstract: Signal peptide is a short peptide located in the N-terminus of proteins. It plays an important role in targeting and transferring transmembrane proteins and secreted proteins to correct positions. Compared with traditional experimental methods to identify and discover signal peptides, the computational methods are faster and more efficient, which are more practical for the analysis of thousands or even millions of protein sequences in reality, especially for the metagenomic data. Therefore, computational tools are recently proposed to classify signal peptides and predict cleavage site positions, but most of them disregard the extreme data imbalance problem in these tasks. In addition, almost all these methods rely on additional group information of proteins to boost their performances, which, however, may not always be available. To deal with these issues, in this paper, we present Unbiased Organism-agnostic Signal Peptide Network (USPNet), a signal peptide prediction and cleavage site prediction model based on deep protein language model. We propose to use label distribution-aware margin (LDAM) loss and evolutionary scale modeling (ESM) embedding to handle data imbalance and object-dependence problems. Extensive experimental results demonstrate that the proposed method significantly outperforms all the previous methods on the classification performance. Additional study on the simulated metagenomic data further indicates that our model is a more universal and robust tool without dependency on additional group information of proteins, with the Matthews correlation coefficient improved by up to 17.5%. The proposed method will be potentially useful to discover new signal peptides from the abundant metagenomic data.


title: Syotti: Scalable Bait Design for DNA Enrichment

abstract: Bait-enriched sequencing is a relatively new sequencing protocol that is becoming increasingly ubiquitous as it has been shown to successfully amplify regions of interest in metagenomic samples. In this method, a set of synthetic probes ("baits") are designed, manufactured, and applied to fragmented metagenomic DNA. The probes bind to the fragmented DNA and any unbound DNA is rinsed away, leaving the bound fragments to be amplified for sequencing. This effectively enriches the DNA for which the probes were designed. Most recently, Metsky et al. (Nature Biotech 2019) demonstrated that bait-enrichment is capable of detecting a large number of human viral pathogens within metagenomic samples. In this work, we formalize the problem of designing baits by defining the Minimum Bait Cover problem, which aims to find the smallest possible set of bait sequences that cover every position of a set of reference sequences under an approximate matching model. We show that the problem is NP-hard, and that it remains NP-hard under very restrictive assumptions. This indicates that no polynomial-time exact algorithm exists for the problem, and that the problem is intractable even for small and deceptively simple inputs. In light of this, we design an efficient heuristic that takes advantage of succinct data structures. We refer to our method as syotti. The running time of syotti shows linear scaling in practice, running at least an order of magnitude faster than state-of-the-art methods, including the recent method of Metsky et al. At the same time, our method produces bait sets that are smaller than the ones produced by the competing methods, while also leaving fewer positions uncovered. Lastly, we show that syotti requires only 25 minutes to design baits for a dataset comprised of 3 billion nucleotides from 1000 related bacterial substrains, whereas the method of Metsky et al. shows clearly super-linear running time and fails to process even a subset of 8% of the data in 24 hours. Our implementation is publicly available at https://github.com/jnalanko/syotti.


title: Toward optimal fingerprint indexing for large scale genomics

abstract:
MotivationTo keep up with the scale of genomic databases, several methods rely on local sensitive hashing methods to efficiently find potential matches within large genome collections. Existing solutions rely on Minhash or Hyperloglog fingerprints and require reading the whole index to perform a query. Such solutions can not be considered scalable with the growing amount of documents to index.

ResultsWe present NIQKI, a novel structure using well-designed fingerprints that lead to theoretical and practical query time improvements, outperforming state-of-the-art by orders of magnitude. Our contribution is threefold. First, we generalize the concept of Hyperminhash fingerprints in (h,m)-HMH fingerprints that can be tuned to present the lowest false positive rate given the expected sub-sampling applied. Second, we provide a structure able to index any kind of fingerprints based on inverted indexes that provide optimal queries, namely linear with the size of the output. Third, we implemented these approaches in a tool dubbed NIQKI that can index and calculate pairwise distances for over one million bacterial genomes from GenBank in a matter of days on a small cluster. We show that our approach can be orders of magnitude faster than state-of-the-art with comparable precision. We believe that this approach can lead to tremendous improvement allowing fast query, scaling on extensive genomic databases.

Availability and implementationWe wrote the NIQKI index as an open-source C++ library under the AGPL3 license available at https://github.com/Malfoy/NIQKI. It is designed as a user-friendly tool and comes along with usage samples


title: The β-lactam Ticarcillin is a Staphylococcus aureus UDP-N-acetylglucosamine 2-epimerase binder.

abstract: Infectious diseases account for 25% of the causes of death worldwide and this rate is expected to increase due to antibiotic resistance. Among the bacteria associated with healthcare infections, Staphylococcus aureus is a prevalent pathogen and about 50% of the isolates are found to be methicillin-resistant. Here we describe the identification of ticarcillin as a weak binder of the S. aureus UDP-N-acetylglucosamine 2-epimerase. After a docking screening, ticarcillin was identified as a ligand in using the recently proposed isothermal analysis of differential scanning fluorimetry data. Finally, an equilibrium MD simulation confirmed the docking binding mode as a stable pose, with large contributions to the binding energy coming from interactions between Arg206 and Arg207 and the carboxylate groups in ticarcillin.

category: biophysics


title: Prediction of broad chemical toxicities using induced pluripotent stem cells and gene networks by transfer learning from embryonic stem cell data

abstract: The assessment of toxic chemicals using animals has limited applicability to humans. Moreover, from the perspective of animal protection, effective alternatives are also desired. Previously, we developed a method that combines developmental toxicity testing based on undifferentiated human embryonic stem (ES) cells (KhES-3) and gene networks. We showed that [≥] 95% accurate predictions could be achieved for neurotoxins, genotoxic carcinogens, and non-genotoxic carcinogens. Here, we expanded this method to predict broad toxicities and predicted the toxicity of 24 chemicals in six categories (neurotoxins, cardiotoxins, hepatotoxins, nephrotoxins [glomerular nephrotoxins/tubular nephrotoxins], and non-genotoxic carcinogens) and achieved high prediction accuracy (AUC = 0.90-1.00) in all categories. Moreover, to develop a testing system with fewer ethical issues, we screened for an induced pluripotent stem (iPS) cell line on the basis of cytotoxic sensitivity and used this line to predict toxicity in the six categories based on the gene networks of iPS cells using transfer learning from the ES cell gene networks. We successfully predicted toxicities in four toxin categories (neurotoxins, hepatotoxins, glomerular nephrotoxins, and non-genotoxic carcinogens) at high accuracy (AUC = 0.82-0.99). These results demonstrate that the prediction of chemical toxicity is possible even with iPS cells by transfer learning once a gene expression database has been developed from an ES cell line. This method holds promise for tailor-made safety evaluations using individual iPS cells.

category: pharmacology and toxicology


title: Salidroside protects lipopolysaccharide-induced acute lung injury by regulating miR-145/ cytosolic phospholipase A2

abstract:
Salidroside is one of the main active components from the root of Rhodiola rosea. Previous reports showed that salidroside exhibits anti-inflammatory properties, but the underlying mechanisms are not fully understood. Here, we observed the effects of salidroside on lipopolysaccharide (LPS)-induced acute lung injury (ALI) both in vivo and in vitro. As revealed by survival study, salidroside reduced mortality of rats and prolonged their survival time. Meanwhile, salidroside significantly improved LPS-induced lung histopathologic changes, decreased lung wet-to-dry and lung-to-body weight ratios, inhibited lung myeloperoxidase (MPO) activity. Salidroside also suppressed the expression of cytosolic PLA2 (cPLA2), the activity of phospholipase A2 (PLA2) in LPS-treated rats and the metabolites of PLA2 in bronchoalveolar lavage fluid (BALF), which was confirmed by results of prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and thromboxane B2 (TXB2) detection. And the expression of microRNA-145 in LPS-treated rats was up-regulated by salidroside. Besides, salidroside raised the level of miR-145and reduced PLA2 activity in LPS-induced A549 cells in a concentration-dependent manner, which was obviously reversed by miR-145 inhibition. In conclusion, the current study demonstrated that salidroside exhibited a protective effect on LPS-induced ALI by inhibiting of the inflammatory response, which may involve in the up-regulation of miR-145 and the suppression of cPLA2.

HighlightsO_LISalidroside reduces acute lung injury by inhibiting the increment and metabolism of phospholipase A2;
C_LIO_LISalidroside inhibits LPS-induced PLA2 increase dependent on miR-145;
C_LIO_LIThe inhibitory effect of Salidroside on Phospholipases A2 provides a link between the identification of new targets and potential new therapeutic agents for the treatment of acute lung injury.
C_LI


title: An astrocytic signaling loop for frequency-dependent control of dendritic integration and spatial learning

abstract: Dendrites of hippocampal CA1 pyramidal cells amplify clustered glutamatergic input by activation of voltage-gated sodium channels and N-methyl-D-aspartate receptors (NMDARs). NMDAR activity depends on the presence of NMDAR co-agonists such as D-serine, but how co-agonists influence dendritic integration is not well understood. Using combinations of whole-cell patch clamp, iontophoretic glutamate application, two-photon excitation fluorescence microscopy and glutamate uncaging we found that exogenous D-serine reduces the threshold of dendritic spikes and increases their amplitude. Triggering an astrocytic mechanism controlling endogenous D-serine supply via endocannabinoid receptors (CBRs) also increased dendritic spiking. Unexpectedly, this pathway was activated by pyramidal cell activity primarily in the theta range, which required HCN channels and astrocytic CB1Rs. Therefore, astrocytes close a positive and frequency-dependent feedback loop between pyramidal cell activity and their integration of dendritic input. Its disruption led to an impairment of spatial memory, which demonstrates its behavioral relevance.

category: neuroscience


title: Organizing Principles of Astrocytic Nanoarchitecture in the Mouse Cerebral Cortex

abstract:
Astrocytes have complex roles in central nervous system (CNS) health and disease. Underlying these roles is an elaborate architecture based on frequent, extremely fine, but seemingly haphazard branches, as well as prominent features including tripartite synaptic complexes and perivascular endfeet. While broad categories of structures in astrocytes are known, the fundamental building blocks that compose them and their organizing principles have yet to be adequately defined. This is largely due to the absence of high-resolution datasets that can reveal nanoscopic features of astrocytes (i.e. 10-20nm diameter in x, y, and z) and a lack of computational approaches that can effectively interrogate astrocyte shape, organization, and nanoarchitecture. Here, we produced and analyzed multiple, high-resolution datasets of layer 2/3 mouse somatosensory cortex using focused ion beam scanning electron microscopy (8nm intervals) and computer vision approaches to provide a principled, quantitative analysis of astrocytic nanoarchitecture. A decomposition of astrocytes into fundamental  parts led to the discovery of unique structural components, recurring structural motifs, and assembly of parts into an organized hierarchy. New relationships were also discerned between astrocytic processes and other CNS microanatomy including mitochondria, tripartite synapses, and cerebrovasculature. By deploying computational resources to quantitatively understand the organizing principles and nanoarchitecture of astrocytes, this study reveals the specialized anatomical adaptations of these complex cells within the CNS.

One Sentence SummaryUsing high-resolution serial electron microscopy datasets and computer vision, this study provides a systematic analysis of astrocytic nanoarchitecture from multiple samples of layer 2/3 of adult mouse neocortex, and presents quantitative evidence that astrocytes organize their morphology into purposeful, classifiable assemblies with unique structural and subcellular organelle adaptations related to their physiological functions.


title: Defining the architecture of cerebrospinal fluid cellular communities in neuroinflammatory diseases

abstract: Cerebrospinal fluid (CSF) biomarkers are important for multiple sclerosis (MS) diagnosis. Moreover, absent of autopsy or biopsy tissue, CSF is the most relevant source for studying the immune cells involved in MS pathophysiology. Single-cell RNA sequencing (scRNA-seq) provides new opportunities to advance our understanding of disease-associated changes in CSF immune cells. Here, using scRNA-seq data generated from 58 CSF and 10 PBMC samples, we provide an updated atlas of the immune cells present in human CSF in MS and other neuroinflammatory conditions, including novel lymphoid and myeloid cell clusters. Our atlas can thus serve as a reference for future studies of immune cells in neuroinflammation. Our further characterization of CSF myeloid cells suggests that most CSF microglia-like cells resemble two of the previously-described brain microglia signatures. Additionally, our data from a sex-mismatched bone marrow transplant recipient suggest that CSF microglia-like cells are of peripheral origin. Our comparisons between MS and other neuroinflammatory disorders show a highly-specific increase in plasma cells, along with reductions in the proportion of microglia-like cells in MS CSF. Furthermore, our analyses on MS patients receiving anti-CD20 therapy ocrelizumab suggest that the treatment effects are not limited to B cell depletion, and ocrelizumab appears to reverse some MS-associated T and myeloid changes in CSF. Finally, we utilized our atlas to prioritize (1) CSF cell types expressing genes associated with MS susceptibility, and (2) ligand-receptor gene pairs that are differentially expressed in MS CSF, providing targets for further mechanistic and causal investigations in pathophysiology and treatment of MS.

category: immunology


title: Rational Designed Hybrid Peptides Show up to a 6-fold Increase in Antimicrobial Activity and Demonstrate Different Ultrastructural Changes as the Parental Peptides measured by BioSAXS

abstract: Antimicrobial peptides (AMPs) are a promising class of compounds being developed against multi-drug resistant bacteria. Hybridization has been reported to increase antimicrobial activity. Here, two proline-rich peptides (consP1: VRKPPYLPRPRPRPL-CONH2 and Bac5-v291: RWRRPIRRRPIRPPFWR-CONH2) were combined with two arginine-isoleucine-rich peptides (optP1: KIILRIRWR-CONH2 and optP7: KRRVRWIIW-CONH2). Proline-rich antimicrobial peptides (PrAMPs) are known to inhibit the bacterial ribosome, shown also for Bac5-v291, whereas it is hypothesized a "dirty drug" model for the arginine-isoleucine-rich peptides. That hypothesis was underpinned by transmission electron microscopy and biological small-angle X-ray scattering (BioSAXS). The hybrid peptides showed a stronger antimicrobial activity compared to the proline-rich peptides, except when compared to Bac5-v291 against E. coli. The increase in activity compared to the arginine-isoleucine-rich peptides was up to 6-fold, however, it was not a general increase but was dependent on the combination of peptides and bacteria. BioSAXS experiments revealed that proline-rich peptides and arginine-isoleucine-rich peptides induce very different ultrastructural changes in E. coli, whereas a hybrid peptide (hyP7B5GK) shows changes, different to both parental peptides and the untreated control. These different ultrastructural changes indicated that the mode of action of the parental peptides is different from each other as well as from the hybrid peptide hyP7B5GK. All peptides showed very low haemolytic activity, some of them showed a 100-fold or larger therapeutic window, demonstrating the potential for further drug development.

category: microbiology


title: Genotype-specific transcriptional responses overshadow salinity effects in a marine diatom sampled along the Baltic Sea salinity cline

abstract: The salinity gradient separating marine and freshwater environments represents a major ecological divide for microbiota, yet the mechanisms by which marine microbes have adapted to and ultimately diversified in freshwater environments are poorly understood. Here, we take advantage of a natural evolutionary experiment: the colonization of the brackish Baltic Sea by the ancestrally marine diatom Skeletonema marinoi. To understand how diatoms respond to low salinity, we characterized transcriptomic responses of S. marinoi grown in a common garden. Our experiment included eight genotypes from source populations spanning the Baltic Sea salinity cline. Changes in gene expression revealed a shared response to salinity across genotypes, where low salinities induced profound changes in cellular metabolism, including upregulation of carbon fixation and storage compound biosynthesis, and increased nutrient demand and oxidative stress. Nevertheless, the genotype effect overshadowed the salinity effect, as genotypes differed significantly in their response, both in the magnitude and direction of gene expression. Intraspecific differences included regulation of transcription and translation, nitrogen metabolism, cell signaling, and aerobic respiration. The high degree of intraspecific variation in gene expression observed here highlights an important but often overlooked source of biological variation associated with how diatoms respond and adapt to environmental change.


title: Prioritising the eradication of invasive species from island archipelagos with high reinvasion risk

abstract: Eradicating invasive species from islands is a proven method for safeguarding threatened and endangered species from extinction. Island eradications can deliver lasting benefits, but require large up-front expenditure of limited conservation resources. The choice of islands must therefore be prioritised. Numerous tools have been developed to prioritise island eradications, but none fully account for the risk of those eradicated species later returning to the island: reinvasion. In this paper, we develop a prioritisation method for island eradications that accounts for the complexity of the reinvasion process. By merging spatially-explicit metapopulation modelling with stochastic dynamic optimisation techniques, we construct a decision-support tool that optimises conservation outcomes in the presence of reinvasion risk. We applied this tool to two different case studies - rat (Rattus rattus) invasions in the Seaforth archipelago in New Zealand, and cane toad (Rhinella marina) invasions in the Dampier archipelago in Australia - to illustrate how state-dependent optimal policies can maximise expected conservation gains. In both case studies, incorporating reinvasion risk dramatically altered the optimal order of island eradications, and improved the potential conservation benefits. The increase in benefits was larger in Dampier than Seaforth (42% improvement versus 6%), as a consequence of both the characteristics of the invasive species, and the arrangement of the islands. Our results illustrate the potential consequences of ignoring reinvasion risk, and demonstrate that including reinvasion in eradication prioritisation can dramatically improve conservation outcomes.

category: ecology


title: Convergent evolution of elaborate nests as structural defences in birds

abstract: The  hanging-basket nests of some weaverbird and icterid species are among the most complex structures built by any animal, but why they have evolved remains to be explained. The precarious attachments and extended entrance tunnels characteristic of these nests are widely speculated to act as structural defences against invasion by nest predators, particularly tree-climbing snakes, but this hypothesis has yet to be systematically tested. We use phylogenetic comparative methods to investigate the role of nest structure in developmental period length, a proxy for offspring mortality, in the weaverbirds (Ploceidae) and icterids (Icteridae), two bird families in which  hanging-basket nests have independently evolved. We find that more elaborate nest designs, particularly those with entrance tunnels, are associated with longer developmental periods in both families. This finding is robust to potentially confounding effects of body mass, phylogenetic relationships and nest location. We provide the first comparative evidence for the role of elaborate nest designs in reducing offspring mortality from nest invasion. More broadly, our findings highlight the importance of offspring mortality risk in shaping diversity in birds nest designs and suggest that complex structures built by animals can buffer against environmental hazards and facilitate the evolution of slower life histories.


title: Multiplex genomic recording of enhancer and signal transduction activity in mammalian cells

abstract: Measurements of gene expression and signal transduction activity are conventionally performed with methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm, termed ENGRAM (ENhancer-driven Genomic Recording of transcriptional Activity in Multiplex), in which the activity and dynamics of multiple transcriptional reporters are stably recorded to DNA. ENGRAM is based on the prime editing-mediated insertion of signal- or enhancer-specific barcodes to a genomically encoded recording unit. We show how this strategy can be used to concurrently genomically record the relative activity of at least hundreds of enhancers with high fidelity, sensitivity and reproducibility. Leveraging synthetic enhancers that are responsive to specific signal transduction pathways, we further demonstrate time- and concentration-dependent genomic recording of Wnt, NF-{kappa}B, and Tet-On activity. Finally, by coupling ENGRAM to sequential genome editing, we show how serially occurring molecular events can potentially be ordered. Looking forward, we envision that multiplex, ENGRAM-based recording of the strength, duration and order of enhancer and signal transduction activities has broad potential for application in functional genomics, developmental biology and neuroscience.

category: genomics


title: A temporally resolved, multiplex molecular recorder based on sequential genome editing

abstract: DNA is naturally well-suited to serve as a digital medium for in vivo molecular recording. However, DNA-based memory devices described to date are constrained in terms of the number of distinct signals that can be concurrently recorded and/or by a failure to capture the precise order of recorded events1. Here we describe DNA Ticker Tape, a general system for in vivo molecular recording that largely overcomes these limitations. Blank DNA Ticker Tape consists of a tandem array of partial CRISPR-Cas9 target sites, with all but the first site truncated at their 5 ends, and therefore inactive. Signals of interest are coupled to the expression of specific prime editing guide RNAs2. Editing events are insertional, and record the identity of the guide RNA mediating the insertion while also shifting the position of the "write head" by one unit along the tandem array, i.e. sequential genome editing. In this proof-of-concept of DNA Ticker Tape, we demonstrate the recording and decoding of complex event histories or short text messages; evaluate the performance of dozens of orthogonal tapes; and construct "long tape" potentially capable of recording the order of as many as 20 serial events. Finally, we demonstrate how DNA Ticker Tape simplifies the decoding of cell lineage histories.


title: An End-to-End Deep Learning Sound Coding Strategy for Cochlear Implants

abstract: Cochlear implant (CI) users struggle to understand speech in noisy conditions. To address this problem, we propose a deep learning speech denoising sound coding strategy that estimates the CI electric stimulation patterns out of the raw audio data captured by the micro-phone, performing end-to-end CI processing. To estimate the relative denoising performance differences between various approaches, we compared this technique to a classic Wiener filter and to a convTasNet. Speech enhancement performance was assessed by means of signal-to-noise-ratio improvement and the short-time objective speech intelligibility measure. Additionally, 5 CI users were evaluated for speech intelligibility in noise to assess the potential benefits of each algorithm. Our results show that the proposed method is capable of replacing a CI sound coding strategy while preserving its general use for every listener and performing speech enhancement in noisy environments, without sacrificing algorithmic latency.


title: An End-to-End Deep Learning Speech Coding and Denoising Strategy for Cochlear Implants


title: An Entropy-based Directed Random Walk for Pathway Activity Inference Using Topological Importance and Gene Interactions

abstract: The integration of microarray technologies and machine learning methods has become popular in predicting pathological condition of diseases and discovering risk genes. The traditional microarray analysis considers pathways as simple gene sets, treating all genes in the pathway identically while ignoring the pathway networks structure information. This study, however, proposed an entropy-based directed random walk (e-DRW) method to infer pathway activity. This study aims (1) To enhance the gene-weighting method in Directed Random Walk (DRW) by incorporating t-test statistic scores and correlation coefficient values, (2) To implement entropy as a parameter variable for random walking in a biological network, and (3) To apply Entropy Weight Method (EWM) in DRW pathway activity inference. To test the objectives, the gene expression dataset was used as input datasets while the pathway dataset was used as reference datasets to build a directed graph. An equation was proposed to assess the connectivity of nodes in the directed graph via probability values calculated from the Shannon entropy formula. A direct proof of calculation based on the proposed mathematical formula was presented using e-DRW with gene expression data. Based on the results, there was an improvement in terms of sensitivity of prediction and accuracy of cancer classification between e-DRW and conventional DRW. The within-dataset experiments indicated that our novel method demonstrated robust and superior performance in terms of accuracy and number of predicted risk-active pathways compared to the other DRW methods. In conclusion, the results revealed that e-DRW not only improved prediction performance, but also effectively extracted topologically important pathways and genes that are specifically related to the corresponding cancer types.


title: Rational control of structural off-state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement

abstract: Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, a key parameter that largely dictates the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.


title: No Correlation Between Pollen Fertility and Viability: Differential Measures of Male Gametophytic Fitness in Cannabis sativa L.

abstract: Pollen grains are male gametophytes, an ephemeral haploid generation of plants, commonly engaging in competition for a limited supply of ovules. Since differential male fertility may influence the direction and pace of population evolution, the relative fitness of pollen is regularly estimated as either pollen viability, the proportion of pollen containing intact cytoplasms and regenerative nuclei, or pollen fertility, the frequency of pollen germinating under standardized conditions. Here, we estimated the relative fitness of pollen in a dioecious, wind-pollinated model system, Cannabis sativa, by characterizing pollen fertility and viability from multiple sires. Pollen fertility quickly declined within two weeks of anther dehiscence, and pollen stored under freezer conditions did not germinate regardless of storage time. In contrast, pollen viability declined slowly and persisted longer than the lifetime of a sporophyte plant under both room temperature and freezer conditions. Pollen samples that underwent both fertility and viability analysis displayed no significant correlation, implying researchers cannot predict pollen fertility from pollen viability, nor infer male gametophytic fitness from a single measure. Our work demonstrates two approaches to measure proxies of male fitness in C. sativa, and identifies new questions around what are valuable estimates of male fitness in plants.

category: plant biology


title: Targeting SerpinE1 reverses cellular features of Hutchinson-Gilford progeria syndrome

abstract: Hutchinson-Gilford progeria syndrome (HGPS) is a rare, fatal disease caused by Lamin A mutation, leading to altered nuclear architecture, loss of perinuclear heterochromatin and deregulated gene expression. HGPS patients eventually die by coronary artery disease and cardiovascular alterations. However, how deregulated transcriptional networks at the cellular level impact on the systemic disease phenotype is currently unclear. We have performed a longitudinal genome-wide analysis of gene expression in primary HGPS fibroblasts from patients at two sequential stages of disease that revealed a progressive activation of Rho signaling and SerpinE1, also known as Plasminogen Activator Inhibitor (PAI-1). siRNA-mediated downregulation or pharmacological inhibition of SerpinE1 by TM5441 could revert key pathological features of HGPS in patient-derived fibroblasts, including re-activation of cell cycle progression, reduced DNA damage signaling, decreased expression of pro-fibrotic genes and recovery of mitochondrial defects. These effects were accompanied by reduced levels of Progerin and correction of nuclear abnormalities. These data point to SerpinE1 as a novel potential effector of HGPS pathogenesis and target for therapeutic interventions.

category: cell biology


title: A trimeric NTD and RBD SARS-CoV-2 subunit vaccine induced protective immunity in CAG-hACE2 transgenic mice and rhesus macaques

abstract: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to significant public health, economic and social problems. Development of effective vaccines is still a priority to contain the virus and end the global pandemic. In this study, we reported that ReCOV, a recombinant trimeric NTD and RBD two-component SARS-CoV-2 subunit vaccine adjuvanted with BFA03 (an AS03-like squalene adjuvant), induced high levels of neutralizing antibodies against SARS-CoV-2 and the circulating variants in mice, rabbits and rhesus macaques. Notably, two-dose immunizations of ReCOV provided complete protection against challenge with SARS-CoV-2 in hACE2 transgenic mice and rhesus macaques, without observable antibody-dependent enhancement of infection. These results support further clinical development of ReCOV and the vaccine is currently being evaluated in a phase I clinical trial in New Zealand (NCT04818801).


title: The oral protease inhibitor (PF-07321332) protects Syrian hamsters against infection with SARS-CoV-2 variants of concern

abstract: There is an urgent need for potent and selective antivirals against SARS-CoV-2. Pfizer developed PF-07321332 (PF-332), a potent inhibitor of the viral main protease (Mpro, 3CLpro) that can be dosed orally and that is in clinical development. We here report that PF-332 exerts equipotent in vitro activity against the four SARS-CoV-2 variants of concerns (VoC) and that it can completely arrest replication of the alpha variant in primary human airway epithelial cells grown at the air-liquid interface. Treatment of Syrian Golden hamsters with PF-332 (250 mg/kg, twice daily) completely protected the animals against intranasal infection with the beta (B.1.351) and delta (B.1.617.2) SARS-CoV-2 variants. Moreover, treatment of SARS-CoV-2 (B.1.617.2) infected animals with PF-332 completely prevented transmission to untreated co-housed sentinels.


title: Towards a Theory of Evolution as Multilevel Learning

abstract:
We apply the theory of learning to physically renormalizable systems in an attempt to develop a theory of biological evolution, including the origin of life, as multilevel learning. We formulate seven fundamental principles of evolution that appear to be necessary and sufficient to render a universe observable and show that they entail the major features of biological evolution, including replication and natural selection. These principles also follow naturally from the theory of learning. We formulate the theory of evolution using the mathematical framework of neural networks, which provides for detailed analysis of evolutionary phenomena. To demonstrate the potential of the proposed theoretical framework, we derive a generalized version of the Central Dogma of molecular biology by analyzing the flow of information during learning (back-propagation) and predicting (forward-propagation) the environment by evolving organisms. The more complex evolutionary phenomena, such as major transitions in evolution, in particular, the origin of life, have to be analyzed in the thermodynamic limit, which is described in detail in the accompanying paper.

Significance statementModern evolutionary theory gives a detailed quantitative description of microevolutionary processes that occur within evolving populations of organisms, but evolutionary transitions and emergence of multiple levels of complexity remain poorly understood. Here we establish correspondence between the key features of evolution, renormalizability of physical theories and learning dynamics, to outline a theory of evolution that strives to incorporate all evolutionary processes within a unified mathematical framework of the theory of learning. Under this theory, for example, natural selection readily arises from the learning dynamics, and in sufficiently complex systems, the same learning phenomena occur on multiple levels or on different scales, similar to the case of renormalizable physical theories.


title: Assessment of AI-based Protein Structure Prediction for the NLRP3 Target

abstract: The recent success of AlphaFold and RoseTTAFold has demonstrated the values of AI methods in predicting highly accurate protein structures. Despite the advances, their roles in the context of small-molecule drug discovery need to be thoroughly explored. In this study, we evaluated the possibility whether the AI-based models can lead to reliable three-dimensional structures of protein-ligand complexes. The structure we predicted was NLRP3, a challenging protein target in terms of obtaining the three-dimensional model both experimentally and computationally. The conformation of the binding pockets generated by the AI models were carefully characterized and compared with the experimental structures.


title: Prolonged and extended impacts of SARS-CoV-2 on the olfactory neurocircuit

abstract: The impact of SARS-CoV-2 on the olfactory pathway was studied over several time points using Syrian golden hamsters. We found an incomplete recovery of the olfactory sensory neurons, prolonged activation of glial cells in the olfactory bulb, and a decrease in the density of dendritic spines within the hippocampus. These data may be useful for elucidating the mechanism underlying long-lasting olfactory dysfunction and cognitive impairment as a post-acute COVID-19 syndrome.


title: Increased Occurrence of Treponema spp. and Double-species Infections in Patients with Alzheimer's Disease

abstract:
ObjectiveAlthough the link between microbial infections and Alzheimers disease (AD) has been demonstrated in multiple studies, the involvement of pathogens in the development of AD remains unclear. Therefore, this theory beckons further systematic investigation. In this study, we have examined the association between the 10 most widely discussed viral and bacterial pathogens found in serum and cerebrospinal fluid (CSF) from patients with AD.

MethodsWe have used an in-house developed multiplex PCR kit for simultaneous detection of five bacterial and five viral pathogens in serum and CSF from 50 AD patients and 53 healthy controls. Data analysis was performed with multiple statistical methods: Fishers exact test, chisquare goodness of fit test, and one-sample proportion test.

ResultsWe observed an increased frequency of AD patients tested positive for Treponema spp. (AD: 62.2%; CTRL: 30.3%; p-value = 0.007). Furthermore, we confirmed a significantly higher prevalence of cases with two and more simultaneous infections in AD patients compared to controls (AD: 24%; CTRL 7.5%; p-value = 0.029). The studied pathogens were widespread equally in serum and CSF. Borrelia burgdorferi, human herpesvirus 7, and human cytomegalovirus were not detected in any of the studied samples.

DiscussionAn increased prevalence of Treponema spp. and double-species infections in AD patients compared to the healthy controls provides further evidence of the association between microbial infections and AD. Paralleled analysis of multiple sample specimens provides complementary information and is advisable for future studies.


title: Stenoparib, an inhibitor of cellular poly (ADP-ribose) polymerases (PARPs), blocks in vitro replication of SARS-CoV-2 variants

abstract: We recently published a preliminary assessment of the activity of a poly (ADP-ribose) polymerase (PARP) inhibitor, stenoparib, also known as 2X-121, which inhibits viral replication by affecting pathways of the host. Here we show that stenoparib effectively inhibits a SARS-CoV-2 wt (BavPat1/2020) strain and four additional variant strains; alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and gamma (P.1) in vitro, with 50% effective concentration (EC50) estimates of 4.1 M, 8.5 M, 24.1 M, 8.2 M and 13.6 M, respectively. A separate experiment focusing on a combination of 10 M stenoparib and 0.5 M remdesivir, an antiviral drug, resulted in over 80% inhibition of the alpha (B.1.1.7) variant, which is substantially greater than the effect achieved with either drug alone, suggesting at least additive effects from combining the different mechanisms of activity of stenoparib and remdesivir.


title: TRIM67 drives tumorigenesis in oligodendrogliomas through Rho GTPase-dependent membrane blebbing

abstract:
Oligodendrogliomas are a subtype of isocitrate dehydrogenase (IDH) mutant gliomas defined by the co-deletion of chromosome arms 1p and 19q. Although the somatic genomic alterations of oligodendrogliomas have been well described, transcriptional changes unique to these tumors are not well studied. Here, we identify Tripartite Motif Containing 67 (TRIM67), an E3 ubiquitin ligase with essential roles during neuronal development, as an oncogene distinctly upregulated in oligodendrogliomas. We characterize the function of TRIM67 using high throughput assays, including RNA sequencing, total lysate-mass spectrometry (MS) and co-immunoprecipitation (IP)-MS using human neural progenitor cells and patient-derived glioma tumorspheres constitutively overexpressing TRIM67. Our high throughput data suggest that TRIM67 overexpression alters the abundance of cytoskeletal proteins, which were validated by functional assays, including immunofluorescence (IF) staining, co-IP and western blotting (WB). Additionally, IF staining results indicate that TRIM67 ectopic expression induces formation of membrane blebs in glioma cells, which could be reverted with the nonmuscle class II myosin inhibitor blebbistatin and selective ROCK inhibitor fasudil. GTP pulldown and WB assays further indicate that Rho GTPase/ROCK2 signaling is altered upon TRIM67 ectopic expression. Phenotypically, TRIM67 expression resulted in higher cell motility in wound healing experiments, reduced cell adherence in adhesion assays, accelerated tumor growth and reduced survival in mouse orthotopic implantation models of an oligodendroglioma-derived patient tumorsphere line. Taken together, our results demonstrate that upregulated TRIM67 induces blebbing-based rounded cell morphology through Rho GTPase/ROCK-mediated signaling thereby contributing to glioma pathogenesis.

SignificanceWe identify TRIM67 as a novel oncogene in oligodendroglioma that leads to increased cell motility, tumor growth, reduced adhesion, and survival in mice. Our results also show that constitutive TRIM67 expression transforms cell morphology from an adherent to a rounded appearance with membrane blebs. Mechanistic alteration of actin cytoskeleton and Rho GTPase signaling upon TRIM67 upregulation underlies the rounded cell structure and the membrane blebbing phenotype. TRIM67-induced blebbing is specifically regulated by RHOA-RAC1-ROCK2 signaling axis. TRIM67 overexpression also alters pathways associated with cell migration and wound healing in various glioma cell lines and human neural progenitor cells, suggesting a general oncogenic mechanism in gliomas. Overall, our study highlights TRIM67 as a novel player orchestrating cytoskeleton, Rho GTPase signaling and bleb-based cell movement, ultimately causing tumorigenic outcomes in oligodendrogliomas.

category: cancer biology


title: Two new threatened species of Rinorea (Violaceae), forest trees from East and South Regions, Cameroon

abstract: Two tree species are described as new to science: Rinorea spongiocarpa Achound. sp. nov (placed in Rinorea [unranked] Brachypetalae) and Rinorea dimakoensis Achound. sp. nov.( placed in Rinorea [unranked] Ilicifolieae). Both species are endemic to Cameroon, occurring south of the Sanaga river, the first from South and East Regions, occurring in evergreen forest from Ebolowa to Dja, while the second occurs in the northern part of East Region in semi-deciduous forest towards the interface with woodland habitats in the Dimako-Bertoua area. The two species are illustrated, and their affinities and conservation status according to the 2012 IUCN categories and criteria are discussed. Both species are threatened with extinction due to habitat destruction, the first is assessed as Vulnerable, the second Endangered.


title: A novel whole yeast-based subunit oral vaccine against Eimeria tenella in chickens

abstract: Cheap, easy-to-produce oral vaccines are needed for control of coccidiosis in chickens to reduce the impact of this disease on welfare and economic performance. Saccharomyces cerevisiae yeast expressing three Eimeria tenella antigens were developed and delivered as heat-killed, freeze-dried whole yeast oral vaccines to chickens in four separate studies. After vaccination, E. tenella replication was reduced following low dose challenge (250 oocysts) in Hy-Line Brown layer chickens (p<0.01). Similarly, caecal lesion score was reduced in Hy-Line Brown layer chickens vaccinated using a mixture of S. cerevisiae expressing EtAMA1, EtIMP1 and EtMIC3 following pathogenic-level challenge (4,000 E. tenella oocysts; p<0.01). Mean body weight gain post-challenge with 15,000 E. tenella oocysts was significantly increased in vaccinated Cobb500 broiler chickens compared to mock-vaccinated controls (p<0.01). Thus, inactivated recombinant yeast vaccines offer cost-effective and scalable opportunities for control of coccidiosis, with relevance to broiler production and chickens reared in low-and middle-income countries (LMICs).


title: Sequence conservation, domain architectures, and phylogenetic distribution of the HD-GYP type c-di-GMP phosphodiesterases

abstract:
The HD-GYP domain, named after two of its conserved sequence motifs, was first described in 1999 as a specialized version of the widespread HD phosphohydrolase domain that had additional highly conserved amino acid residues. Domain associations of HD-GYP indicated its involvement in bacterial signal transduction and distribution patterns of this domain suggested that it could serve as a hydrolase of the bacterial second messenger c-di-GMP, in addition to or instead of the EAL domain. Subsequent studies confirmed the ability of various HD-GYP domains to hydrolyze c-di-GMP to linear pGpG and/or GMP. Certain HD-GYP-containing proteins hydrolyze another second messenger, cGAMP, and some HD-GYP domains participate in regulatory protein-protein interactions. The recently solved structures of HD-GYP domains from four distinct organisms clarified the mechanisms of c-di-GMP binding and metal-assisted hydrolysis. However, the HD-GYP domain is poorly represented in public domain databases, which causes certain confusion about its phylogenic distribution, functions, and domain architectures. Here, we present a refined sequence model for the HD-GYP domain and describe the roles of its most conserved residues in metal and/or substrate binding. We also calculate the numbers of HD-GYPs encoded in various genomes and list the most common domain combinations involving HD-GYP, such as the RpfG (REC-HD-GYP), Bd1817 (DUF3391- HD-GYP), and PmGH (GAF-HD-GYP) protein families. We also provide the descriptions of six HD-GYP-associated domains, including four novel integral membrane sensor domains. This work is expected to stimulate studies of diverse HD-GYP-containing proteins, their N-terminal sensor domains and the signals to which they respond.

IMPORTANCEThe HD-GYP domain forms class II of c-di-GMP phosphodiesterases that control the cellular levels of the universal bacterial second messenger c-di-GMP and therefore affect flagellar and/or twitching motility, cell development, biofilm formation, and, often, virulence. Despite more than 20 years of research, HD-GYP domains are insufficiently characterized; they are often confused with  classical HD domains that are involved in various housekeeping activities and may participate in signaling, hydrolyzing (p)ppGpp and c-di-AMP. This work provides an updated description of the HD-GYP domain, including its sequence conservation, phylogenetic distribution, domain architectures, and the most widespread HD-GYP-containing protein families. This work shows that HD-GYP domains are widespread in many environmental bacteria and are predominant c-di-GMP hydrolases in many lineages, including clostridia and deltaproteobacteria.


title: Sexual reproduction contributes to the evolution of resistance breaking isolates of the spinach pathogen Peronospora effusa

abstract:
Peronospora effusa causes downy mildew, the economically most important disease of cultivated spinach worldwide. To date, 19 P. effusa races have been denominated based on their capacity to break spinach resistances, but their genetic diversity and the evolutionary processes that contribute to race emergence are unknown. Here, we performed the first systematic analysis of P. effusa races showing that those emerge by both asexual and sexual reproduction. Specifically, we studied the diversity of 26 P. effusa isolates from 16 denominated races based on mitochondrial and nuclear comparative genomics. Mitochondrial genomes based on long-read sequencing coupled with diversity assessment based on short-read sequencing uncovered two mitochondrial haplogroups, each with distinct genome organization. Nuclear genome-wide comparisons of the 26 isolates revealed that ten isolates from six races could clearly be divided into three asexually evolving groups, in concordance with their mitochondrial phylogeny. The remaining isolates showed signals of reticulated evolution and discordance between nuclear and mitochondrial phylogenies, suggesting that these evolved through sexual reproduction. Increased understanding of this pathogens reproductive modes will provide the framework for future studies into the molecular mechanisms underlying race emergence and into the P. effusa-spinach interaction, thus assisting in sustainable production of spinach through knowledge-driven resistance breeding.

Significance statementMany microbial plant pathogens depend on the successful colonization of their hosts to complete their life cycle, thereby damaging food crops worldwide. The most effective way of disease control is to deploy genetic disease resistances. However, the extensive use of resistant crop varieties exerts strong selective pressure on microbial plant pathogens to adapt in order to escape resistance. Through yet unknown mechanisms, the spinach pathogen Peronospora effusa can rapidly break the resistance of newly introduced varieties, often within a single growing season. Thus, there is an urgent need to better understand the mechanisms driving adaptation in P. effusa. This information will lead the way to knowledge-driven resistance breeding. Here, we capture for the first time the genetic variation of 26 P. effusa, 16 of which can break a different combination of host resistances. We demonstrate that P. effusa isolates evolve by both asexual and sexual reproduction, and thereby provide the framework to study the molecular mechanisms of the interactions between P. effusa and spinach.


title: Single-Cell RNA Sequencing Characterizes the Molecular Heterogeneity of the Larval Zebrafish Optic Tectum

abstract: The optic tectum (OT) is a multilaminated midbrain structure that acts as the primary retinorecipient in the zebrafish brain. Homologous to the mammalian superior colliculus, the OT is responsible for the reception and integration of stimuli, followed by elicitation of salient behavioral responses. While the OT has been the focus of functional experiments for decades, less is known concerning specific cell types, microcircuitry, and their individual functions within the OT. Recent efforts have contributed substantially to the knowledge of tectal cell types; however, a comprehensive cell catalog is incomplete. Here we contribute to this growing effort by applying single-cell RNA-sequencing (scRNA-seq) to characterize the transcriptomic profiles of tectal cells labeled by the transgenic enhancer trap line y304Et(cfos:Gal4;UAS:Kaede). We sequenced 13,320 cells, a 4X cellular coverage, and identified 25 putative OT cell populations. Within those cells, we identified several mature and developing neuronal populations, as well as non-neuronal cell types including oligodendrocytes, microglia, and radial glia. Although most mature neurons demonstrate GABAergic activity, several glutamatergic populations are present, as well as one glycinergic population. We also conducted Gene Ontology analysis to identify enriched biological processes, and computed RNA velocity to infer current and future transcriptional cell states. Finally, we conducted in situ hybridization to validate our bioinformatic analyses and spatially map select clusters. In conclusion, the larval zebrafish OT is a complex structure containing at least 25 transcriptionally distinct cell populations. To our knowledge, this is the first time scRNA-seq has been applied to explore the OT alone and in depth.


title: Scrutiny of human lung infection by SARS-CoV-2 and associated human immune responses in humanized mice

abstract: There is an urgent need for animal models of COVID-19 to study immunopathogenesis and test therapeutic intervenes. In this study we showed that NSG mice engrafted with human lung (HL) tissue (NSG-L mice) could be infected efficiently by SARS-CoV-2, and that live virus capable of infecting Vero cells was found in the HL grafts and multiple organs from infected NSG-L mice. RNA-seq examination identified a series of differentially expressed genes, which are enriched in viral defense responses, chemotaxis, interferon stimulation, and pulmonary fibrosis between HL grafts from infected and control NSG-L mice. Furthermore, when infecting humanized mice with human immune system (HIS) and autologous HL grafts (HISL mice), the mice had bodyweight loss and hemorrhage and immune cell infiltration in HL grafts, which were not observed in immunodeficient NSG-L mice, indicating the development of anti-viral immune responses in these mice. In support of this possibility, the infected HISL mice showed bodyweight recovery and lack of detectable live virus at the later time. These results demonstrate that NSG-L and HISL mice are susceptible to SARS-CoV-2 infection, offering a useful in vivo model for studying SARS-CoV-2 infection and the associated immune response and immunopathology, and testing anti-SARS-CoV-2 therapies.


title: Turnover in life strategies recapitulates microbial succession on synthetic marine particles

abstract: Particulate organic matter (POM) in the ocean sustains diverse communities of bacteria that mediate the remineralization of organic complex matter. However, the variability of these particles and of the environmental conditions surrounding them present a challenge to the study of the ecological processes shaping particle-associated communities and their function. In this work, we utilise data from experiments in which coastal water communities were grown on synthetic particles to ask which are the most important ecological drivers of their assembly and associated traits. Combining 16S rRNA amplicon sequencing with shotgun metagenomics, together with an analysis of the full genomes of a subset of isolated strains, we were able to identify two-to-three distinct community classes, corresponding to early vs. late colonizers. We show that these classes are shaped by environmental selection (early colonizers) and facilitation (late colonizers), and find distinctive traits associated with each class. While early colonizers have a larger proportion of genes related to uptake of nutrients, motility and environmental sensing with few pathways enriched for metabolism, late colonizers devote a higher proportion of genes for metabolism, comprising a wide array of different pathways including metabolism of carbohydrates, amino acids and xenobiotics We find evidence in selected metabolic pathways for the existence of a trophic-chain topology connecting both classes. The interpretation of these traits suggests a distinction between early and late colonizers analogous to other classifications found in the literature, and we discuss connections with the classical distinction between r- and K-strategists.


title: SARS-CoV-2 infection in free-ranging white-tailed deer (Odocoileus virginianus)

abstract:
Human-to-animal spillover of SARS-CoV-2 virus has occurred in a wide range of animals, but thus far, the establishment of a new natural animal reservoir has not been detected. Here, we detected SARS-CoV-2 virus using rRT-PCR in 129 out of 360 (35.8%) free-ranging white-tailed deer (Odocoileus virginianus) from northeast Ohio (USA) sampled between January-March 2021. Deer in 6 locations were infected with at least 3 lineages of SARS-CoV-2 (B.1.2, B.1.596, B.1.582). The B.1.2 viruses, dominant in Ohio at the time, spilled over multiple times into deer populations in different locations. Deer-to-deer transmission may have occurred in three locations. The establishment of a natural reservoir of SARS-CoV-2 in white-tailed deer could facilitate divergent evolutionary trajectories and future spillback to humans, further complicating long-term COVID-19 control strategies.

One-Sentence SummaryA significant proportion of SARS-CoV-2 infection in free-ranging US white-tailed deer reveals a potential new reservoir.

category: molecular biology


title: Pharmacologic rescue of circadian β-cell failure through P2Y1 purinergic receptor identified by small-molecule screen

abstract: The mammalian circadian clock drives daily oscillations in physiology and behavior through an autoregulatory transcription feedback loop present in central and peripheral cells. Ablation of the core clock within the endocrine pancreas of adult animals impairs the transcription and splicing of genes involved in hormone exocytosis and causes hypoinsulinemic diabetes. However, identification of druggable proteins and pathways to ameliorate the burden of circadian metabolic disease remains a challenge. Here, we generated {beta} cells expressing a nano-luciferase reporter within the proinsulin polypeptide to screen 2,640 pharmacologically-active compounds and identify insulinotropic molecules that bypass the secretory defect in clock mutant {beta} cells. We validated lead compounds in primary mouse islets and identified known modulators of ligandgated ion channels and G-protein coupled receptors, including the antihelmintic ivermectin. Single-cell electrophysiology in circadian mutant mouse and human cadaveric islets validated ivermectin as a glucose-dependent secretagogue. Genetic, genomic, and pharmacologic analyses established that the molecular clock controls the expression of the purinergic P2Y1 receptor to mediate the insulinotropic activity of ivermectin. These findings identify the P2Y1 purinergic receptor as a target to rescue circadian {beta}-cell failure and establish a chemical genetic screen for endocrine therapeutics.


title: Identification and characterization of BEND2 as a novel and key regulator of meiosis during mouse spermatogenesis

abstract:
The chromatin state undergoes global and dynamic changes during spermatogenesis, and is critical to chromosomal synapsis, meiotic recombination, and transcriptional regulation. However, the key regulators involved and the underlying molecular mechanisms remain poorly understood. Herein we report that mouse BEND2, one of the BEN-domain-containing proteins conserved in vertebrates, was specifically expressed in spermatogenic cells within a short time-window spanning meiotic initiation, and that it plays an essential role in the progression of prophase in meiosis I. Bend2 gene knockout in male mice arrested meiosis at the transition from zygonema to pachynema, disrupted synapsis and DNA double-strand break repair, and induced non-homologous chromosomal pairing. BEND2 interacted with a number of chromatin-associated proteins--including ZMYM2, LSD1, CHD4, and ADNP-- which are components of certain transcription-repressor complexes. BEND2-binding sites were identified in diverse chromatin states and enriched in simple sequence repeats. BEND2 contributed to shutting down the mitotic gene-expression program and to the activation of meiotic and post-meiotic gene expression, and it regulated chromatin accessibility as well as the modification of H3K4me3. Therefore, our study identified BEND2 as a novel and key regulator of meiosis, gene expression, and chromatin state during mouse spermatogenesis.

TeaserMeiosis is a highly complex yet poorly understood process that involves the concerted actions of an increasing number of regulators, of which the list remains incomplete. Ma et al. identified BEND2 as a novel and key regulator of meiosis and showed that it interacts with critical chromatin modulators and specific genomic elements to control the expression of mitotic and meiotic genes.

category: developmental biology


title: A novel enhancer of Agouti contributes to parallel evolution of cryptically colored beach mice

abstract: Identifying the genetic basis of repeatedly evolved traits provides a way to reconstruct their evolutionary history and ultimately investigate the predictability of evolution. Here, we focus on the oldfield mouse (Peromyscus polionotus), which occurs in the southeastern United States, where it exhibits considerable coat-color variation. Dorsal coats range from dark brown in mice inhabiting mainland habitat to near white on the white-sand beaches of the southeastern US, where light pelage has evolved independently on Floridas Gulf and Atlantic coasts as an adaptation to visually hunting predators. To facilitate genomic analyses in this species, we first generated a high-quality, chromosome-level genome assembly of P. polionotus subgriseus. Next, in a uniquely variable mainland population that occurs near beach habitat (P. p. albifrons), we scored 23 pigment traits and performed targeted resequencing in 168 mice. We find that variation in pigmentation is strongly associated with a ~2 kb region approximately 5 kb upstream of the Agouti-signaling protein (ASIP) coding region. Using a reporter-gene assay, we demonstrate that this regulatory region contains an enhancer that drives expression in the dermis of mouse embryos during the establishment of pigment prepatterns. Moreover, extended tracts of homozygosity in this region of Agouti indicate that the light allele has experienced recent and strong positive selection. Notably, this same light allele appears fixed in both Gulf and Atlantic coast beach mice, despite these populations being separated by >1,000km. Given the evolutionary history of this species, our results suggest that this newly identified Agouti enhancer allele has been maintained in mainland populations as standing genetic variation and from there has spread to, and been selected in, two independent beach mouse lineages, thereby facilitating their rapid and parallel evolution.


title: Mitochondrial DNA variants segregate during human preimplantation development into genetically different cell lineages that are maintained postnatally

abstract: Humans present remarkable mitochondrial DNA (mtDNA) variant mosaicism, not only across tissues but even across individual cells within one person. The timing of the first appearance of this mosaicism has not yet been established. In this study, we hypothesized it occurs during preimplantation development. To investigate this, we deep-sequenced the mtDNA of 254 oocytes from 85 donors, 158 single blastomeres of 25 day-3 embryos, 17 inner cell mass and trophectoderm samples of 7 day-5 blastocysts, 142 bulk DNA and 68 single cells of different adult tissues. We found that day-3 preimplantation embryos already present blastomeres that carry variants unique to that cell, showing that the first events of mtDNA mosaicism happen very early in human development. We classified the mtDNA variants based on their recurrence or uniqueness across sibling oocytes and embryos, and between single cells and samples from the same embryos or adult individuals. Variants that recurred across samples had higher heteroplasmic loads and more frequently resulted in synonymous changes or were located in non-coding regions than variants that were unique to one oocyte or single embryonic cell. These differences were maintained through developmental stages, suggesting that the mtDNA mosaicism arising in preimplantation development is maintained into adulthood. Further, the results support a model in which close clustering of mitochondria carrying specific mtDNA variants in the ooplasm leads to asymmetric distribution of these mitochondria throughout the cell divisions of the preimplantation embryo, resulting in the appearance of the first form of mtDNA mosaicism in human development.

category: genetics


title: Correlation scan: identifying genomic regions that affect genetic correlations applied to fertility traits

abstract: Reproductive traits are often genetically correlated. Yet, we dont fully understand the complexities, synergism, or trade-offs between male and female fertility. Here, we introduce correlation scan, a novel framework for identifying the drivers or antagonizers of the genetic correlation between male and female fertility traits across the bovine genome. The identification of these regions facilitates the understanding of the complexity of these traits. Although the methodology was applied to cattle phenotypes, using high-density SNP genotypes, the general framework developed can be applied to any species or traits, and it can easily accommodate genome sequence data.


title: CALDERA: Finding all significant de Bruijn subgraphs for bacterial GWAS

abstract: Genome wide association studies (GWAS), aiming to find genetic variants associated with a trait, have widely been used on bacteria to identify genetic determinants of drug resistance or hypervirulence. Recent bacterial GWAS methods usually rely on k-mers, whose presence in a genome can denote variants ranging from single nucleotide polymorphisms to mobile genetic elements. Since many bacterial species include genes that are not shared among all strains, this approach avoids the reliance on a common reference genome. However, the same gene can exist in slightly different versions across different strains, leading to diluted effects when trying to detect its association to a phenotype through k-mer based GWAS. Here we propose to overcome this by testing covariates built from closed connected subgraphs of the De Bruijn graph defined over genomic k-mers. These covariates are able to capture polymorphic genes as a single entity, improving k-mer based GWAS in terms of power and interpretability. As the number of subgraphs is exponential in the number of nodes in the DBG, a method naively testing all possible subgraphs would result in very low statistical power due to multiple testing corrections, and the mere exploration of these subgraphs would quickly become computationally intractable. The concept of testable hypothesis has successfully been used to address both problems in similar contexts. We leverage this concept to test all closed connected subgraphs by proposing a novel enumeration scheme for these objects which fully exploits the pruning opportunity offered by testability, resulting in drastic improvements in computational efficiency. We illustrate this on both real and simulated datasets and also demonstrate how considering subgraphs leads to a more powerful and interpretable method. Our method integrates with existing visual tools to facilitate interpretation. We also provide an implementation of our method, as well as code to reproduce all results at https://github.com/HectorRDB/Caldera_Recomb.


title: A truncated reverse transcriptase enhances prime editing by split AAV vectors

abstract: Prime editing is a new CRISPR-based genome editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with AAV viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4x fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. This novel minimized PE provides great value to AAV-based delivery applications in vivo.


title: Regulation of ERK2 activity by dynamic S-acylation

abstract: The extracellular signal-regulated kinases (ERK1/2) are key effector proteins of the mitogen-activated protein kinase pathway, choreographing essential processes of cellular physiology. Critical in regulating these regulators are a patchwork of mechanisms, including post-translational modifications (PTMs) such as MEK-mediated phosphorylation. Here, we discover that ERK1/2 are subject to S-palmitoylation, a reversible lipid modification of cysteine residues, at C271/C254. Moreover, the levels of ERK1/2 S-acylation are modulated by epidermal growth factor (EGF) signaling, mirroring its phosphorylation dynamics, and palmitoylation-deficient ERK2 displays altered phosphorylation patterns at key sites. We find that chemical inhibition of either lipid addition or removal significantly alters ERK1/2s EGF-triggered transcriptional program. We also identify a subset of "writer" protein acyl transferases (PATs) and an "eraser" acyl protein thioesterase (APT) that drive ERK1/2s cycle of palmitoylation and depalmitoylation. Finally, we examine ERK1/2 S-acylation in a mouse model of metabolic syndrome, correlating changes in its lipidation levels with alterations in writer/eraser expression and solidifying the link between ERK1/2 activity, ERK1/2 lipidation, and organismal health. This study not only presents a previously undescribed mode of ERK1/2 regulation and a node to modulate MAPK pathway signaling in pathophysiological conditions, it also offers insight into the role of dynamic S-palmitoylation in cell signaling more generally.


title: Spread of Xanthomonas vasicola pv. musacearum within banana mats: implications for Xanthomonas wilt management

abstract: Xanthomonas wilt (XW) of banana caused by Xanthomonas vasicola pv. musacearum (Xvm) does not spread to all plants physically interconnected through the rhizome when one or a few are diseased. However, the factors behind this incomplete systemic spread of Xvm are not fully known yet could inform XW management. This study explored the effect of Xvm inoculum amounts, number and size of suckers, sucker positioning on mother plant corms and other mother plant corm attributes on sucker colonization. A shorter (p <0.05) incubation period (17.9 vs 21.1 days) and higher (p<.001) cumulative number of symptomatic leaves (5.2 vs 1.6 leaves) was observed when all (high inoculum) compared to two leaves (low inoculum) were inoculated. Xvm was recovered in corms at 29 days post inoculation (dpi) in both treatments with no differences (p >0.05) in proportions of corms with Xvm between the treatments. However, Xvm was recovered earlier and at a higher frequency in suckers when all leaves were inoculated. Lower Xvm recoveries occurred in the lower corm sections to which most suckers were attached relative to the middle and upper corm sections. Xvm incidence in corms increased with the number of attached maiden suckers, and the dpi while it declined with increasing mother plant and corm height. Thus, Xvm spread within mats is influenced by the amount of inoculum and the physiological stage (e.g., height) of the plant and attached suckers. The position of suckers, predominantly at the bottom of corms also protects them from infection. Measures that reduce Xvm inoculum build-up in mats are thus crucial for minimizing within mat XW spread.

category: pathology


title: Discovery of imidazole-based inhibitors of P. falciparum cGMP-dependent protein kinase

abstract: The discovery of new targets for treatment of malaria and in particular those aimed at the pre-erythrocytic stage in the life cycle, advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, this manuscript presents initial structure-activity relationships in an imidazole scaffold at four positions, representative in vitro ADME, hERG characterization and cell-based anti-parasitic activity. This series of PfPKG inhibitors has good in vitro PfPKG potency, low hERG activity and cell-based anti-parasitic activity against multiple Plasmodium species that appears to correlate with in vitro potency.


title: Brilacidin, a COVID-19 Drug Candidate, demonstrates broad-spectrum antiviral activity against human coronaviruses OC43, 229E and NL63 through targeting both the virus and the host cell

abstract: Brilacidin, a mimetic of host defense peptides (HDPs), is currently in phase 2 clinical trial as an antibiotic drug candidate. A recent study reported that brilacidin has antiviral activity against SARS-CoV-2 by inactivating the virus. In this work, we discovered an additional mechanism of action of brilacidin by targeting heparan sulfate proteoglycans (HSPGs) on host cell surface. Brilacidin, but not acetyl brilacidin, inhibits the entry of SARS-CoV-2 pseudovirus into multiple cell lines, and heparin, a HSPG mimetic, abolishes the inhibitory activity of brilacidin on SARS-CoV-2 pseudovirus cell entry. In addition, we found that brilacidin has broad-spectrum antiviral activity against multiple human coronaviruses (HCoVs) including HCoV-229E, HCoV-OC43, and HCoV-NL63. Mechanistic studies revealed that brilacidin has a dual antiviral mechanism of action including virucidal activity and binding to coronavirus attachment factor HSPGs on host cell surface. Brilacidin partially loses its antiviral activity when heparin was included in the cell cultures, supporting the host-targeting mechanism. Drug combination therapy showed that brilacidin has a strong synergistic effect with remdesivir against HCoV-OC43 in cell culture. Taken together, this study provides appealing findings for the translational potential of brilacidin as a broad-spectrum antiviral for coronaviruses including SARS-CoV-2.


title: Validation and invalidation of SARS-CoV-2 papain-like protease inhibitors

abstract: SARS-CoV-2 encodes two viral cysteine proteases, the main protease (Mpro) and the papain-like protease (PLpro), both of which are validated antiviral drug targets. The PLpro is involved in the cleavage of viral polyproteins as well as immune modulation through removing ubiquitin and interferon-stimulated gene product 15 (ISG15) from host proteins. Therefore, targeting PLpro might be a two-pronged approach. Several compounds including YM155, cryptotanshinone, tanshinone I, dihydrotanshinone I, tanshinone IIA, SJB2-043, 6-thioguanine, and 6-mercaptopurine were recently identified as SARS-CoV-2 PLpro inhibitors through high-throughput screening. In this study, we aim to validate/invalidate the reported PLpro inhibitors using a combination of PLpro target specific assays including enzymatic FRET assay, thermal shift binding assay (TSA), and the cell based FlipGFP assay. Collectively, our results showed that all compounds tested either did not show binding or led to denaturation of the PLpro in the TSA binding assay, which might explain their weak enzymatic inhibition in the FRET assay. In addition, none of the compounds showed cellular inhibition of PLpro as revealed by the FlipGFP assay. Therefore, more efforts are needed to search for specific and potent SARS-CoV-2 PLpro inhibitors.


title: Erythropoietin and miRNA profiles during the menstrual cycle in relation to hematological and lipid biomarkers

abstract:
BackgroundCirculatory micro RNAs (miRNA) have been discussed as complementary diagnostic markers in cardiovascular diseases, and in anti-doping testing. MiR-144 and miR-486 have been associated with cholesterol homeostasis and hematopoiesis, respectively. In addition, they have been suggested as putative biomarkers for autologous blood transfusion and erythropoietin (EPO) doping. The aim of the present study was to assess the variability of miR-144-3p/5p, miR-486-5p/3p and EPO during the menstrual cycle. Secondary aim was to study the correlations between miRNAs, EPO and hematological parameters and lipids.

Methods13 healthy women with regular menses were followed with weekly blood sampling during two whole menstrual cycles. MiRNAs were analyzed using TaqMan and PCR followed by calculation of the relative expression for each miRNA using ddCT approach.

ResultsThere was no menstrual cycle variability in miRNAs and EPO. MiRNA-144-3p was associated with HDL-C (rs=-0.34, p=0.036) and miRNA-486-5p with Hb (rs=0.32, p=0.046). EPO concentrations correlated to lymphocytes (rs=-0.062, p=0.0002)), Hb (rs= -0.42, p=0.0091), HDL-C (rs=0.36, p=0.030) and triglycerides (rs=-0.54, p=0.0006).

ConclusionsThe results of this study may increase the understanding of how miR486-5p and miR144-3p as well as EPO correlate to hematopoietic and lipid biomarkers.

category: physiology


title: Enteroendocrine cell types that drive food reward and aversion

abstract: Animals must learn through experience which foods are nutritious and should be consumed, and which are toxic and should be avoided. Enteroendocrine cells (EECs) are the principal chemosensors in the GI tract, but investigation of their role in behavior has been limited by the difficulty of selectively targeting these cells in vivo. Here we describe an intersectional genetic approach for manipulating EEC subtypes in behaving mice. We show that multiple EEC subtypes inhibit food intake but have different effects on learning. Conditioned flavor preference is driven by release of cholecystokinin whereas conditioned taste aversion is mediated by serotonin and substance P. These positive and negative valence signals are transmitted by vagal and spinal afferents, respectively. These findings establish a cellular basis for how chemosensing in the gut drives learning about food.


title: Design, construction and optimization of a synthetic RNA polymerase operon in Escherichia coli.

abstract: Prokaryotic genes encoding functionally related proteins are often clustered in operons. The compact structure of operons allows for co-transcription of the genes, and for co-translation of the polycistronic messenger RNA to the corresponding proteins. This leads to reduced regulatory complexity and enhanced gene expression efficiency, and as such to an overall metabolic benefit for the protein production process in bacteria and archaea. Interestingly, the genes encoding the subunits of one of the most conserved and ubiquitous protein complexes, the RNA polymerase, are not clustered in a single operon. Rather, its genes are scattered in all known prokaryotic genomes, generally integrated in different ribosomal operons. To analyze the impact of this genetic organization on the fitness of Escherichia coli, we constructed a bacterial artificial chromosome harboring the genes encoding the RNA polymerase complex in a single operon. Subsequent deletion of the native chromosomal genes led to a reduced growth on minimal medium. However, by using adaptive laboratory evolution the growth rate was restored to wild-type level. Hence, we show that a highly conserved genetic organization of core genes in a bacterium can be reorganized by a combination of design, construction and optimization, yielding a well-functioning synthetic genetic architecture.

category: synthetic biology


title: Single cell RNA-seq uncovers the nuclear decoy lincRNA PIRAT as a regulator of systemic monocyte immunity during COVID-19

abstract: The systemic immune response to viral infection is shaped by master transcription factors such as NF{kappa}B or PU.1. Although long non-coding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA-seq approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9 - key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling characterized PIRAT as a nuclear decoy RNA, diverting the PU.1 transcription factor from alarmin promoters to dead-end pseudogenes in naive monocytes. NF{kappa}B-dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Our results suggest a major role of nuclear noncoding RNA circuits in systemic antiviral responses to SARS-CoV-2 in humans.


title: Prevalence of bovine tuberculosis and characterization of the members of the Mycobacterium tuberculosis complex from slaughtered cattle in Rwanda

abstract:
BackgroundBovine tuberculosis (bTB) is an endemic disease in Rwanda, but little is known about its prevalence and causative mycobacterial species. The disease causes tremendous losses in livestock and wildlife and remains a significant threat to public health.

Materials and methodsA cross-sectional study employing a systematic random sampling of cattle (n=300) with the collection of retropharyngeal lymph nodes and tonsils (n=300) irrespective of granulomatous lesions was carried out in six abattoirs to investigate the prevalence and identify mycobacterial species using culture, acid-fast bacteria staining, polymerase chain reaction, and GeneXpert assay. Individual risk factors and the origin of samples were analysed for association with the prevalence.

FindingsOf the 300 samples, six were collected with visible TB-like lesions. Our findings demonstrated the presence of Mycobacterium tuberculosis complex (MTBC) in 1.7% (5/300) of sampled slaughtered cattle. M. bovis was isolated from 1.3% (4/300) animals while one case was caused by a rifampicin-resistance (RR) M. tuberculosis. Non-tuberculous mycobacteria were identified in 12.0% (36/300) of the sampled cattle. There were no significant associations between the prevalence and abattoir category, age, sex, and breeds of slaughtered cattle.

ConclusionsThis study is the first in Rwanda to isolate both M. bovis and RR M. tuberculosis in slaughtered cattle indicating that bTB is prevalent in Rwanda with a low prevalence. The isolation of RR M. tuberculosis from cattle indicates possible zooanthroponotic transmission of M. tuberculosis and close human-cattle contact. To protect humans against occupational zoonotic diseases, it is essential to control bTB in cattle and raise the awareness among all occupational groups as well as reinforce biosafety at the farm level and in the abattoirs.

Authors summaryTuberculosis in cattle (bTB) causes financial losses to livestock owners and is a disease transmissible to humans especially those with an occupational risk through exposure to infected animals and animal products. This study aimed to identify the prevalence of bTB and characterize the mycobacterial species from cattle slaughtered in the six abattoirs in Rwanda. Four M. bovis, as well as one rifampicin-resistant (RR) M. tuberculosis, were identified from slaughtered cattle and, thus, the apparent bTB prevalence was 1.7% (5/300). Likely, the RR M. tuberculosis isolate was mostly likely of human origin and transmitted to cattle during close human-cattle contact. It is therefore essential to control bTB in cattle and reinforce the protection of farmworkers and abattoir workers who are always exposed to infected animals.


title: Outer Membrane Vesicles from the gut microbiome contribute to tumor immunity by eliciting cross-reactive T cells

abstract: The gut microbiome plays a key role in cancer immunity. One proposed mechanism is through the elicitation of T cells, which incidentally recognize neo-epitopes arising from cancer mutations ("molecular mimicry (MM)" hypothesis). To support MM, Escherichia coli Nissle was engineered with the SIINFEKL epitope (OVA) and orally administered to C57BL/6 mice. The treatment elicited OVA-specific CD8+ T cells in the lamina propria and inhibited the growth of OVA-B16F10 tumors. Importantly, the administration of Outer Membrane Vesicles (OMVs) engineered with different T cell epitopes elicited epitope-specific T cells and inhibited tumor growth. Microbiome shotgun sequencing and TCR sequencing provided evidence that cross-reacting T cells were induced at the mucosal level and subsequently reached the tumor site. Overall, our data support the role of MM in tumor immunity, assign a new role to OMVs and pave the way to new probiotics/OMV-based anti-cancer immunotherapies.