title: Slc9a6 mutation causes Purkinje cell loss and ataxia in the spontaneous shaker rat

abstract:
BackgroundThe shaker rat carries a naturally occurring mutation leading to progressive ataxia characterized by Purkinje cell (PC) loss. We previously reported on fine-mapping the shaker locus to the long arm of the rat X chromosome. In this work, we sought to identify the mutated gene underlying the shaker phenotype and confirm its identity by functional complementation.

MethodsWe fine-mapped the candidate region and analyzed cerebellar transcriptomes to identify deleterious variants. We generated an adeno-associated virus (AAV) targeting solute carrier family 9, member A6 (Slc9a6) expression to PCs using a mouse L7-6 (L7) promoter, as well as a control green fluorescent protein (GFP)-expressing virus. We administered AAVs prior to the onset of PC degeneration through intracerebroventricular injection and evaluated the molecular, cellular, and motor phenotypes.

ResultsWe identified a XM_217630.9 (Slc9a6):c.[191_195delinsA] variant in the Slc9a6 gene that segregated with disease. This mutation is predicted to generate a truncated sodium-hydrogen exchanger 6 (NHE6) protein, p.(Ala64Glufs*23). Administration of AAV9-PHP.eB expressing rat Slc9a6 prior to symptom onset reduced the shaker motor, molecular, and cellular phenotypes.

InterpretationSlc9a6 is mutated in shaker and also in human Christianson syndrome, an epileptic encephalopathy. AAV-based gene therapy may be a viable therapeutic strategy for Christianson syndrome, and the shaker rat model may aid in therapeutic development.


title: Slc9a6 mutation causes Purkinje cell loss and ataxia in the shaker rat

category: neuroscience


title: An atypical ABC transporter is involved in antifungal resistance and host interactions in the pathogenic fungus Cryptococcus neoformans

abstract:
ATP-binding cassette (ABC) transporters represent one of the largest protein superfamilies. Functionally diverse, ABC transporters have been implicated in many aspects of microbial physiology. The genome of the human fungal pathogen Cryptococcus neoformans encodes 54 putative ABC transporters and the majority of them remain uncharacterized. In a previous genetic screen for fungal regulators of phagocytosis, we identified an uncharacterized gene, CNAG_06909, that modulates host interactions. This gene encodes a half-size ABC transporter of the PDR-type, and phenotypic studies of a strain missing this gene revealed an altered antifungal profile, including hypersensitivity to fluconazole (FLC). This gene, which we have named PDR6, localizes to the endoplasmic reticulum (ER) and plasma membrane (PM), and when absent, less ergosterol was observed in the PM. Additionally, we observed that the pdr6{Delta} strain displays a reduction in secreted polysaccharide capsular material. These changes to the cellular surface may explain the observed increased uptake by macrophages and the reduced intracellular survival. Finally, studies in mice demonstrate that PDR6 function is required for normal progression of cryptococcal infection. Taken together, this study demonstrates a novel dual role for PDR transporters in C. neoformans, which could represent a potential target for antifungal therapeutics. Furthermore, the atypical half-size transporter encoded by PDR6 is conserved in many fungal pathogens, but absent in non-pathogenic fungal models. Hence, this study provides for the first time, a function for this unique group of fungal half-size PDR transporters that, although conserved, remain largely understudied.

IMPORTANCEConserved across all kingdoms of life, ABC transporters comprise one of the largest protein families. They are associated with multidrug resistance, affecting aspects such as resistance to antimicrobials or cancer treatments. Despite their importance, they are understudied in fungal pathogens. In the environmental fungus Cryptococcus neoformans, a leading cause of fungal infections, only few ABC transporters have been studied. Here we characterize an atypical, half-size, ABC transporter of the PDR-type, that affects both antifungal resistance and host-pathogen interactions. PDR-type transporters are only present in fungi and plants, and this subgroup of half-size transporters is conserved in fungal pathogens, yet their function was completely unknown. Because the current treatments for cryptococcal infection are suboptimal, understanding the mechanisms of antifungal resistance and the host interactions that drive the infection is critical to improve the management of this disease. Here we provide insights into these important aspects of cryptococcal pathogenesis.

category: microbiology


title: The connection between the gut mycobiome community of natural black soldier fly populations and their environment

abstract:
In contrast to interactions between prokaryotic organisms (e.g. bacteria) and insects, an understanding of the interactions between eukaryotic microorganisms (e.g. fungi) and insects is lacking. In this study, black soldier fly larvae were collected from natural populations in household composts. The mycobiome composition of the larval gut and environment was identified using next generation sequencing. The nutrient composition of the larvaes environment was analyzed. The mycobiome composition was divided into three groups: the first included larvae from Kfar HaHoresh that comprised mostly Meyerozyma and were influenced by the fiber and carbohydrate composition of the compost; the second included the compost samples from Kfar HaHoresh and Misgav Am that mainly comprised the Dipodascaceae family and were influenced by the fiber composition of the compost. The third group contained all the remaining compost and larvae samples and mainly comprised the genus Candida. Candida spp. are the most common in the environment and may thus affect their abundance in the insect gut. However, this species may be adapted to the insects gut, providing the insect with a benefit that allows the insect to disperse Candida to new resource patches and thereby help increase its abundance in the environment. In cases where dominant species are not present, it seems that the important factor determining fungal composition in the insect gut is its diet. Understanding the metabolic effect of fungi, e.g. Candida, on the BSF larvae is still a black box which requires further study.

IMPORTANCEThe research analyzed the mycobiome composition of BSF larvae from natural environments. It was found that the most dominant fungi in these communities is the Candida genus, which was found also dominant in the BSF environment (household compost) that differ in its nutrient composition. Understanding the metabolic effect of these fungi on the BSF larvae have the potential to dramatically affect the physiological condition of the insect. Revealing these metabolic interactions may help to increase the rearing efficiency of insects, such as the BSF, which is currently being reared at large scales as an alternative protein source.


title: Microtubule-Connexin-43 regulation suppresses arrhythmias and fibrosis in Duchenne muscular dystrophy mice.

abstract: Dilated cardiomyopathy is the leading cause of death in Duchenne muscular dystrophy (DMD) patients due to advancements in skeletal muscle therapies yet limited presence of cardiac treatments. The phosphorylation status of gap junction protein Connexin-43 (Cx43) drives Cx43 remodeling and the development of arrhythmias and fibrosis. Based on evidence that Colchicine drug treatment improves Cx43 phosphorylation and remodeling, we compared the microtubule cytoskeleton in DMD mice (mdx) versus mdx mice genetically altered to be Cx43-phosphorylation-deficient (mdxS3A). Reciprocally, we analyzed the microtubule cytoskeleton in mdx mice genetically altered to be Cx43-phospho-mimicking (mdxS3E). We found a link between the phospho-status of Connexin-43 and regulation of microtubule organization, in which phospho-dead Cx43 (S3A) inhibits improvements seen with Colchicine treatment in mdx mice, and phospho-mimic S3E promotes microtubule reorganization in mdx mice. A reduction in arrhythmias and fibrosis suggests an unsuspecting Cx43-microtubule link for translational corrective activities for DMD cardiomyopathy.

category: cell biology


title: Canonical Wnt Signaling and the Regulation of Divergent Mesenchymal Fgf8 expression in Axolotl Limb Development and Regeneration

abstract: The expression of Fibroblast growth factors (Fgf) ligands in a specialized epithelial compartment, the Apical Ectodermal Ridge (AER), is a conserved feature of limb development across vertebrate species. In vertebrates, Fgf 4, 8, 9, and 17 are all expressed in the AER. An exception to this paradigm is the salamander (axolotl) developing and regenerating limb, where key Fgf ligands are expressed in the mesenchyme. The mesenchymal expression of Amex.Fgf8 in axolotl has been suggested to be critical for regeneration. To date, there is little knowledge regarding what controls Amex.Fgf8 expression in the axolotl limb mesenchyme. A large body of mouse and chick studies have defined a set of transcription factors and canonical Wnt signaling as the main regulators of epidermal Fgf8 expression in these organisms. In this study, we address the hypothesis that alterations to one or more of these components during evolution has resulted in mesenchymal Amex.Fgf8 expression in the axolotl. To sensitively quantify gene expression with spatial precision, we combined optical clearing of whole-mount axolotl limb tissue with single molecule fluorescent in situ hybridization and a semi-automated quantification pipeline. Several candidate upstream components were found expressed in the axolotl ectoderm, indicating that they are not direct regulators of Amex.Fgf8 expression. We found that Amex.Wnt3a is expressed in axolotl limb epidermis, similarly to chicken and mouse. However, unlike in amniotes, Wnt target genes are activated preferentially in limb mesenchyme rather than in epidermis. Inhibition and activation of Wnt signaling results in downregulation and upregulation of mesenchymal Amex.Fgf8 expression respectively. These results implicate a shift in tissue responsiveness to canonical Wnt signaling from epidermis to mesenchyme as one step contributing to the unique mesenchymal Amex.Fgf8 expression seen in the axolotl.

category: developmental biology


title: 2-deoxyglucose inhibits yeast AMPK signaling and triggers glucose transporter endocytosis, potentiating the drug toxicity

abstract: 2-deoxyglucose is a glucose analog that impacts many aspects of cellular physiology. After its uptake and its phosphorylation into 2-deoxyglucose-6-phosphate (2DG6P), it interferes with several metabolic pathways including glycolysis and protein N-glycosylation. Despite this systemic effect, resistance can arise through strategies that are only partially understood. In yeast, 2DG resistance is often associated with mutations causing increased activity of the yeast 5-AMP activated protein kinase (AMPK), Snf1. Here we focus on the contribution of a Snf1 substrate in 2DG resistance, namely the alpha-arrestin Rod1 involved in nutrient transporter endocytosis. We report that 2DG triggers the endocytosis of many plasma membrane proteins, mostly in a Rod1-dependent manner. Rod1 is central to 2DG-induced endocytosis because 2DG, following its phosphorylation by hexokinase Hxk2, triggers changes in Rod1 post-translational modifications and promotes its function in endocytosis. Mechanistically, this is explained by a transient, 2DG-induced activation of protein phosphatase 1 (PP1) and a concomitant inactivation of Snf1/AMPK. We show that 2DG-induced endocytosis is detrimental to cells, and the lack of Rod1 counteracts this process by stabilizing glucose transporters at the plasma membrane. This facilitates glucose uptake, which may help override the metabolic blockade caused by 2DG, and 2DG export - thus terminating the process of 2DG detoxification. Altogether, these results shed a new light on the regulation of AMPK signaling in yeast and highlight a remarkable strategy to bypass 2DG toxicity involving glucose transporter regulation.

category: genetics


title: A DEAD-box helicase drives the partitioning of a pro-differentiation NAB protein into nuclear foci

abstract: How cells regulate gene expression in a precise spatiotemporal manner during organismal development is a fundamental question in biology. Recent studies have demonstrated the role of transcriptional condensates in gene regulation1-5. However, little is known about the function and regulation of these transcriptional condensates in the context of animal development and physiology. We found that the evolutionarily conserved DEAD-box helicase DDX-23 controls stem cell fate in C. elegans at least in part by binding to and facilitating the condensation of MAB-10, the C. elegans homolog of mammalian NAB protein. MAB-10 is a transcriptional cofactor that functions with the EGR protein LIN-29 to regulate the transcription of genes required for exiting the cell cycle, terminal differentiation, and the larval-to-adult transition6. We suggest that DEAD-box helicase proteins function more generally during animal development to control the condensation of NAB proteins important in cell-fate decisions and that this mechanism is evolutionarily conserved. In mammals, a comparable mechanism might underlie terminal cell differentiation and when misregulated might promote cancerous growth.


title: The Modular μSiM: a Mass Produced, Rapidly Assembled, and Reconfigurable Platform for the Study of Barrier Tissue Models In Vitro

abstract: Advanced in vitro tissue chip models can reduce and replace animal experimentation and may eventually support  on-chip clinical trials. To realize this potential, however, tissue chip platforms must be both mass-produced and reconfigurable to allow for customized design. To address these unmet needs, we introduce an extension of our {micro}SiM (microdevice featuring a silicon-nitride membrane) platform. The modular {micro}SiM (m-{micro}SiM) uses mass-produced components to enable rapid assembly and reconfiguration by laboratories without knowledge of microfabrication. We demonstrate the utility of the m-{micro}SiM by establishing an hiPSC-derived blood-brain barrier (BBB) in bioengineering and non-engineering, brain barriers focused laboratories. We develop and validate in situ and sampling-based assays of small molecule diffusion as a measure of barrier function. BBB properties show excellent interlaboratory agreement and match expectations from literature, validating the m-{micro}SiM as a platform for barrier models and demonstrating successful dissemination of components and protocols. We then demonstrate the ability to quickly reconfigure the m-{micro}SiM for co-culture and immune cell transmigration studies through addition of accessories and/or quick exchange of components. Because the development of modified components and accessories is easily achieved, custom designs of the m-{micro}SiM should be accessible to any laboratory desiring a barrier-style tissue chip platform.

category: bioengineering


title: Reference-based cell type matching of spatial transcriptomics data

abstract: With the advent of multiplex fluorescence in situ hybridization (FISH) and in situ RNA sequencing technologies, spatial transcriptomics analysis is advancing rapidly. Spatial transcriptomics provides spatial location and pattern information about cells in tissue sections at single cell resolution. Cell type classification of spatially-resolved cells can also be inferred by matching the spatial transcriptomics data to reference single cell RNA-sequencing (scRNA-seq) data with cell types determined by their gene expression profiles. However, robust cell type matching of the spatial cells is challenging due to the intrinsic differences in resolution between the spatial and scRNA-seq data. In this study, we systematically evaluated six computational algorithms for cell type matching across four spatial transcriptomics experimental protocols (MERFISH, smFISH, BaristaSeq, and ExSeq) conducted on the same mouse primary visual cortex (VISp) brain region. We find that while matching results of individual algorithms vary to some degree, they also show agreement to some extent. We present two ensembl meta-analysis strategies to combine the individual matching results and share the consensus matching results in the Cytosplore Viewer (https://viewer.cytosplore.org) for interactive visualization and data exploration. The consensus matching can also guide spot-based spatial data analysis using SSAM, allowing segmentation-free cell type assignment.

category: bioinformatics


title: The Codon Statistics Database: a Database of Codon Usage Bias

abstract:
MotivationMost amino acids can be encoded by a set of synonymous codons. Often, for any given amino acid, certain codons are significantly more used than others, a phenomenon known as codon usage bias. The genomes of different species differ in the frequencies at which they use each codon (e.g., a codon that is highly used in one species may be lowly used in another species). In addition, within any given genome, genes differ in their degree of codon bias, with highly expressed genes being more likely to use preferred codons. Knowing the codons that are preferred by a certain genome, and the amount of codon bias exhibited by each gene, has multiple applications (e.g., in heterologous expression, gene prediction, or phylogenetic inference).

ResultsWe have developed the Codon Statistics Database, an online database that contains codon usage statistics for all the species with reference or representative genomes in RefSeq. The user can search for any species and access two sets of tables. One set lists, for each codon, the frequency, the Relative Synonymous Codon Usage (RSCU), and whether the codon is preferred. Another set of tables lists, for each gene, its GC content, Effective Number of Codons (ENC), Codon Adaptation Index (CAI), and frequency of optimal codons (Fop). Equivalent tables can be accessed for 1) all nuclear genes, 2) nuclear genes encoding ribosomal proteins, 3) mitochondrial genes and 4) chloroplastic genes (if available in the relevant assembly). The user can also search for any taxonomic group (e.g., "primates") and obtain a table comparing all the species in the group.

AvailabilityThe database is free to access without registration at http://codonstatsdb.unr.edu.


title: Fast model-free standardization and integration of single-cell transcriptomics data

abstract: Single-cell transcriptomics datasets from the same anatomical sites generated by different research labs are becoming mainstream. However, fast, and computationally inexpensive tools for standardization of cell-type annotation and data integration are still needed to increase research inclusivity. To standardize cell-type annotation and integrate single-cell transcriptomics datasets, we have built a fast, model-free integration method called MASI (Marker-Assisted Standardization and Integration). MASI can run integrative annotation on a personal laptop for approximately one million cells, providing a cheap computational alternative for the single-cell data analysis community. MASI has an average macro F1/overall accuracy of 0.79/0.89 over the 4 benchmark datasets. We demonstrate that MASI outperforms other methods based on speed, and its performance for the tasks of data integration and cell-type annotation is comparable or even superior to other existing methods. We apply MASI for integrative lineage analysis and show that it preserves the underlying biological signal in datasets tested. Finally, to harness knowledge from single-cell atlases, we demonstrate three case studies that cover integration across research groups, biological conditions, and surveyed participants, respectively.


title: Hip osteoarthritis: A novel network analysis of subchondral trabecular bone structures

abstract: Hip osteoarthritis (HOA) is a degenerative joint disease that leads to the progressive destruction of subchondral bone and cartilage at the hip joint. Development of effective treatments for HOA remains an open problem, primarily due to the lack of knowledge of its pathogenesis and a typically late-stage diagnosis. We describe a novel network analysis methodology for micro-computed tomography (micro-CT) images of human trabecular bone. We explored differences between the trabecular bone microstructure of femoral heads with and without HOA. Large-scale automated extraction of the network formed by trabecular bone revealed significant network properties not previously reported for bone. Profound differences were discovered, particularly in the proximal third of the femoral head, where HOA networks demonstrated elevated numbers of edges, vertices and graph components. When further differentiating healthy joint and HOA networks, the latter showed fewer small-world network properties, due to decreased clustering coefficient and increased characteristic path length. Furthermore, we found that HOA networks had reduced length of edges, indicating the formation of compressed trabecular structures. In order to assess our network approach, we developed a deep learning model for classifying HOA and control cases, and we fed it with two separate inputs: (i) micro-CT images of the trabecular bone, and (ii) the network extracted from them. The model with plain micro-CT images achieves 74.63% overall accuracy while the trained model with extracted networks attains 96.47% accuracy. We anticipate our findings to be a starting point for a novel description of bone microstructure in HOA, by considering the phenomenon from a graph theory viewpoint.

category: biophysics


title: Paclitaxel-loaded PEGylated Cationic Lipid Nanodiscs and Small Liposomes with Protruding Brush Conformation PEG-chains Show Tumor Penetration and Proapoptotic Caspase-3 Activation

abstract: Novel approaches are required to address the urgent need to develop lipid-based carriers of paclitaxel (PTX) and other hydrophobic drugs for cancer chemotherapy. Carriers based on cationic liposomes (CLs) (e.g. EndoTAG-1(R)) have shown promise in animal models and advanced to late-stage clinical trials. We recently found that the addition of cone-shaped poly(ethylene glycol)-lipid (PEG-lipid) to PTX-loaded CLs (CLsPTX) drives a transition to a phase of sterically stabilized, higher curvature (smaller) lipid nanoparticles consisting of coexisting PEGylated CLsPTX and PTX-containing nanodiscs (bicelles) (nanodiscsPTX). At PEG coverages in the brush regime, these PEGylated CLsPTX and nanodiscsPTX showed significantly improved delivery and cytotoxicity to human cancer cells in vitro. Here, we investigated the PTX loading and stability of the CLsPTX and nanodiscsPTX at high concentration and assessed their circulation half-life and biodistribution in vivo. At higher content of PEG-lipid (5 and 10 mol% of a PEG-lipid with PEG molecular weight 5000 g/mol), the mixture of PEGylated CLsPTX and nanodiscsPTX formed almost exclusively particles of sub-micron size and incorporated up to 2.5 mol% PTX without crystallization for at least 20 h. Remarkably, compared to lipid vector preparations containing 2 and 5 mol% PEG5K-lipid (with the PEG chains in the mushroom regime), those at 10 mol% with PEG chains in the extended brush conformation showed significantly more tumor penetration and increased proapoptotic caspase-3 activation in 4T1 breast cancer lesions modeled in immunocompetent mice.


title: Cell-free prototyping enables implementation of optimized reverse beta-oxidation pathways in heterotrophic and autotrophic bacteria

abstract: Carbon-negative synthesis of biochemical products has the potential to mitigate global CO2 emissions. An attractive route to do this is the reverse {beta}-oxidation (r-BOX) pathway coupled to the Wood-Ljungdahl pathway. Here, we optimized and implemented r-BOX for the synthesis of C4-C6 acids and alcohols. With a high-throughput in vitro prototyping workflow, we screened 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity. Implementation of these pathways into Escherichia coli generated designer strains for the selective production of butanoic acid (4.9 {+/-}0.1 gL-1), hexanoic acid (3.06 {+/-} 0.03 gL-1) and 1-hexanol (1.0 {+/-} 0.1 gL-1) at the best performance reported to date in this bacterium. We also generated Clostridium autoethanogenum strains able to produce 1-hexanol from syngas, achieving a titer of 0.26 gL-1 in a 1.5-L continuous fermentation. Our strategy enables optimization of rBOX derived products for biomanufacturing and industrial biotechnology.

category: synthetic biology


title: Validation of DeepLabCut as a tool for markerless 3D pose estimation

abstract: Deep learning-based approaches to markerless 3D pose estimation have taken neuroscience by storm. Yet many of these tools remain unvalidated. Here, we report on the validation of one increasingly popular tool (DeepLabCut) against simultaneous measurements obtained from a reference measurement system (Fastrak) with well-known performance characteristics. Our results confirm close (mm range) agreement between the two, indicating that deep learning-based approaches can be used by the research community with confidence.


title: Molecular Control of Circuit Plasticity and the Permanence of Imprinted Odor Memory

abstract: Behavioral imprinting is a distinct form of learning that has a lifelong impact on social interactions and affectional behaviors1-4. Unlike other forms of memory, imprinting does not require conspicuous association of stimuli; exposure per se appears sufficient to induce memories that neither undergo extinction nor are altered by experience later in life. The site of storage of imprinted memory and the mechanisms that control its formation and permanence are unknown. Here we uncover a molecular mechanism that controls olfactory imprinting, which underlies behaviors including kin and nest recognition, maternal attachment, and homing5-10. We show that odor exposure during the perinatal period converts an innately aversive odor into a homing signal. The behavioral change is associated with odor-induced changes in the projection patterns of olfactory sensory neuron (OSN) expressing the cognate receptors for the exposed odor. We show that the Wnt signaling receptor Frizzled1 (Fzd1) acts as a master regulator of the critical period of OSN development and is responsible for closing the critical period to prevent further changes in the neural circuit. In Fzd1 knockout mice axon projection patterns are continually modified by sensory experience. As Fzd1 knockout abolishes the developmental critical period, it also abolishes odor imprinting. Specific knockout of Fzd1 in the OSNs have the same effect. Mechanistically, Fzd1 controls the critical period through an autoregulated shutdown and by controlling an activity-driven regulon in the OSNs. The transient expression and the subsequent downregulation of Fzd1 leads to the irreversible closure of the critical period to lock in circuits established during the critical period. The evidence suggests that imprinted odor memory is stored in the patterns of connectivity at the first synapse in the olfactory bulb. Early odor experience induces changes in the OSN projection to alter connectivity with innate circuits to establish a life-long memory.


title: Made together, replayed together: Context reinstatement during sleep guides memory consolidation

abstract: Newly formed memories arent quarantined one from another when first encoded; rather, they are interlinked with other memories that were encoded in temporal proximity or share semantic features. In this study, we selectively biased memory processing during sleep in humans to test the hypothesis that memories contexts influence their consolidation. Participants formed 18 idiosyncratic narratives, each linking four objects together. They then memorized random on-screen spatial positions assigned to each of the 72 objects. During sleep, 12 object-specific sounds were unobtrusively presented, thereby cuing the associated memories and improving spatial recall for those objects. As predicted, memory for uncued objects that were contextually linked with cued objects benefited as well. The correlational structure of spectral power for electrophysiological responses revealed that sleep spindles supported context reinstatement and predicted context-related memory benefits. Moreover, context-specific electrophysiological activity patterns emerged during sleep. We conclude that when an individual memory is reactivated during sleep, the corresponding encoding context is also reinstated, impacting consolidation and enhancing subsequent retrieval of associated knowledge.


title: Comprehensive profiling of antibody responses to the human anellome using programmable phage display

abstract:
Viruses belonging to the diverse Anelloviridae family represent a major constituent of the commensal human virome. Aside from their widespread prevalence and persistence in humans and their absence of detectable pathologic associations, little is known about the immunobiology of the human anellome. In this study, we employed the Phage ImmunoPrecipitation Sequencing (PhlP-Seq) assay for comprehensive analyses of antibody binding to 56 amino acid long anellovirus peptides. We designed and constructed a large and diverse "AnelloScan" T7 phage library comprising more than 32,000 non-redundant peptides representing the ORF1, ORF2, ORF3 and TTV-derived apoptosis-inducing protein (TAIP) sequences of more than 800 human anelloviruses (spanning three genera). We used this library to profile the antibody reactivities of serum samples from 156 subjects. The vast majority of anellovirus peptides were not reactive in any of the subjects tested (n=~28,000; ~85% of the library). Antibody reactive peptides were largely restricted to the C-terminal region of the putative capsid protein, ORF1. To characterize antibody responses to newly acquired anellovirus infections, we screened a longitudinal cohort of matched blood-transfusion donors and recipients. Most transmitted anelloviruses did not elicit detectable antibody reactivity in the recipient (29 out of a total of 40 transmitted anelloviruses) and the remainder demonstrated delayed reactivity (~100-150 days after transfusion). This study represents the first large-scale epitope-level serological survey of the antibody response to the human anellome.

Graphical Abstract

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category: immunology


title: Costimulation of TLR8 responses by CXCL4 in Human Monocytes Mediated by TBK1-IRF5 Signaling and Epigenomic Remodeling

abstract: Regulation of endosomal TLR responses by the chemokine CXCL4 is implicated in inflammatory and fibrotic diseases. The current paradigm is that CXCL4 potentiates TLR responses by binding and facilitating endosomal delivery of nucleic acid TLR ligands. We report that in human monocytes/macrophages, CXCL4 initiates signaling cascades and downstream epigenomic reprogramming that change the profile of the TLR8 response by selectively and dramatically amplifying inflammatory gene transcription and IL-1{beta} production, while partially attenuating the IFN response. Mechanistically, costimulation by CXCL4 and TLR8 synergistically activated TBK1/IKK{varepsilon} and repurposed these kinases towards an inflammatory response via coupling with IRF5, and by activating the NLRP3 inflammasome without the need for an exogenous activator of a second signal for IL-1{beta} maturation. CXCL4 signaling strongly induced chromatin remodeling and de novo enhancers associated with inflammatory genes in a cooperative and synergistic manner with TLR8. These findings identify new mechanisms of regulation of TLR responses relevant for cytokine storm, and suggest targeting the TBK1/IKK{varepsilon}-IRF5 axis may be beneficial in inflammatory diseases.


title: Reproducible and sensitive micro-tissue RNA-sequencing from formalin-fixed paraffin-embedded tissue for spatial gene expression analysis

abstract: Spatial transcriptome analysis of formalin-fixed paraffin-embedded (FFPE) tissues using RNA-sequencing (RNA-seq) provides interactive information on morphology and gene expression, which is useful for clinical applications. However, despite the advantages of long-term storage at room temperature, FFPE tissues may be severely damaged by methylene crosslinking and provide less gene information than fresh-frozen tissues. In this study, we proposed a sensitive FFPE micro-tissue RNA-seq method that combines the punching of tissue sections (diameter: 100 m) and the direct construction of RNA-seq libraries. We evaluated a method using mouse liver tissues at 2 years after fixation and embedding and detected approximately 7,000 genes in micro-punched tissue-spots (thickness: 10 m), similar to that detected with purified total RNA (2.5 ng) equivalent to the several dozen cells in the spot. We applied this method to clinical FFPE specimens of lung cancer that had been fixed and embedded 6 years prior, and found that it was possible to determine characteristic gene expression in the microenvironment containing tumor and non-tumor cells of different morphologies. This result indicates that spatial gene expression analysis of the tumor microenvironment is feasible using FFPE tissue sections stored for extensive periods in medical facilities.

category: molecular biology


title: Estimating interaction matrices from performance data for diverse systems

abstract:
O_LINetwork theory allows us to understand complex systems by evaluating how their constituent elements interact with one another. Such networks are built from matrices which describe the effect of each element on all others. Quantifying the strength of these interactions from empirical data can be difficult, however, because the number of potential interactions increases non-linearly as more elements are included in the system, and not all interactions may be empirically observable when some elements are rare.
C_LIO_LIWe present a novel modelling framework which estimates the strength of pairwise interactions in diverse horizontal systems, using measures of species performance in the presence of varying densities of their potential interaction partners.
C_LIO_LIOur method allows us to directly estimate pairwise effects when they are statistically identifiable and approximate pairwise effects when they would otherwise be statistically unidentifiable. The resulting interaction matrices can include positive and negative effects, the effect of a species on itself, and are non-symmetrical.
C_LIO_LIThe advantages of these features are illustrated with a case study on an annual wildflower community of 22 focal and 52 neighbouring species, and a discussion of potential applications of this framework extending well beyond plant community ecology.
C_LI

category: ecology


title: Automated identification and registration of anatomical landmarks in C. elegans

abstract: The physiology of the nematode C. elegans can be visualized with many microscopy techniques. However, quantitative microscopy of C. elegans is complicated by the flexible and deformable nature of the nematode. These differences in posture and shape must be addressed in some fashion in any automated or manual analysis. Manual approaches are time intensive and require hand-labeling anatomical regions of interest. Automated tools exist, but generally rely on high-magnification imaging using labeled nuclei as fiducial markers. Here we describe a suite of new tools that allows for high-throughput analysis of whole-body images, aligned using anatomical landmarks identified from brightfield images. We show how these tools can be used in basic morphometric tasks and examine anatomical variation and morphological changes in a population over time.


title: Solvation Dynamics of a Single Triglyceride as a Function of its Chain Length

abstract: Triglycerides (TG) are transported packaged inside lypophillic particles. Several lipid exchange/transfer proteins interact with these lipoproteins and facilitate lipid exchange amongst lipoproteins, to maintain a constant flux in RCT. During this process, these neutral lipids are inadvertently exposed to the bulk water. Previous studies have elucidated the behavior of triglycerides in the bulk (on the surface of bilayer or inside a lipid droplet). However, isolated TGs during lipid exchange behave differently than when in bulk, due to an increased exposure to water. We studied the solvation dynamics of a single TG in a polar (water) and a non-polar (cyclohexane) medium to elucidate its solvated structure while drawing parallels with its structural organization in bulk (lipid droplet). We also examine the role of acyl chain length and its contribution to the free energy of solvation. Finally, we have established the predominant conformation of TG in water and cyclohexane and discuss the thermodynamics for such a preference.


title: Spatial transcriptomic analysis of childhood ependymoma implicates unresolved wound healing as a driver of tumor progression

abstract:
Childhood ependymoma (EPN) is a brain tumor that has seen limited improvements in outcome over past decades. The underlying cellular components of EPN have recently been revealed by single cell RNA-sequencing (scRNAseq), providing biological insights. Here we use spatial transcriptomics to comprehensively chart gene expression across the cellular landscape of posterior fossa subgroup A (PFA) EPN, the commonest and most deadly EPN variant, providing novel resolution of cellular heterogeneity and cellular interaction. We reveal that PFA are comprised of epithelial and mesenchymal histological zones each containing a diversity of cellular states. These include co-existing and spatially distinct undifferentiated progenitor-like clusters - a quiescent mesenchymal zone population, and a second highly mitotic progenitor population that is restricted to hypercellular epithelial zones. We show that myeloid cell interaction is the leading cause of mesenchymal transition in PFA, occurring in zones spatially distinct from hypoxia-induced mesenchymal transition, and these distinct EMT-initiating processes were replicated in in-vitro models of PFA. Collectively, our transcriptomic and functional analyses mirror the processes of normal wound healing where PFA mesenchymal and epithelial zones interact with immune subpopulations in cycle of persistent tissue damage response and mitogenic re-epithelialization signals. Spatial transcriptomics advances our understanding of PFA biology, implicating a "wound that will not heal" process as a driver of tumor progression, a new concept that could provide novel targets for effective therapeutic intervention.

SignificanceSpatial transcriptomic analysis of the ependymoma tumor microenvironment identifies novel cell populations and interactions, implicating unresolved wound healing as a clinically actionable driver of tumor progression in this refractory childhood brain tumor.

category: cancer biology


title: 3'UTR-directed, kinase proximal mRNA decay inhibits C/EBPβ phosphorylation/activation to suppress senescence in tumor cells

abstract: C/EBP{beta} is a potent regulator of RAS-induced senescence (RIS) and the SASP. C/EBP{beta} is post- translationally activated in RIS cells by the effector kinases ERK1/2 and CK2, but in tumor cells activation is suppressed by the CEBPB 3UTR. 3'UTR regulation of protein activity (UPA) requires a G/U-rich element (GRE) and its cognate binding protein, HuR. These components segregate CEBPB transcripts away from a perinuclear compartment harboring ERK1/2 and CK2, restricting C/EBP{beta} from its activating kinases. We report here that the mRNA decay proteins UPF1 and Staufen1/2 are essential UPA factors enriched within the perinuclear cytoplasm. STAU1/2 colocalize with CK2 on perinuclear signaling endosomes where they promote localized CEBPB mRNA decay. UPF1 or STAU1/2 depletion in tumor cells increased CEBPB transcripts adjacent to CK2 foci, coinciding with C/EBP{beta} activation and senescence. The GRE and an adjacent STAU binding site act to suppress C/EBP{beta}-mediated senescence, while a separate 3UTR region inhibits SASP induction. Accordingly, KrasG12D-driven lung tumors in mice carrying a Cebpb GRE deletion rarely progressed to malignant adenocarcinomas. Our findings identify kinase-proximal mRNA decay as a novel mechanism to inhibit C/EBP{beta} activating modifications in tumor cells to facilitate senescence bypass.


title: A mosaic-type trimeric RBD-based COVID-19 vaccine candidate induces potent neutralization against Omicron and other SARS-CoV-2 variants

abstract: Large-scale populations in the world have been vaccinated with COVID-19 vaccines, however, breakthrough infections of SARS-CoV-2 are still growing rapidly due to the emergence of immune-evasive variants, especially Omicron. It is urgent to develop effective broad-spectrum vaccines to better control the pandemic of these variants. Here, we present a mosaic-type trimeric form of spike receptor-binding domain (mos-tri-RBD) as a broad-spectrum vaccine candidate, which carries the key mutations from Omicron and other circulating variants. Tests in rats showed that the designed mos-tri-RBD, whether used alone or as a booster shot, elicited potent cross-neutralizing antibodies against not only Omicron but also other immune-evasive variants. Neutralizing antibody titers induced by mos-tri-RBD were substantially higher than those elicited by homo-tri-RBD (containing homologous RBDs from prototype strain) or the inactivated vaccine BBIBP-CorV. Our study indicates that mos-tri-RBD is highly immunogenic, which may serve as a broad-spectrum vaccine candidate in combating SARS-CoV-2 variants including Omicron.


title: Vomocytosis of Cryptococcus neoformans cells from murine, bone marrow-derived dendritic cells

abstract: Cryptococcus neoformans (CN) cells survive within the acidic phagolysosome of macrophages for extended times, then escape without impacting the viability of the host cell via a phenomenon that has been coined  vomocytosis. Through this mechanism, CN disseminate throughout the body, sometimes resulting in a potentially fatal condition - Cryptococcal Meningitis (CM). Justifiably, vomocytosis studies have focused primarily on macrophages, as alveolar macrophages within the lung act as first responders that ultimately expel this fungal pathogen. Herein, we hypothesize that dendritic cells (DCs), an innate immune cell with attributes that include phagocytosis and antigen presentation, can also act as  vomocytes. Presciently, this report shows that vomocytosis of CN indeed occurs from DCs. Primarily through time-lapse microscopy imaging, we show that rates of vomocytosis events from DCs are comparable to those seen from macrophages and further, are independent of the presence of the CN capsule and infection ratios. Moreover, phagosome-altering drugs such as chloroquine and bafilomycin A, as well as the actin-modifying drug, cytochalasin B inhibit this phenomenon from DCs. Although DC immunophenotype does not affect the total number of vomocytic events, we observed differences in the numbers of CN per phagosome and expulsion times. Interestingly, these observations were similar in primary, murine macrophages. Understanding the vomocytic behavior of different phagocytes and their phenotypic subtypes is needed to help elucidate the full picture of the dynamic interplay between CN and the immune system. Critically, deeper insight into vomocytosis could reveal novel approaches to treat CM, as well as other immune-related conditions.


title: The impact of the rice production system (irrigated vs lowland) on root-associated microbiome from farmer's fields in western Burkina Faso

abstract: As a consequence of its potential applications for food safety, there is a growing interest in rice root-associated microbial communities, but some systems remain understudied. Here, we compare the assemblage of root-associated microbiota in rice sampled in 19 small farmers fields from irrigated and rainfed lowlands in western Burkina Faso, using an amplicon metabarcoding approach 16S (Prokaryotes, three plant sample per field) and ITS (fungi, one sample per field). In addition to the expected structure according to the root compartment (root vs. rhizosphere) and geographical zones, we show that the rice production system is a major driver of microbiome structure, both for prokaryotes and fungi. In irrigated systems, we found a higher diversity of prokaryotic communities from rhizosphere and more complex co-occurrence networks, compared to rainfed lowlands. Core taxa were different between the two systems, and indicator species were identified: mostly within Bacillaceae and Bradyrhizobiaceae families in rainfed lowlands, and within Burkholderiaceae and Moraxellaceae in irrigated areas. Finally, phylotypes assigned to putative phytobeneficial and pathogen species were found. Mycorrhizal fungi Glomeromycetes abundance was higher in rainfed lowlands. Our results highlight deep microbiome differences induced by contrasted rice production systems that should consequently be considered for potential microbial engineering applications.


title: Nectar compounds impact bacterial and fungal growth and shift community dynamics in a nectar analog

abstract:
Floral nectar is frequently colonized by fungi and bacteria. However, within individual flowers, nectar microbial communities are typically species-poor and dominated by few cosmopolitan genera. One hypothesis is that nectar constituents may act as a strong environmental filter. Non-sugar constituents in nectar could affect species composition via broad antimicrobial activity or differential effects on nectar microbial species. Here, we tested how five non-sugar nectar compounds as well as elevated sugar impacted the growth of 12 fungal and bacterial species isolated from flowers, pollinators, and the environment. We hypothesized that microbes isolated from nectar would be better able to grow in the presence of these compounds. Additionally, to test if nectar compounds could affect the outcome of competition among microbial taxa, we grew a subset of microbes in co-culture assays across a subset of treatments.

We found that some compounds such as H2O2 broadly suppressed microbial growth across many but not all microbes tested. Other tested compounds were more specialized in the microbes they impacted. As hypothesized, the nectar specialist Metschnikowia reukaufii was unaffected by most nectar compounds assayed. However, many non-nectar specialist microbes remained unaffected by compounds thought to reduce microbial growth in nectar. Our results show that nectar chemistry can influence nectar microbial communities but that microbe-specific responses to nectar compounds are common. Nectar chemistry also affected the outcome of species interactions among microbial taxa, suggesting that non-sugar compounds in nectar can affect microbial community assembly and abundance in flowers.


title: Superfast Evolution of Multi-drug Antibiotic Resistance

abstract: In the presence of antibiotics, SOS response supports bacteria survival by activating the DNA repair system. Here, we find that the reduction of the SOS response by deletion of its master regulator recA can cause an even superfast establishment of antibiotic resistance (20-fold MIC) in Escherichia coli, which only takes 8 hours after a single exposure to ampicillin. And the gene acrB mutations were observed with a multi-drug resistance to other classes of antibiotics. This process is accompanied with the rapidly occurring DNA mutations, but orthogonal to the SOS response.


title: The outer mitochondrial membrane protein TMEM11 is a novel negative regulator of BNIP3/BNIP3L-dependent receptor-mediated mitophagy

abstract: Mitochondria play critical roles in cellular metabolism and to maintain their integrity, they are regulated by several quality control pathways, including mitophagy. During BNIP3/BNIP3L-dependent receptor-mediated mitophagy, mitochondria are selectively degraded by the direct recruitment of the autophagosome biogenesis protein LC3. BNIP3 and/or BNIP3L are upregulated situationally, for example during hypoxia and developmentally during erythrocyte maturation. However, it is not well understood how they are regulated at steady-state. Here, we find that the poorly characterized mitochondrial cristae morphology regulator TMEM11 unexpectedly localizes to the outer membrane where it forms a complex with BNIP3 and BNIP3L. Loss of TMEM11 causes mitochondrial morphology defects in a BNIP3/BNIP3L-dependent manner and, further, we find that mitophagy is hyper-active in the absence of TMEM11 during both normoxia and hypoxia. Our results reveal a non-canonical role for TMEM11 as a negative regulator of BNIP3/BNIP3L-mediated mitophagy and suggest that the TMEM11/BNIP3/BNIP3L complex coordinately regulates mitochondrial quality control.


title: Discovery of a druggable copper-signaling pathway that drives cell plasticity and inflammation

abstract: Inflammation is a complex physiological process triggered in response to harmful stimuli. It involves specialized cells of the immune system able to clear sources of cell injury and damaged tissues to promote repair. Excessive inflammation can occur as a result of infections and is a hallmark of several diseases. The molecular basis underlying inflammatory responses are not fully understood. Here, we show that the cell surface marker CD44, which characterizes activated immune cells, acts as a metal transporter that promotes copper uptake. We identified a chemically reactive pool of copper(II) in mitochondria of inflammatory macrophages that catalyzes NAD(H) redox cycling by activating hydrogen peroxide. Maintenance of NAD+ enables metabolic and epigenetic programming towards the inflammatory state. Targeting mitochondrial copper(II) with a rationally-designed dimer of metformin triggers distinct metabolic and epigenetic states that oppose macrophage activation. This drug reduces inflammation in mouse models of bacterial and viral (SARS-CoV-2) infections, improves well-being and increases survival. Identifying mechanisms that regulate the plasticity of immune cells provides the means to develop next-generation medicine. Our work illuminates the central role of copper as a regulator of cell plasticity and unveils a new therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.


title: Subcellular mapping of the protein landscape of SARS-CoV-2 infected cells for target-centric drug repurposing

abstract: The COVID-19 pandemic has resulted in millions of deaths and affected socioeconomic structure worldwide and the search for new antivirals and treatments are still ongoing. In the search for new drug target and to increase our understanding of the disease, we used large scale immunofluorescence to explore the host cell response to SARS-CoV-2 infection. Among the 602 host proteins studied in this host response screen, changes in abundance and subcellular localization were observed for 97 proteins, with 45 proteins showing increased abundance and 10 reduced abundances. 20 proteins displayed changed localization upon infection and an additional 22 proteins displayed altered abundance and localization, together contributing to diverse reshuffling of the host cell protein landscape. We then selected existing and approved small-molecule drugs (n =123) against our identified host response proteins and identified 3 compounds - elesclomol, crizotinib and rimcazole, that significantly reduced antiviral activity. Our study introduces a novel, targeted and systematic approach based on host protein profiling, to identify new targets for drug repurposing. The dataset of [~]75,000 immunofluorescence images from this study are published as a resource available for further studies.


title: A multidisciplinary analysis of the as-isolated Escherichia coli SufBC2D complex reveals the presence of two new iron-sulfur clusters

abstract: Iron-sulfur (Fe-S) clusters are essential inorganic cofactors dedicated to a wide range of biological functions including electron transfer and catalysis. Specialized multi-protein machineries present in all types of organisms support their biosynthesis. These machineries encompass a scaffold protein on which Fe-S clusters are assembled before being transferred to cellular targets. Here, we describe the first characterization of the native Fe-S cluster of the anaerobically purified SufBC2D scaffold from Escherichia coli by XAS, Mossbauer, UV-visible absorption and EPR spectroscopy. Interestingly, we propose that SufBC2D harbors two types of Fe-S cluster, a [2Fe-2S] cluster with an unprecedented usual coordination and a previously unreported [3Fe-3S] cluster. These data combined with mutagenesis and biochemistry allow to propose ligands for these clusters. These results support the hypothesis that both SufB and SufD are involved in Fe-S cluster ligation and are discussed in the context of Fe-S cluster biogenesis where both [2Fe-2S] and [4Fe-4S] clusters need to mature cellular Fe-S protein targets.

category: biochemistry


title: Predicting Protein Function and Orientation on a Gold Nanoparticle Surface Using a Residue-Based Affinity Scale

abstract: The orientation adopted by proteins on nanoparticle surfaces determines the nanoparticles bioactivity and its interactions with living systems. Here, we present a residue-based affinity scale for predicting protein orientation on citrate-gold nanoparticles (AuNPs). Competitive binding between protein variants accounts for thermodynamic and kinetic aspects of adsorption in this scale. For hydrophobic residues, the steric considerations dominate, whereas electrostatic interactions are critical for hydrophilic residues. The scale rationalizes the well-defined binding orientation of the small GB3 protein, and it subsequently predicts the orientation and active site accessibility of two enzymes on AuNPs. Additionally, our approach accounts for the AuNP-bound activity of five out of six additional enzymes from the literature. The model developed here enables high-throughput predictions of protein behavior on nanoparticles, and it enhances our understanding of protein orientation in the biomolecular corona, which should greatly enhance the performance and safety of nanomedicines used in vivo.


title: Reactive oxygen species generated by infrared laser light in optical tweezers inhibits the germination of bacterial spores

abstract: Bacterial spores are highly resistant to heat, radiation, and various disinfection chemicals. These impacts on the biophysical and physicochemical properties of spores can be studied on the single-cell level using optical tweezers. However, the effect of the trapping laser on spores germination rate is not fully understood. In this work, we assess the impact of 1064 nm laser light on the germination of Bacillus thuringiensis spores. The results show that the germination rate of spores after laser exposure follows a sigmoid dose-response relationship, with only 15 % of spores germinating after 20 J of laser light. Under anaerobic growth conditions, the percentage of germinating spores at 20 J increased to 65 %. The results thereby indicate that molecular oxygen is a major contributor to the germination-inhibiting effect observed. Thus, our study highlights the risk for optical trapping of spores and ways to mitigate it.


title: Inositol hexakisphosphate (IP6) accelerates immature HIV-1 Gag protein assembly towards kinetically-trapped morphologies

abstract: During the late stages of the HIV-1 lifecycle, immature virions are produced by the concerted activity of Gag polyproteins, primarily mediated by the capsid (CA) and spacer peptide 1 (SP1) domains, which assemble into a spherical lattice, package viral genomic RNA, and deform the plasma membrane. Recently, inositol hexakisphosphate (IP6) has been identified as an essential assembly cofactor that efficiently produces both immature virions in vivo and immature virus-like particles in vitro. To date, however, several distinct mechanistic roles for IP6 have been proposed on the basis of independent functional, structural, and kinetic studies. In this work, we investigate the molecular influence of IP6 on the structural outcomes and dynamics of CA/SP1 assembly using coarse-grained (CG) molecular dynamics (MD) simulations and free energy calculations. Here, we derive a bottom-up, low-resolution, and implicit-solvent CG model of CA/SP1 and IP6, and simulate their assembly under conditions that emulate both in vitro and in vivo systems. Our analysis identifies IP6 as an assembly accelerant that promotes curvature generation and fissure-like defects throughout the lattice. Our findings suggest that IP6 induces kinetically-trapped immature morphologies, which may be physiologically important for later stages of viral morphogenesis and potentially useful for virus-like particle technologies.


title: Base editing screens map mutations affecting IFNγ signalling in cancer

abstract: IFN{gamma}signalling underpins host responses to infection, inflammation and anti-tumour immunity. Mutations in the IFN{gamma}signalling pathway cause immunological disorders, haematological malignancies, and resistance to immune checkpoint blockade (ICB) in cancer, however the function of most clinically observed variants remain unknown. Here, we systematically investigate the genetic determinants of IFN{gamma}response in colorectal cancer cells using CRISPR-Cas9 screens and base editing mutagenesis. Deep mutagenesis of JAK1 with cytidine and adenine base editors, combined with pathway-wide screens, reveal loss-of-function and gain-of-function mutations with clinical precedence, including causal variants in haematological malignancies and mutations detected in patients refractory to ICB. We functionally validate variants of uncertain significance in primary tumour organoids, where engineering missense mutations in JAK1 enhanced or reduced sensitivity to autologous tumour-reactive T cells. By classifying > 300 missense variants altering IFN{gamma} pathway activity, we demonstrate the utility of base editing for mutagenesis at scale, and generate a resource to inform genetic diagnosis.


title: spongEffects: ceRNA modules offer patient-specific insights into the miRNA regulatory landscape

abstract:
MotivationCancer is one of the leading causes of death worldwide. Despite significant improvements in prevention and treatment, mortality remains high for many cancer types. Hence, innovative methods that use molecular data to stratify patients and identify biomarkers are needed. Promising biomarkers can also be inferred from competing endogenous RNA (ceRNA) networks that capture the gene-miRNA-gene regulatory landscape. Thus far, the role of these biomarkers could only be studied globally but not in a sample-specific manner. To mitigate this, we introduce spongEffects, a novel method that infers subnetworks (or modules) from ceRNA networks and calculates patient- or sample-specific scores related to their regulatory activity.

ResultsWe show how spongEffects can be used for downstream interpretation and machine learning tasks such as tumor classification and for identifying subtype-specific regulatory interactions. In a concrete example of breast cancer subtype classification, we prioritize modules impacting the biology of the different subtypes. In summary, spongEffects prioritizes ceRNA modules as biomarkers and offers insights into the miRNA regulatory landscape. Notably, these module scores can be inferred from gene expression data alone and can thus be applied to cohorts where miRNA expression information is lacking.

Availabilityhttps://bioconductor.org/packages/devel/bioc/html/SPONGE.html spongEffects scores calculated for TCGA datasets: https://doi.org/10.6084/m9.figshare.19328885.v1

Contactmarkus.list@tum.de

Supplementary informationSupplementary data are available at Bioinformatics online.

category: systems biology


title: Glia-neuron coupling via a bipartite sialylation pathway promotes neural transmission and stress tolerance in Drosophila

abstract:
Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila, CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. We found that these genes function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. Our results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance.

Graphical Abstract

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title: Neuron-specific transcriptomic signatures indicate neuroinflammation and altered neuronal activity in ASD temporal cortex

abstract: Autism spectrum disorder (ASD) is a highly heterogeneous disorder, yet transcriptomic profiling of bulk brain tissue has identified substantial convergence among dysregulated genes and pathways in ASD. However, this approach lacks cell-specific resolution. We performed comprehensive transcriptomic analyses on bulk tissue and laser-capture microdissected (LCM) neurons of 59 postmortem human brains (27 ASD and 32 matched controls) in the superior temporal gyrus (STG) ranging from 2-73 years of age. In bulk tissue, synaptic signaling, heat shock protein-related pathways and RNA splicing were significantly altered in ASD. There was age-dependent dysregulation of genes involved in GABA (GAD1 and GAD2) and glutamate (SLC38A1) signaling pathways. In LCM neurons, AP-1 mediated neuroinflammation and insulin/IGF-1 signaling pathways were upregulated in ASD, while mitochondrial function, ribosome and spliceosome components were downregulated. GABA synthesizing enzymes GAD1 and GAD2 were both downregulated in ASD neurons. Alterations in small nucleolar RNAs (snoRNAs) associated with splicing events suggested interplay between snoRNA dysregulation and splicing disruption in neurons of individuals with ASD. Our findings supported the fundamental hypothesis of altered neuronal communication in ASD, demonstrated that inflammation was elevated at least in part in ASD neurons, and may reveal windows of opportunity for biotherapeutics to target the trajectory of gene expression and clinical manifestation of ASD throughout the human lifespan.


title: Convergent epitope specificities, V gene usage and public clones elicited by primary exposure to SARS-CoV-2 variants

abstract:
While humoral immune responses to infection or vaccination with ancestral SARS-CoV-2 have been well-characterized, responses elicited by infection with variants are less understood. Here we characterized the repertoire, epitope specificity, and cross-reactivity of antibodies elicited by Beta and Gamma variant infection compared to ancestral virus. We developed a high-throughput approach to obtain single-cell immunoglobulin sequences and isolate monoclonal antibodies for functional assessment. Spike-, RBD- and NTD-specific antibodies elicited by Beta- or Gamma-infection exhibited a remarkably similar hierarchy of epitope immunodominance for RBD and convergent V gene usage when compared to ancestral virus infection. Additionally, similar public B cell clones were elicited regardless of infecting variant. These convergent responses may account for the broad cross-reactivity and continued efficacy of vaccines based on a single ancestral variant.

One Sentence SummaryWA1, Beta and Gamma variants of SARS-CoV-2 all elicit antibody responses targeting similar RBD epitopes; public and cross-reactive clones are common.


title: Phylogenetic analysis of the origin and spread of plague in Madagascar

abstract:
BackgroundPlague is a zoonotic disease caused by the bacterium Yersinia pestis, highly prevalent in the Central Highlands, a mountainous region in the center of Madagascar. After a plague-free period of over 60 years in the northwestern coast city of Mahajanga, the disease reappeared in 1991 and caused several outbreaks until 1999. Previous research indicates that the disease was reintroduced to the city of Mahajanga from the Central Highlands instead of reemerging from a local reservoir. However, it is not clear how many reintroductions occurred and when they took place.

Methodology/Principal findingsIn this study we applied a Bayesian phylogeographic model to detect and date migrations of Y. pestis between the two locations that could be linked to the re-emergence of plague in Mahajanga. Genome sequences of 300 Y. pestis strains sampled between 1964 and 2012 were analyzed. Four migrations from the Central Highlands to Mahajanga were detected. Two resulted in persistent transmission in humans, one was responsible for most of the human cases recorded between 1995 and 1999, while the other produced plague cases in 1991 and 1992. We dated the emergence of the Y. pestis sub-branch 1.ORI3, which is only present in Madagascar and Turkey, to the beginning of the 20th century, using a Bayesian molecular dating analysis. The split between 1.ORI3 and its ancestor lineage 1.ORI2 was dated to the second half of the 19th century.

Conclusions/SignificanceOur results indicate that two independent migrations from the Central Highlands caused the plague outbreaks in Mahajanga during the 1990s, with both introductions occurring during the early 1980s. They happened over a decade before the detection of human cases, thus the pathogen likely survived in wild reservoirs until the spillover to humans was possible. This study demonstrates the value of Bayesian phylogenetics in elucidating the re-emergence of infectious diseases.

Author summaryIn 1991 human cases of plague were reported in the city of Mahajanga, located in the west coast of Madagascar, after 60 years without human cases. Existing evidence suggests that Yersinia pestis, the causal agent of the disease, was reintroduced to the city from a mountainous region known as the Central Highlands. We performed a phylogeographic analysis on 300 Y. pestis genome sequences to determine how many migrations of the pathogen between the two locations were related to the reappearance of the disease in Mahajanga. The results revealed that two migrations from the Central Highlands were the cause of the outbreaks of plague in the west coast of the country during the 1990s. We also aimed to date the emergence of the Y.pestis variant that circulates in Madagascar and is also present in Turkey. To do this, we conducted a molecular dating analysis using an extended data set of 445 sequences, which contained sequences from Turkey and India (the country from which the pathogen was exported to Madagascar for the first time). The analysis indicated that this particular variant emerged in the first decade of the 20th century.


title: Characterization of the growth and morphology of a BSL-2 Coccidioides posadasii strain that persists in the parasitic life cycle at ambient CO2.

abstract: Coccidioides is a dimorphic fungus responsible for Valley Fever and is the cause of severe morbidity and mortality in the infected population. Although there is some insight into the genes, pathways, and growth media involved in the parasitic to saprophytic growth transition, the exact determinants that govern this switch are largely unknown. In this work, we examined the growth and morphology of a novel Coccidioides posadasii strain (C. posadasii S/E) that efficiently produces spherules and endospores and persists in the parasitic life cycle at ambient CO2. We demonstrated that C. posadasii S/E remains virulent in an insect infection model. Surprisingly, under spherule-inducing conditions, C. posadasii S/E culture was found to be completely hyphal. Differential interference contrast (DIC) and transmission electron microscopy (TEM) revealed unexpected cellular changes in this strain including cell wall remodeling and formation of septal pores with Woronin bodies. Our study suggests that the C. posadasii S/E strain is a useful BSL-2 model for studying the molecular factors that are active during the parasitic life cycle and the mechanisms underlying the parasitic to saprophytic growth transition - a morphological switch that can impact the pathogenicity of the organism in the host.


title: PinR as a Stress Inducible Inhibitory Transcription Factor in Stopping Stress-induced Mutagenesis Bacterial Evolution of Antibiotic Resistance

abstract: PinR is a lambdoid prophage Rac existing in many different bacteria, but its function is yet to be discovered. Here, we report PinR as an effective "anti-evolution" target towards combating antibiotic resistant bacteria. We show the function of PinR as a stress inducible inhibitory transcription factor in the bacterial evolution of antibiotic resistance, which represses the transcriptions of many oxidative stress response genes and consequently orchestrate the ROS burst-induced evolution of antibiotic resistance. We show that deletion of pinR gene can avoid the accelerated evolution of antibiotic resistance induced by the deficiency of DNA repair, while the strategies to target RecA, e.g., the deletion of recA gene, can be safely implemented to disarm the bacterial resistances to a variety of antibiotics.


title: Tuberculosis treatment failure associated with evolution of antibiotic resilience

abstract: Antibiotics are a cornerstone of medicine, placing bacterial pathogens under intense pressure to evolve new survival mechanisms. Analysis of 51,229 Mycobacterium tuberculosis (Mtb) clinical isolates identified an essential transcriptional regulator, Rv1830 (here named resR) as a frequent target of positive (adaptive) selection. resR mutants do not demonstrate canonical drug resistance or drug tolerance but instead have significantly faster recovery after drug treatment across all antibiotics and combinations tested, a phenotype which we term antibiotic resilience. ResR acts in a regulatory cascade with other growth-controlling transcriptional regulators WhiB2 and WhiA, which are also under positive selection in Mtb clinical isolates. Mutations of these genes are associated with treatment failure and the acquisition of canonical drug resistance.


title: DNA methylation in human gastric epithelial cells allows cell type-related plasticity and defines regional identity

abstract:
BackgroundEpigenetic modifications in mammalian DNA are commonly manifested by DNA methylation. In the stomach, altered DNA methylation patterns have been observed following chronic Helicobacter pylori infections and in gastric cancer. In the context of epigenetic regulation, the regional nature of the stomach has been rarely considered in detail.

ResultsHere, we describe the DNA methylation landscape across the phenotypically different regions of the healthy human stomach (i.e., antrum, corpus, fundus) together with the corresponding transcriptomes. We show that stable regional DNA methylation differences translate to a limited extent into regulation of the transcriptomic phenotype, indicating a largely permissive epigenetic regulation. We identify a small number of transcription factors with novel region-specific activity and likely epigenetic impact in the stomach, including GATA4, IRX5, IRX2, PDX1, and CDX2. Detailed analysis of the Wnt pathway reveals differential regulation along the craniocaudal axis, which involves non-canonical Wnt signaling in determining cell fate in the proximal stomach.

ConclusionsBy extending our analysis to pre-neoplastic lesions and gastric cancers, we conclude that epigenetic dysregulation already characterizes intestinal metaplasia as a founding basis for functional changes in gastric cancer. Finally, our study provides a well-defined resource of regional stomach transcription and epigenetics as a starting point for further studies.


title: Regulation of EBNA1 Protein Stability by PLOD1 Lysine Hydroxylase

abstract:
Epstein-Barr virus (EBV) is a ubiquitous human {gamma}-herpesvirus that is causally associated with various malignancies and autoimmune disease. Epstein-Barr Nuclear Antigen 1 (EBNA1) is the viral-encoded DNA binding protein required for viral episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 is known to be a highly stable protein, but its mechanism of protein stability is not completely understood. Proteomic analysis of EBNA1 revealed interaction with Procollagen Lysine-2 Oxoglutarate 5 Dioxygenase (PLOD) family of proteins. Depletion of PLOD1 by shRNA or inhibition with small molecule inhibitors 2,-2 dipyridyl resulted in the loss of EBNA1 protein levels, along with a selective growth inhibition of EBV-positive lymphoid cells. PLOD1 depletion also caused a loss of EBV episomes from latently infected cells and inhibited oriP-dependent DNA replication. We used mass spectrometry to identify EBNA1 peptides with lysine hydroxylation at K460 or K461. Mutation of K460 to alanine or arginine abrogates EBNA1-driven DNA replication of oriP, while K461 mutations enhanced replication. These findings suggest that PLOD1 is a novel post-translational regulator of EBNA1 protein stability and function in viral plasmid replication, episome maintenance and host cell survival.

ImportanceEBNA1 is essential for EBV latent infection and implicated in viral pathogenesis. We found that EBNA1 interacts with PLOD family of lysine hydroxylases and that this interaction is required for EBNA1 protein stability and function in viral persistence during viral latent infection. Identification of PLOD1 regulation of EBNA1 protein stability provide new opportunity to target EBNA1 for degradation in EBV associated disease.


title: Structural basis of actin filament assembly and aging

abstract: The dynamic turnover of actin filaments (F-actin) controls cellular motility in eukaryotes and is coupled to changes in the F-actin nucleotide state. It remains unclear how F-actin hydrolyzes ATP and subsequently undergoes subtle conformational rearrangements that ultimately lead to filament depolymerization by actin-binding proteins. Here, we present cryo-EM structures of F-actin in all nucleotide states, polymerized in the presence of Mg2+ or Ca2+, at resolutions ([~]2.2 [A]) that allow for the visualization of hundreds of water molecules. The structures reveal that the G- to F-actin transition induces the relocation of water molecules in the nucleotide binding pocket, activating one of them for the nucleophilic attack of ATP. Unexpectedly, the back door for the subsequent release of inorganic phosphate (Pi) is closed in all structures, indicating that the F-actin conformation that allows for Pi release occurs transiently. The small changes in the nucleotide-binding pocket after ATP hydrolysis and Pi release are sensed by a key amino acid, amplified and transmitted to the filament periphery. Furthermore, differences in the positions of waters in the nucleotide binding pocket explain why Ca2+-actin exhibits slower polymerization rates than Mg2+-actin. Our work elucidates the solvent-driven rearrangements that govern actin filament assembly and aging and lays the foundation for the rational design of drugs and small molecules for imaging and therapeutic applications.


title: Driving Forces underlying Selectivity Filter Gating in the MthK Potassium Channel

abstract: K+ channel activity can be limited by C-type inactivation, which is likely initiated in part by dissociation of K+ ions from the selectivity filter, and modulated by side chains surrounding the selectivity filter. Whereas crystallographic and computational studies have linked inactivation to a "collapsed" selectivity filter conformation in the KcsA channel, the structural basis for selectivity filter gating in other K+ channels has been less clear. Here, we combined electrophysiological recordings with molecular dynamics based, in silico electrophysiology simulations to study selectivity filter gating in the model potassium channel MthK and the MthK mutant V55E (analogous to KcsA E71) in the pore-helix. We find that MthK V55E has lower open probability than the WT channel, due to decreased stability of the open state. Simulations account for aspects of these observations on the atomistic scale, showing that ion permeation in V55E is differentially altered in two distinct orientations of the E55 side chain. In the  vertical orientation of E55, in which E55 forms a hydrogen bond with D64 (as observed with KcsA WT channels), the filter displays reduced conductance compared to MthK WT. In contrast, with  horizontal orientation, K+ conductance is similar to MthK WT; however the filter is less stable in the conductive conformation. Surprisingly, transitions of MthK WT and V55E channels to the non-conducting (inactivated) state observed in simulations are associated with a widening selectivity filter, unlike the narrowing selectivity filter observed with KcsA, and similar to recent structures of stably-inactivated Shaker W434F and Kv1.2 W362F mutants.


title: Repeated ethanol exposure and withdrawal alters ACE2 expression in discrete brain regions: Implications for SARS-CoV-2 infection

abstract: Emerging evidence suggests that people with alcohol use disorders are at higher risk for SARS-CoV-2. SARS-CoV-2 engages angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) receptors for cellular entry. While ACE2 and TMPRSS2 genes are upregulated in the cortex of alcohol-dependent individuals, information on expression in specific brain regions and neural populations implicated in SARS-CoV-2 neuroinvasion, particularly monoaminergic neurons, is limited. We sought to clarify how chronic alcohol exposure affects ACE2 and TMPRSS2 expression in monoaminergic brainstem circuits and other putative SARS-CoV-2 entry points. C57BL/6J mice were exposed to chronic intermittent ethanol (CIE) vapor for 4 weeks and brains were examined using immunofluorescence. We observed increased ACE2 levels in the olfactory bulb and hypothalamus following CIE, which are known to mediate SARS-CoV-2 neuroinvasion. Total ACE2 immunoreactivity was also elevated in the raphe magnus (RMG), raphe obscurus (ROB), and locus coeruleus (LC), while in the dorsal raphe nucleus (DRN), ROB, and LC we observed increased colocalization of ACE2 with monoaminergic neurons. ACE2 also increased in the periaqueductal gray (PAG) and decreased in the amygdala. Whereas ACE2 was detected in most brain regions, TMPRSS2 was only detected in the olfactory bulb and DRN but was not significantly altered after CIE. Our results suggest that previous alcohol exposure may increase the risk of SARS-CoV-2 neuroinvasion and render brain circuits involved in cardiovascular and respiratory function as well as emotional processing more vulnerable to infection, making adverse outcomes more likely. Additional studies are needed to define a direct link between alcohol use and COVID-19 infection.


title: CD169-mediated restrictive SARS-CoV-2 infection of macrophages induces pro-inflammatory responses

abstract:
Exacerbated and persistent innate immune response marked by pro-inflammatory cytokine expression is thought to be a major driver of chronic COVID-19 pathology. Although macrophages are not the primary target cells of SARS-CoV-2 infection in humans, viral RNA and antigens in activated monocytes and macrophages have been detected in post-mortem samples, and dysfunctional monocytes and macrophages have been hypothesized to contribute to a protracted hyper-inflammatory state in COVID-19 patients. In this study, we demonstrate that CD169, a myeloid cell specific I-type lectin, facilitated ACE2-independent SARS-CoV-2 fusion and entry in macrophages. CD169- mediated SARS-CoV-2 entry in macrophages resulted in expression of viral genomic and sub-genomic (sg) RNAs with minimal viral protein expression and no infectious viral particle release, suggesting a post-entry restriction of the SARS-CoV-2 replication cycle. Intriguingly this post-entry replication block was alleviated by exogenous ACE2 expression in macrophages. Restricted expression of viral gRNA and sgRNA in CD169+ macrophages elicited a pro-inflammatory cytokine expression (TNF, IL-6 and IL-1{beta}) in a RIG-I, MDA-5 and MAVS-dependent manner, which was suppressed by remdesivir pre- treatment. These findings suggest that de novo expression of SARS-CoV-2 RNA in macrophages contributes to the pro-inflammatory cytokine signature and that blocking CD169-mediated ACE2 independent infection and subsequent activation of macrophages by viral RNA might alleviate COVID-19-associated hyperinflammatory response.

Author SummaryOver-exuberant production of pro-inflammatory cytokine expression by macrophages has been hypothesized to contribute to severity of COVID-19 disease. Molecular mechanisms that contribute to macrophage-intrinsic immune activation during SARS- CoV-2 infection are not fully understood. Here we show that CD169, a macrophage- specific sialic-acid binding lectin, facilitates abortive SARS-CoV-2 infection of macrophages that results in innate immune sensing of viral replication intermediates and production of proinflammatory responses. We identify an ACE2-independent, CD169- mediated endosomal viral entry mechanism that results in cytoplasmic delivery of viral capsids and initiation of virus replication, but absence of infectious viral production. Restricted viral replication in CD169+ macrophages and detection of viral genomic and sub-genomic RNAs by cytoplasmic RIG-I-like receptor family members, RIG-I and MDA5, and initiation of downstream signaling via the adaptor protein MAVS, was required for innate immune activation. These studies uncover mechanisms important for initiation of innate immune sensing of SARS-CoV-2 infection in macrophages, persistent activation of which might contribute to severe COVID-19 pathophysiology.


title: ZccE is a Novel P-type ATPase That Protects Streptococcus mutans Against Zinc Intoxication

abstract:
Zinc is a trace metal that is essential to all forms of life, but that becomes toxic at high concentrations. Because it has both antimicrobial and anti-inflammatory properties and low toxicity to mammalian cells, zinc has been used as a therapeutic agent for centuries to treat a variety of infectious and non-infectious conditions. While the usefulness of zinc-based therapies in caries prevention is controversial, zinc is incorporated into toothpaste and mouthwash formulations to prevent gingivitis and halitosis. Despite this widespread use of zinc in oral healthcare, the mechanisms that allow Streptococcus mutans, a keystone pathogen in dental caries and prevalent etiological agent of infective endocarditis, to overcome zinc toxicity are largely unknown. Here, we discovered that S. mutans is inherently more tolerant to high zinc stress than all other species of streptococci tested, including commensal streptococci associated with oral health. Using a transcriptome approach, we uncovered several potential strategies utilized by S. mutans to overcome zinc toxicity. Among them, we identified a previously uncharacterized P-type ATPase transporter and cognate transcriptional regulator, which we named ZccE and ZccR respectively, as responsible for the remarkable high zinc tolerance of S. mutans. In addition to zinc, we found that ZccE, which was found to be unique to S. mutans strains, mediates tolerance to at least three additional metal ions, namely cadmium, cobalt, and copper. Loss of the ability to maintain zinc homeostasis when exposed to high zinc stress severely disturbed Zinc:Manganese ratios, leading to heightened peroxide sensitivity that was alleviated by manganese supplementation. Finally, we showed that the ability of the {Delta}zccE strain to stably colonize the rat tooth surface after topical zinc treatment was significantly impaired, providing proof of concept that ZccE and ZccR are suitable targets for the development of antimicrobial therapies specifically tailored to kill S. mutans.

Author summaryDental caries is an overlooked infectious disease affecting more than 50% of the adult population. While several bacteria that reside in dental plaque have been associated with caries development and progression, Streptococcus mutans is deemed a keystone caries pathogen due to its capacity to modify the dental plaque environment in a way that is conducive with disease development. Zinc is an essential trace metal to life but very toxic when encountered at high concentrations, to the point that it has been used as an antimicrobial for centuries. Despite the widespread use of zinc in oral healthcare products, little is known about the mechanisms utilized by oral bacteria to overcome its toxic effects. In this study, we discovered that S. mutans can tolerate exposure to much higher levels of zinc than closely related streptococcal species, including species that antagonize S. mutans and are associated with oral health. In this study, we identified a new metal transporter, named ZccE, as directly responsible for the inherently high zinc tolerance of S. mutans. Because ZccE is not present in other bacteria, our findings provide a new target for the development of a zinc-based therapy specifically tailored to kill S. mutans.


title: Dynamic de novo adipose tissue development during metamorphosis in Drosophila melanogaster

abstract:
Adipose tissue is a central organ for controlling systemic metabolism both in invertebrates and vertebrates. Here, we have investigated the cellular mechanisms of the adult-type fat body (AFB) development in Drosophila. We have established genetic tools that allow visualization and genetic manipulations of cells in the AFB lineage from early in metamorphosis. We identified precursor cells that give rise to the AFB and delineated dynamic cellular mechanisms underlying AFB formation. These precursor cells displayed polarized cell shapes and oriented motility, with emigration from the thorax and subsequent dispersal to the abdomen and head. After the migration period, these cells adhered to each other, assembling into the AFB with a sheet-like architecture. Continuous cell proliferation occurred during and after the large-scale migration to achieve appropriate fat tissue mass. Homotypic cell fusion after the sheet formation contributed to the establishment of multinucleated cells in the AFB. We also examined candidate gene functions, and our results argue that Rac1, ecdysone signaling, and the transcription factor Serpent support adult fat body organogenesis.

Brief Summary StatementDrosophila adult fat body precursor cells form adult adipose tissue during metamorphosis by directional migration, continuous cell proliferation, and homotypic cell fusion.


title: The addition of antibiotics to embryo culture media caused altered expression of genes in pathways governing DNA integrity in mouse blastocysts

abstract: Antibiotics are common components of embryo culture media and minimize the risk of microbial contamination and infection during assisted reproductive technology procedures (ART). The effects of two aminoglycoside antibiotics (gentamicin, streptomycin) and penicillin on the global profiles of gene expression (DE) were assessed by RNA-seq of individual mouse blastocysts. Zygotes were cultured in an optimized defined medium formulation (KSOM) to which a dose range of each antibiotic was added. A dose-dependent retardation of the rate of zygote development to morphologically normal blastocyst was observed and this was accompanied by a reduction in the number of cells present within the resulting blastocysts. The lowest dose of each antibiotic tested (similar to the concentrations used in clinical grade media) caused significant differential expression of approximately 1800 genes. In most cases antibiotic treatment caused a reduction in gene expression and gene ontology analysis showed that down regulated genes were enriched for several biological processes related to the maintenance of genomic integrity. All three antibiotics caused the downregulation of Brca2, Blm, Rad51c and Rad54l, genes involved DNA homologous recombination pathways and also several p53-dependent genes. Immunolocalization studies showed that each antibiotic also reduced level of BRCA2 and RAD51C detected within blastocysts. The present study shows that the supplementing embryo culture media with antibiotics is associated with wide ranging alterations in gene expression in a manner that could potentially compromise the genomic integrity of the resulting embryos.


title: Lack of universal mutational biases in a fungal phylum

abstract: Mutations fuel the diversity of life forms on earth through changes of nucleotides in DNA sequence. Patterns of mutations are biased; for example, mutational biases toward adenine and thymine have been previously noted in bacteria and biases for transitions over transversions are observed in diverse groups of organisms. However, the mutational biases in fungi, whose genomes vary widely in their GC content, remain poorly understood. Here, we characterized patterns of single nucleotide polymorphisms among 537 strains from 30 species and four classes from Ascomycota, the most species-rich fungal phylum. We found that mutational biases vary across Ascomycota; for example, some species in the class Saccharomycetes, in particular the bipolar budding yeast Hanseniaspora uvarum and the emerging pathogen Candida auris, show strong mutational bias toward A|T substitutions whereas the black mold Stachybotrys chartarum in the class Sordariomycetes shows substantial mutational bias toward G|C substitutions. Examination of GC content and GC equilibrium content, a measure that represents the GC content under selective neutrality and accounts for rates of G|C > A|T and A|T > G|C substitutions, revealed that fungal species vary in how their genome nucleotide composition is affected by neutral processes, mutational biases, and external evolutionary forces, such as selection. Whereas genome nucleotide composition is consistent with neutral expectations and is mainly driven by mutational bias in some species (e.g., Zymoseptoria tritici), the composition of others is influenced by both mutational bias and selection (e.g., H. uvarum and S. chartarum). These results highlight the variation of patterns of mutations across a fungal phylum and suggest that both neutral and selective processes shape the nucleotide composition of fungal genomes.

category: evolutionary biology


title: THRONCAT: Efficient metabolic labeling of newly synthesized proteins using a bioorthogonal threonine analog

abstract: Profiling the nascent cellular proteome and capturing early proteomic changes in response to external stimuli provides valuable insight into cellular physiology. Existing metabolic protein labeling approaches based on bioorthogonal methionine-or puromycin analogs allow for the selective visualization and enrichment of the newly synthesized proteins. However, their applications are limited as they require methionine-free conditions, auxotrophic cells and/or are toxic to cells. Here, we introduce THRONCAT, a novel threonine-derived non-canonical amino acid tagging method based on bioorthogonal threonine analog {beta}-ethynylserine ({beta}ES) that enables efficient and non-toxic labeling of the nascent proteome in complete growth media within minutes. We used THRONCAT for the visualization and enrichment of nascent proteins in bacteria, mammalian cells and Drosophila melanogaster. We profiled immediate proteome dynamics of Ramos B-cells in response to receptor activation, demonstrating the ease-of-use of the method and its potential to address diverse biological questions. In addition, using a Drosophila model of Charcot-Marie-Tooth peripheral neuropathy, we show that THRONCAT enables visualization and quantification of relative protein synthesis rates in vivo.


title: Evolution of decreased aggression in honey bees targeted brain cell gene regulatory networks

abstract:
A major challenge in biology is to understand how inherited variation results in phenotypic differences, especially for complex polygenic traits like behavior. Integrating genetic variants associated with evolutionary differences in honey bee aggression with genes identified via brain single-cell (sc) transcriptomics and sc gene regulatory network (GRN) modeling, we achieved robust genotype-to-phenotype mapping. We identified genetic differences in GRN structure associated with evolutionary differences in aggression, located in specific brain cell populations related to olfaction and vision. Some involve serotonin, which already was known to influence bee aggression, but not yet from a genetic perspective. These results demonstrate that, as theory predicts, GRN analysis can integrate evolutionary and systems biology to identify molecular mechanisms underlying adaptive polygenic traits.

One Sentence SummaryGenetic variation affects behavioral variation by causing changes in brain cell gene regulatory networks.

category: genomics


title: TOR acts as metabolic gatekeeper for auxin-dependent lateral root initiation in Arabidopsis thaliana

abstract: Plants post-embryonic organogenesis requires matching the available metabolic resources to the developmental programs. The root system is determined by the formation of lateral roots (LR), which in Arabidopsis thaliana entails the auxin-induced activation of founder cells located in the pericycle. While the allocation of sugars to roots influences root branching, how sugar availability is sensed for auxin-triggered formation of LRs remains unknown. Here, we combine metabolic profiling with cell-specific genetic interference to show that LR formation is an important sink for carbohydrate accompanied by a switch to glycolysis. We show that the target-of-rapamycin (TOR) kinase is locally activated in the pericycle and the founder cells and that both chemical and genetic inhibition of TOR kinase lead to a block of LR initiation. TOR marginally affects the auxin-induced transcriptional response of the pericycle but modulates the translation of ARF19, ARF7 and LBD16, three key targets of auxin signalling. These data place TOR as a gatekeeper for post-embryonic LR formation that integrates local auxin-dependent pathways with systemic metabolic signals, modulating the translation of auxin induced gene expression.

category: plant biology


title: Xanthomonas infection transforms the apoplast into an accessible and habitable niche for Salmonella enterica

abstract:
The physiology of plant hosts can be dramatically altered by phytopathogens. Xanthomonas hortorum pv. gardneri is one such pathogen that creates an aqueous niche within the leaf apoplast by manipulating the plant via the transcription activator-like effector AvrHah1. Simultaneous immigration of X. gardneri and Salmonella enterica to healthy tomato leaves results in increased survival of S. enterica as Xanthomonas infection progresses. However, the fate of S. enterica following arrival on actively infected leaves has not been examined. We hypothesized that the water-soaking caused by X. gardneri could facilitate the ingression of S. enterica into the apoplast, and that this environment would be conducive for growth. We found that a water-soaked apoplast, abiotically or Xanthomonas-infected, enabled surface S. enterica to passively localize to the protective apoplast and facilitated migration of S. enterica to distal sites within the aqueous apoplast. AvrHah1 contributed to the protection and migration of S. enterica early in X. gardneri infection. Xanthomonas-infected apoplasts facilitated prolonged survival and promoted S. enterica replication compared to healthy apoplasts. Access to an aqueous apoplast in general protects S. enterica from immediate exposure to irradiation whereas, the altered environment created by Xanthomonas infection provides growth-conducive conditions for S. enterica. Overall, we have characterized an ecological relationship in which host infection converts an unreachable niche to a habitable environment.

ImportanceBacterial spot disease caused by Xanthomonas species devastates tomato production worldwide. Salmonellosis outbreaks from consumption of raw produce have been linked to the arrival of Salmonella enterica on crop plants in the field via contaminated irrigation water. Considering that Xanthomonas is difficult to eradicate, it is highly likely that S. enterica arrives on leaves pre-colonized by Xanthomonas with infection underway. Our study demonstrates that infection and disease fundamentally alter the leaf, resulting in redistribution and change in abundance of a phyllosphere bacterial member. These findings contribute to our understanding of how S. enterica manages to persist on leaf tissue despite lacking the ability to liberate nutrients from plant cells. More broadly, this study reveals a mechanism by which physiochemical changes to a host environment imposed by a plant pathogen can convert an uninhabitable leaf environment into a hospitable niche for select epiphytic microbes.

category: microbiology