title: The phytonutrient cinnamaldehyde limits intestinal inflammation and enteric parasite infection

abstract: Phytonutrients such as cinnamaldehyde (CA) have been studied for their effects on metabolic diseases, but their influence on mucosal inflammation and immunity to enteric infection are not well documented. Here, we show that consumption of CA significantly down-regulates transcriptional pathways connected to inflammation in the small intestine of mice. During infection with the enteric helminth Heligomosomoides polygyrus, CA-treated mice displayed higher growth rates and less worms, concomitant with altered T-cell populations in mesenteric lymph nodes. Furthermore, infection-induced changes in gene pathways connected to cell cycle and mitotic activity were counteracted by CA. Mechanically, CA did not appear to exert activity through a prebiotic effect, as CA treatment did not significantly change the composition of the gut microbiota. Instead, in vitro experiments showed that CA directly induced xenobiotic metabolizing pathways in intestinal epithelial cells and suppressed endotoxin-induced inflammatory responses in macrophages. Thus, CA down-regulates inflammatory pathways in the intestinal mucosa and regulates host responses to enteric infection. These properties appear to be largely independent of the gut microbiota and instead connected to CAs ability to induce antioxidant pathways in intestinal cells. Our results encourage further investigation into the use of CA and related phytonutrients as functional food components to promote intestinal health in humans and animals.


title: In vitro evaluation of the activity of terpenes and cannabidiol against Human Coronavirus E229

abstract: The activity of a new, terpene-based formulation, code-named NT-VRL-1, against Human Coronavirus (HCoV) strain 229E was evaluated in human lung fibroblasts (MRC-5 cells), with and without the addition of cannabidiol (CBD). The tested formulation exhibited an antiviral effect when it was pre-incubated with the host cells prior to virus infection. The combination of NT-VRL-1 with CBD potentiated the antiviral effect better than the positive controls pyrazofurin and glycyrrhizin. There was a strong correlation between the quantitative results from a cell-viability assay and the cytopathic effect seen under the microscope after 72 h. To the best of our knowledge, this is the first report of activity of a combination of terpenes and CBD against a coronavirus.

category: microbiology


title: Microbial composition, diversity, and activity varies across different types of basal ice

abstract:
Glaciers and ice sheets possess layers of basal ice characterized by high amounts of entrained debris that can serve as sources of nutrients and organic matter, providing habitat for microorganisms adapted to the frozen conditions. Basal ice forms through various mechanisms and is classified based on its ice and debris content; however, little is known about variation in microbial composition, diversity, and activity across different basal ice types. We investigated these parameters in four different types of basal ice from a cold-based and temperate glacier and used a meta-analysis to compare our findings with microbiome studies from other frozen environments. We found basal ice environments harbor a diverse range of microbiomes whose composition and activity can vary significantly between basal ice types, even within adjacent facies from the same glacier. In some debris-rich basal ices, elevated ATP concentrations, isotopic gas signatures, and high ratios of amplified sequences for 16S rRNA relative to that for 16S rRNA genes implicated certain bacterial taxa (e.g., Paenisporosarcina, Desulfocapsa, Syntrophus, Desulfosporosinus) as being potentially active, with ice temperature appearing to be an important predictor for the diversity of taxa inferred to be active. Compared to those of other sympagic environments, these microbiomes often resembled those found in permafrost or perennial cave ice rather than other glacial ice environments. In contrast, debris-poor basal ices harbored microbiomes more like those found in oligotrophic englacial ice. Collectively, these results suggest that different basal ice types contain distinct microbiomes that are actively structured by the diagenesis of their habitat.

IMPORTANCEThe discharge of material from the basal ice zones of glaciers and ice sheets is an important source of dissolved organic matter, nutrients, and trace metals to downstream environments. Microorganisms with the ability to remain metabolically active while frozen have the potential to alter these substrates before they are released and may thus be a vital biogeochemical component of proglacial environments. However, basal ice environments are among the least explored areas of the cryosphere and information on their microbiology is limited. Here, we examine several basal ice types that were collected from different glaciers to investigate how their microbiomes varied. Our results indicate that sediment-rich basal ice from cold-based and temperate glaciers harbor microbiomes that conduct biogeochemical transformations at subzero temperatures and raises the intriguing possibility that subsurface regions of the cryosphere may be more biogeochemically active than previously thought.


title: Variation in bacterial composition, diversity, and activity across different subglacial basal ice types


title: Inter-tissue convergence of gene expression and loss of cellular identity during ageing

abstract: Developmental trajectories of gene expression may reverse in their direction during ageing, a phenomenon previously linked to cellular identity loss. Our analysis of cerebral cortex, lung, liver and muscle transcriptomes of 16 mice, covering development and ageing intervals, revealed widespread but tissue-specific ageing-associated expression reversals. Cumulatively, these reversals create a unique phenomenon: mammalian tissue transcriptomes diverge from each other during postnatal development, but during ageing, they tend to converge towards similar expression levels, a process we term Divergence followed by Convergence, or DiCo. We found that DiCo was most prevalent among tissue-specific genes and associated with loss of tissue identity, which is confirmed using data from independent mouse and human datasets. Further, using publicly available single-cell transcriptome data, we showed that DiCo could be driven both by alterations in tissue cell type composition and also by cell-autonomous expression changes within particular cell types.

category: genomics


title: Inter-tissue convergence of gene expression during ageing suggests age-related loss of tissue and cellular identity


title: ORT: A workflow linking genome-scale metabolic models with reactive transport codes

abstract:
MotivationNutrient and contaminant behavior in the subsurface are governed by multiple coupled hydrobiogeochemical processes which occur across different temporal and spatial scales. Accurate description of macroscopic system behavior requires accounting for the effects of microscopic and especially microbial processes. Microbial processes mediate precipitation and dissolution and change aqueous geochemistry, all of which impacts macroscopic system behavior. As  omics data describing microbial processes is increasingly affordable and available, novel methods for using this data quickly and effectively for improved ecosystem models are needed.

ResultsWe propose a workflow ( Omics to Reactive Transport - ORT) for utilizing metagenomic and environmental data to describe the effect of microbiological processes in macroscopic reactive transport models. This workflow utilizes and couples two open-source software packages: KBase (a software platform for systems biology) and PFLOTRAN (a reactive transport modeling code). We describe the architecture of ORT and demonstrate an implementation using metagenomic and geochemical data from a river system. Our demonstration uses microbiological drivers of nitrification and denitrification to predict nitrogen cycling patterns which agree with those provided with generalized stoichiometries. While our example uses data from a single measurement, our workflow can be applied to spatiotemporal metagenomic datasets to allow for iterative coupling between KBASE and PFLOTRAN.

Availability and ImplementationInteractive models available at https://pflotranmodeling.paf.subsurfaceinsights.com/pflotran-simple-model/. Microbiological data available at NCBI via BioProject ID PRJNA576070. ORT Python code available at https://github.com/subsurfaceinsights/ort-kbase-to-pflotran. KBase narrative available at https://narrative.kbase.us/narrative/71260 or static narrative (no login required) at https://kbase.us/n/71260/258

Contactrebecca.rubinstein@subsurfaceinsights.com or roelof.versteeg@subsurfaceinsights.com

Supplementary informationSupplementary data are available online.

category: bioinformatics


title: A CTDNEP1-lipin 1-mTOR regulatory network restricts ER membrane biogenesis to enable chromosome motions necessary for mitotic fidelity

abstract: The endoplasmic reticulum (ER) dramatically restructures in open mitosis to become excluded from the mitotic spindle; however, the significance of ER reorganization to mitotic progression is not known. Here, we demonstrate that limiting ER membrane biogenesis enables mitotic chromosome movements necessary for chromosome biorientation and prevention of micronuclei formation. Aberrantly expanded ER membranes increase the effective viscosity of the mitotic cytoplasm to physically restrict chromosome dynamics - slowed chromosome motions impede correction of mitotic errors induced by transient spindle disassembly, leading to severe micronucleation. We define the mechanistic link between regulation of ER membrane biogenesis and mitotic fidelity by demonstrating that a CTDNEP1-lipin 1-mTOR regulatory network limits ER lipid synthesis to prevent chromosome missegregation. Together, this work shows that ER membranes reorganize in mitosis to enable chromosome movements necessary for mitotic error correction and reveal dysregulated lipid metabolism as a potential source of aneuploidy in cancer cells.

category: cell biology


title: The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation

abstract:
BackgroundA hallmark of heart failure is cardiac fibrosis, which results from the injury-induced differentiation response of resident fibroblasts to myofibroblasts that deposit extracellular matrix. During myofibroblast differentiation, fibroblasts progress through polarization stages of early pro-inflammation, intermediate proliferation, and late maturation, but the regulators of this progression are poorly understood. Planar cell polarity receptors, receptor tyrosine kinase like orphan receptor 1 and 2 (Ror1/2), can function to promote cell differentiation and transformation. In this study, we investigated the role of the Ror1/2 in a model of heart failure with emphasis on myofibroblast differentiation.

Methods and ResultsThe role of Ror1/2 during cardiac myofibroblast differentiation was studied in cell culture models of primary murine cardiac fibroblast activation and in knockout mouse models that underwent transverse aortic constriction (TAC) surgery to induce cardiac injury by pressure overload. Expression of Ror1 and Ror2 were robustly and exclusively induced in fibroblasts in hearts after TAC surgery, and both were rapidly upregulated after early activation of primary murine cardiac fibroblasts in culture. Cultured fibroblasts isolated from Ror1/2-KO mice displayed a pro-inflammatory phenotype indicative of impaired myofibroblast differentiation. Although the combined ablation of Ror1/2 in mice did not result in a detectable baseline phenotype, TAC surgery led to the death of all mice by day 6 that was associated with myocardial hyper-inflammation and vascular leakage.

ConclusionsTogether, these results show that Ror1/2 are essential for the progression of myofibroblast differentiation and for the adaptive remodeling of the heart in response to pressure overload.


title: Implications of the evolution of agriculture and resource foraging for the maintenance of species diversity and community structure

abstract: Agriculture is found in numerous taxa such as humans, ants, beetles, fishes and even bacteria. This type of niche construction has evolved independently from hunting, though many species remain primarily predators. When a consumer has a positive effect on its resource, we can expect an allocative cost of agriculture, as the agricultural care diverts time and energy from other activities. Defending the resource against predators may divert time from its consumption (exploitation cost). The cost may also occur on the foraging of alternative resources, for instance if the consumer spends more time nearby the farmed resource and underexploiting resources elsewhere (opportunity cost). We here investigate transitions from predation to agriculture in a simple three-species model of a farmer that consumes two resources and has a positive effect on one. We study the conditions for the (co)evolution of the investment into agriculture and specialization on the two resources, and its consequences on the ecological dynamics of the community. Eco-evolutionary dynamics generate a feedback between the evolution of agriculture and specialization on the helped resource, that can lead to varying selected intensity of agriculture, from generalist strategies with no agriculture, to specialist farmers, with possible coexistence between these two extreme strategies.

category: ecology


title: Implications of the (co)evolution of agriculture and resource foraging for the maintenance of species diversity and community structure


title: Limited selection towards lighter seeds in 1 Danish grasslands over an eight-year period

abstract: Time-series analysis of cover data from 236 Danish grassland sites demonstrated community selection on seed mass. Across four grassland habitat types and during an eight-year period there was significant community selection towards lighter seeds. In the analysis, the continuous seed size variable was used to group plant species into functional types. This method was chosen in order to account for the sampling process of the abundance data, and is in contrast to most other analyses of trait selection, where the community weighted mean of the traits is used as the dependent variable. Generally, little is known on the underlying causes of possible plant trait community selection in semi-natural grasslands that are not subjected to successional processes.


title: Limited selection towards lighter seeds in Danish grasslands over an eight-year period


title: Selection towards lighter seeds in Danish grasslands over an eight-year period


title: " Idol with feet of clay ": reliable predictions of forest ecosystem functioning require flawless climate forcings.

abstract:
Climate change affects various aspects of the functioning of ecosystem, especially photosynthesis, respiration and carbon storage. We need accurate modelling approaches (impact models) to simulate the functioning, vitality and provision of ecosystem services of forests in a warmer world. These impact models require climate data as forcings, which are often produced by climate models comparing more or less well with observational climate data. The bias percentage of the climate forcings propagates throughout the modeling chain from the climate model to the impact model.

In this study, we aimed to quantify these bias percentage, addressing three questions: (1) Do the impact model predictions vary when forcing it with different climate models, and how do the predictions under climate model vs. observational climate forcing differ? (2) Does the variability in the impact climate simulations caused by climate forcings fade out at large spatial scale? (3) How the fact of using simulated climatic data affects the process-based model predictions in the case of stressful events?

To answer these questions, we present results obtained over the historical period (e.g. 1970-2010) with the CASTANEA ecophysiological forest model and use the data from three climate models. Our analysis focuses on French forests, studying European beech (Fagus sylvatica), temperate deciduous oaks (Quercus robur and Q. petraea), Scots pine (Pinus sylvestris) and spruce (Picea abies) monospecific stands.

We show that prediction of photosynthesis, respiration and wood growth highly depends on the climate model used, whether debiased or not, and also on species and region considered. Overall, we observed an improvement of prediction after a monthly mean bias or monthly quantile mapping correction for three model considered, but not with the same success. Then we highlighted a large variability in the processes simulated by the impact model under different climate forcings when considering the plot (i.e. scale of a few hectares) scale. This variability fades out at larger scale (e.g. the scale of an ecological region, i.e. 100 km2), owing to an aggregation effect. Moreover, process predictions obtained under different climate forcings are more variable during driest years. These results highlight the necessity to quantify bias and uncertainties in climate forcings before predicting fluxes dynamics with process-based model.


title: Multiple targets of balancing selection in Leishmania donovani complex parasites

abstract: The Leishmania donovani species complex are the causative agents of visceral leishmaniasis, which cause 20-40,000 fatalities a year. Here, we conduct a screen for balancing selection in this species complex. We used 387 publicly-available L. donovani and L. infantum genomes, and sequence 93 isolates of L. infantum from Brazil to describe the global diversity of this species complex. We identify five genetically-distinct populations that are sufficiently represented by genomic data to search for signatures of selection. We find that signals of balancing selection are generally not shared between populations, consistent with transient adaptive events, rather than long-term balancing selection. We then apply multiple diversity metrics to identify candidate genes with robust signatures of balancing selection, identifying a curated set of 19 genes with robust signatures. These include zeta toxin, nodulin-like and flagellum attachment proteins. This study highlights the extent of genetic divergence between L. donovani complex parasites and provides genes for further study.

category: evolutionary biology


title: Deriving accurate molecular indicators of protein synthesis through Raman-based sparse classification

abstract: Raman spectroscopy has the ability to retrieve molecular information from live biological samples non-invasively through optical means. Coupled with machine learning, it is possible to use the large amount of information contained in a Raman spectrum to create models that can predict the state of new samples based on statistical analysis from previous measurements. Furthermore, in case of linear models, the separation coefficients can be used to interpret which bands are contributing to the discrimination between experimental conditions, which correspond here to single-cell measurements of macrophages under in vitro immune stimulation. We here evaluate a typical linear method using discriminant analysis and PCA, and compare it to regularized logistic regression (Lasso). We find that the use of PCA is not beneficial to the classification performance. Furthermore, the Lasso approach yields sparse separation vectors, since it suppresses spectral coefficients which do not improve classification, making interpretation easier. To further evaluate the approach, we apply the Lasso technique to a well-defined case where protein synthesis is inhibited, and show that the separating features are consistent with RNA accumulation and protein levels depletion. Surprisingly, when Raman features are selected purely in terms of their classification power (Lasso), the selected coefficients are contained in side bands, while typical strong Raman peaks are not present in the discrimination vector. We propose that this occurs because large Raman bands are representative of a wide variety of cellular molecules and are therefore less suited for accurate classification.

category: biochemistry


title: Structural basis for the hyperthermostability of an archaeal glutaminase induced by post-translational succinimide formation

abstract: Stability of proteins from hyperthermophiles enabled by reduction of conformational flexibility is realized through various mechanisms. Presence of a stable, hydrolysis-resistant succinimide arising from cyclization of the side chains of aspartyl/asparaginyl residues with backbone amide -NH of the succeeding residue would restrain the torsion angle {Psi}. Here, we describe the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase (MjGATase) and address the mechanism of a succinimide-induced increased thermostability using molecular dynamics simulations. This study reveals the interplay of negatively charged electrostatic shield and n[->]{pi}* interactions in preventing succinimide hydrolysis. The stable succinimidyl residue induces formation of a  conformational-lock, reducing protein flexibility. Protein destabilization upon replacement with the {Phi}-restricted prolyl residue highlights the specificity of the conformationally restrained succinimidyl residue in imparting hyperthermostability. The conservation of succinimide-forming tripeptide sequence (E(N/D)(E/D)) in a group of archaeal GATases suggests an adaptation of this otherwise detrimental post-translational modification as an inducer of thermostability.


title: AMPylation is a specific lysosomal protein posttranslational modification in neuronal maturation.

abstract: Protein AMPylation is a pervasive posttranslational modification with an emerging role in neurodevelopment. In metazoans the two highly conserved protein AMP-transferases together with a diverse group of AMPylated proteins have been identified using chemical proteomics and biochemical techniques. However, the function of this modification remains largely unknown. Particularly problematic is the localization of thus far identified AMPylated proteins and putative AMP-transferases. Here, we uncover protein AMPylation as a novel posttranslational modification of luminal lysosomal proteins characteristic in differentiating neurons. Through a combination of chemical proteomics, advanced gel-based separation of modified and unmodified proteins and activity assay, we show that an AMPylated, lysosomal soluble form of exonuclease PLD3 increases dramatically during neuronal maturation and that AMPylation inhibits its catalytic activity. Together, our findings unveil so far unknown lysosomal posttranslational modification, its connection to neuronal differentiation and putatively provide a novel molecular rationale to design of therapeutics for lysosomal storage diseases.


title: Injury-Free In Vivo Delivery and Engraftment into the Cornea Endothelium Using Extracellular Matrix Shrink-Wrapped Cells

abstract:
Cell injection is a common clinical approach for therapeutic delivery of cells into diseased and damaged tissues in order to achieve regeneration. However, cell retention, viability, and engraftment at the injection site has generally been poor, driving the need for improved approaches. Here, we developed a technique to shrink-wrap micropatterned islands of corneal endothelial cells in a basement membrane-like layer of extracellular matrix (ECM) that enables the cells to maintain their cell-cell junctions and cytoskeletal structure while in suspension. These Monolayers exhibited the ability to rapidly engraft into intact, high-density corneal endothelial monolayers in both in vitro and in vivo model systems. Importantly, the engrafted Monolayers increased local cell density, something that the clinical-standard single cells in suspension failed to do. These results show that shrink-wrapping cells in ECM dramatically improves engraftment and provides a potential alternative to cornea transplant when low endothelial cell density is the cause of corneal blindness.

One Sentence SummaryShrink-wrapped patches of endothelial cells can rapidly attach and integrate into an intact cornea endothelium when injected into the anterior chamber, increasing cell density.

category: bioengineering


title: Human stem cell-based retina-on-chip as new translational model for validation of AAV retinal gene therapy vectors

abstract: Gene therapies using adeno-associated viruses (AAVs) are amongst the most promising strategies to treat or even cure hereditary and acquired retinal diseases. However, the development of new efficient AAV vectors is slow and costly, largely because of the lack of suitable non-clinical models. By faithfully recreating structure and function of human tissues, human induced pluripotent stem cell (iPSC)-derived retinal organoids could become an essential part of the test cascade addressing translational aspects. Organ-on-Chip (OoC) technology further provides the capability to recapitulate microphysiological tissue environments as well as a precise control over structural and temporal parameters. By employing our recently developed Retina-on-chip that merges organoid and OoC-technology, we analyzed the efficacy, kinetics and cell tropism of seven first and second generation AAV vectors. The presented data demonstrate the potential of iPSC-based OoC models as the next generation of screening platforms for future gene therapeutic studies.


title: Adaptation to DNA damage as a bet-hedging mechanism in a fluctuating environment

abstract: In response to DNA damage, efficient repair is essential for cell survival and genome integrity. In eukaryotes, the DNA damage checkpoint is a signalling pathway that coordinates this response and arrests the cell cycle to provide time for repair. However, when repair fails or when the damage is not repairable, cells can eventually bypass the DNA damage checkpoint and undergo cell division despite persistent damage, a process called adaptation to DNA damage. Interestingly, adaptation occurs with a delayed timing compared to repair and shows a large variation in time, two properties that may provide a survival advantage at the population level without interfering with repair. Here, we explore this idea by mathematically modelling cell survival in response to DNA damage and focusing on adaptation parameters. We find that the delayed adaptation timing indeed maximizes survival, but its heterogeneity is beneficial only in a fluctuating damage-inducing environment. Finally, we show that adaptation does not only contribute to survival but also to genome instability and mutations, which might represent another criterion for its selection through-out evolution. Overall, we propose that adaptation can act as a bet-hedging mechanism for cell survival in response to DNA damage.


title: Towards a global investigation of transcriptomic signatures through co-expression networks and pathway knowledge for the identification of disease mechanisms

abstract: In this work, we attempt to address a key question in the joint analysis of transcriptomic data: can we correlate the patterns we observe in transcriptomic datasets to known molecular interactions and pathway knowledge to broaden our understanding of disease pathophysiology? We present a systematic approach that sheds light on the patterns observed in hundreds of transcriptomic datasets from over sixty indications by using pathways and molecular interactions as a template. Our analysis employs transcriptomic datasets to construct dozens of disease specific co-expression networks, alongside a human interactome network of protein-protein interactions described in the literature. Leveraging the interoperability between these two network templates, we explore patterns both common and particular to these diseases on three different levels. Firstly, at the node-level, we identify the most and least common proteins in these diseases and evaluate their consistency against the interactome as a proxy for their prevalence in the scientific literature. Secondly, we overlay both network templates to analyze common correlations and interactions across diseases at the edge-level. Thirdly, we explore the similarity between patterns observed at the disease level and pathway knowledge to identify pathway signatures associated with specific diseases and indication areas. Finally, we present a case scenario in the context of schizophrenia, where we show how our approach can be used to investigate disease pathophysiology.


title: Transcription elongation through lac repressor-mediated DNA loops

abstract: Some proteins, like the lac repressor (LacI), mediate long-range loops that alter DNA topology and create torsional barriers. During transcription, RNA polymerase generates supercoiling that may facilitate passage through such barriers. We monitored E. coli RNA polymerase progress along templates in conditions that prevented, or favored, LacI-mediated DNA looping. Tethered particle motion measurements revealed that RNA polymerase paused longer at unlooped LacI obstacles or those barring entry to a loop than those barring exit from the loop. Enhanced dissociation of a LacI roadblock by the positive supercoiling generated ahead of a transcribing RNA polymerase within a torsion-constrained DNA loop may be responsible. In support of this, RNA polymerase transcribed a looped segment more slowly than an unlooped segment and paused more briefly at LacI obstacles on positively supercoiled templates, especially under increased tension (torque). Positive supercoiling propagating ahead of polymerase appears to facilitate elongation along topologically-complex, protein-coated templates.

category: biophysics


title: Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops


title: Intracellular Oxidative Stress Levels are Significantly Associated with the Green Autofluorescence Intensity of Buthionine Sulfoximine-Treated B16-F10 Cells

abstract: Since oxidative stress is a critical common pathological factor of numerous diseases, it is critical to find biomarkers for non-invasive evaluations of the levels of oxidative stress in the body. Our previous studies have indicated that epidermal green autofluorescence (AF) is a novel biomarker of this type: The oxidative stress inducer buthionine sulfoximine (BSO) can dose-dependently increase the epidermal green AF of mice, with BSO doses being significantly associated with the AF intensity. However, it is necessary to use skin cell cultures to investigate the mechanisms underlying the relationships between BSO and the green AF intensity. In our current study we found that BSO concentration-dependently increased the green AF intensity of B16-F10 cells a skin cell line, with BSO concentrations being significantly associated with the AF intensity. BSO also concentration-dependently increased the intracellular DCF signals an index of ROS levels. The green AF intensity of the cells was also significantly associated with the intracellular ROS levels. Moreover, we found that the green AF intensity was significantly associated with the cell death induced by BSO. Collectively, our study has provided first evidence indicating that the green AF intensity of skin cells is significantly associated with both intracellular ROS levels and cell death of the skin cells exposed to oxidative stress, which has indicated that green AF is a novel biomarker for both oxidative stress and cell death.


title: Perturbation of ACE2 structural ensembles by SARS-CoV-2 spike protein binding

abstract: The human ACE2 enzyme serves as a critical first recognition point of coronaviruses, including SARS-CoV-2. In particular, the extracellular domain of ACE2 interacts directly with the S1 tailspike protein of the SARS-CoV-2 virion through a broad protein-protein interface. Although this interaction has been characterized by X-ray crystallography and Cryo-EM, these structures do not reveal significant differences in ACE2 structure upon S1 protein binding. In this work, using several all-atom molecular dynamics simulations, we show persistent differences in ACE2 structure upon binding. These differences are determined with the Linear Discriminant Analysis (LDA) machine learning method and validated using independent training and testing datasets, including long trajectories generated by D. E. Shaw Research on the Anton 2 supercomputer. In addition, long trajectories for 78 potent ACE2-binding compounds, also generated by D. E. Shaw Research, were projected onto the LDA classification vector in order to determine whether the ligand-bound ACE2 structures were compatible with S1 protein binding. This allows us to predict which compounds are "apo-like" vs "complex-like", as well as to pinpoint long-range ligand-induced allosteric changes of ACE2 structure.


title: The glycocalyx affects force loading-dependent mechanotransductive topography sensing at the nanoscale

abstract:
The cell/microenvironment interface is the starting point of integrin-mediated mechanotransduction, but many details of mechanotransductive signal integration remain elusive due to the complexity of the involved (extra)cellular structures, such as the glycocalyx.

We used nano-bio-interfaces reproducing the complex nanotopographical features of the extracellular matrix to analyse the glycocalyx impact on PC12 cell mechanosensing at the nanoscale (e.g., by force spectroscopy with functionalised probes). Our data demonstrates that the glycocalyx configuration affects spatio-temporal nanotopography-sensitive mechanotransductive events at the cell/microenvironment interface. Opposing effects of glycocalyx removal were observed, when comparing flat and specific nanotopographical conditions. The excessive retrograde actin flow speed and force loading are strongly reduced on certain nanotopographies upon removal of the native glycocalyx, while on the flat substrate we observe the opposite trend.

Our results highlight the importance of the glycocalyx configuration in a molecular clutch force loading-dependent cellular mechanism for mechanosensing of microenvironmental nanotopographical features.


title: The glycocalyx affects the mechanotransductive perception of the topographical microenvironment


title: Chromatin Network Retards Droplet Coalescence

abstract: Nuclear bodies are membraneless condensates that may form via liquid-liquid phase separation. The viscoelastic chromatin network could impact their stability and may hold the key for understanding experimental observations that defy predictions of classical theories. However, quantitative studies on the role of the chromatin network in phase separation have remained challenging. Using a diploid human genome model parameterized with chromosome conformation capture (Hi-C) data, we studied the thermodynamics and kinetics of droplet formation inside the nucleus. Dynamical simulations predicted the formation of multiple droplets for protein particles that experience specific interactions with nucleolus-associated domains (NADs). Coarsening dynamics, surface tension, and coalescence kinetics of the simulated droplets are all in quantitative agreements with experimental measurements for nucleoli. Free energy calculations further supported that a two-droplet state, which is often observed for nucleoli seen in somatic cells, is metastable and separated from the single-droplet state with an entropic barrier. Our study suggests that protein-chromatin interactions facilitate the nucleation of droplets, but hinders their coarsening due to the correlated motion between droplets and the chromatin network: as droplets coalesce, the chromatin network becomes increasingly constrained. Therefore, protein-chromatin interactions arrest phase separation in multi-droplet states and may drive the variation of nuclear body numbers across cell types.


title: Multi-dimensional and spatiotemporal correlative imaging at the plasma membrane of live cells to determine the continuum nano-to-micro scale lipid adaptation and collective motion

abstract: The primary cilium is a specialized plasma membrane protrusion with important receptors for signalling pathways. In polarized epithelial cells, the primary cilium assembles after the midbody remnant (MBR) encounters the centrosome at the apical surface. The membrane surrounding the MBR, namely remnant associated membrane patch (RAMP) once situated next to the centrosome, releases some of its lipid components to form a centrosome-associated membrane patch (CAMP) from which the ciliary membrane stems. The RAMP undergoes a spatiotemporal membrane refinement during the formation of the CAMP, which becomes highly enriched in condensed membranes with low lateral mobility. To better understand this process, we have developed a correlative imaging approach that yields quantitative information about the lipid lateral packing, its mobility and collective assembly at the plasma membrane at different spatial scales over time. Our work paves the way towards a quantitative understanding of lipid collective assembly at the plasma membrane spatiotemporally as a functional determinant in cell biology and its direct correlation with the membrane physicochemical state. These findings allowed us to gain a deeper insight into the mechanisms behind the biogenesis of the ciliary membrane of polarized epithelial cells.


title: Slow, heterogeneous NFκB single molecule conformational dynamics and implications for function

abstract: Binding and unbinding of transcription factors to DNA are kinetically controlled to regulate the transcriptional outcome. Control of the release of the transcription factor NF-{kappa}B from DNA is achieved through accelerated dissociation by the inhibitor protein I{kappa}B. Using single-molecule FRET, we observe a continuum of conformations of NF-{kappa}B in free and DNA-bound states interconverting on the subseconds to minutes timescale, comparable to in vivo binding on the seconds timescale, suggesting that structural dynamics directly control binding kinetics. Much of the DNA-bound NF-{kappa}B is partially bound, allowing I{kappa}B invasion to facilitate DNA dissociation. I{kappa}B induces a locked conformation where the DNA-binding domains of NF-{kappa}B are too far apart to bind DNA, whereas the loss-of-function I{kappa}B mutant retains the NF-{kappa}B conformational ensemble. Overall, our results suggest a novel mechanism with a continuum of binding modes for controlling association and dissociation of transcription factors.


title: Single-molecule conformational dynamics of a transcription factor reveals a continuum of binding modes controlling association and dissociation


title: Olanzapine leads to nonalcoholic fatty liver disease through the apolipoprotein A5 pathway

abstract: The antipsychotic drug olanzapine was reported to induce nonalcoholic fatty liver disease (NAFLD), whereas the underlying mechanism remains incompletely understood. This study investigated whether apolipoprotein A5 (apoA5) and sortilin, two interactive factors involved in NAFLD pathogenesis, are implicated in olanzapine-induced NAFLD. In the present study, at week 8, olanzapine treatment successfully induced hepatic steatosis in male C57 BL/6J mice, even when fed a normal chow diet, which was independent of animal body weight gain. Likewise, olanzapine effectively mediated hepatocyte steatosis in human HepG2 cells in vitro, characterized by substantially elevated intracellular lipid droplets. Increased plasma triglyceride concentration and decreased plasma apoA5 levels were observed in mice treated for 8 weeks with olanzapine. Surprisingly, olanzapine markedly enhanced hepatic apoA5 protein levels in mice, without a significant effect on rodent hepatic ApoA5 mRNA expression. Our in vitro study showed that olanzapine reduced apoA5 protein levels in the medium and enhanced apoA5 protein expression in hepatocytes, whereas this drug exerted no effect on hepatocyte APOA5 mRNA expression. By transfecting APOA5 siRNA into HepG2 cells, it was demonstrated that APOA5 knockdown effectively reversed olanzapine-induced hepatocyte steatosis. In addition, olanzapine drastically increased sortilin mRNA and protein levels in vivo and in vitro. Interestingly, SORT1 knockdown reduced intracellular apoA5 protein expression and increased medium apoA5 protein levels in vitro, without affecting intracellular APOA5 mRNA expression. Furthermore, SORT1 knockdown greatly ameliorated hepatocyte steatosis in vitro. This study provides the first evidence that sortilin inhibits the hepatic apoA5 secretion that is attributable to olanzapine-induced NAFLD, which provides new insight into effective strategies against NAFLD for patients with schizophrenia administered olanzapine.

category: pathology


title: The anti-immune dengue subgenomic flaviviral RNA is found in vesicles in mosquito saliva and associated with increased infectivity

abstract: Mosquito transmission of dengue viruses to humans starts with infection of skin resident cells at the biting site. There is great interest in identifying transmission-enhancing factors in mosquito saliva in order to counteract them. Here we report the discovery of high levels of subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We show that salivary sfRNA is protected in detergent-sensitive, protease-resistant compartments. Furthermore, we show that incubation with mosquito saliva containing higher sfRNA levels results in higher virus infectivity in human cells. Since sfRNA potently inhibits innate immunity in human cells, we posit that sfRNA in mosquito saliva is present in extracellular vesicles that deliver it to cells at the biting site to inhibit innate immunity and enhance dengue virus transmission.

category: physiology


title: Geminiviral genomes encode additional proteins with specific subcellular localizations and virulence function

abstract: Geminiviruses are plant viruses with limited coding capacity. Geminivirus-encoded proteins were identified applying a 10-kDa arbitrary threshold; however, it is increasingly clear that small proteins play relevant roles in biological systems, which calls for the reconsideration of this criterion. Here, we show that geminiviral genomes contain additional ORFs. Using tomato yellow leaf curl virus, we demonstrate that some of these novel ORFs are expressed during the infection, and that the encoded proteins display specific subcellular localizations. We prove that the largest of these new ORFs, which we name V3, is required for full viral infection, and that the V3 protein localizes in the Golgi apparatus and functions as an RNA silencing suppressor. These results imply that the repertoire of geminiviral proteins can be expanded, and that getting a comprehensive overview of the molecular plant-geminivirus interactions will require the detailed study of small ORFs so far neglected.

category: plant biology


title: Predicting biological pathways of chemical compounds with a profile-inspired aproach

abstract: Assignment of chemical compounds to biological pathways is a crucial step to understand the relationship between the chemical repertory of an organism and its biology. Protein sequence profiles are very successful in capturing the main structural and functional features of a protein family, and can be used to assign new members to it based on matching of their sequences against these profiles. In this work, we extend this idea to chemical compounds, constructing a profile-inspired model for a set of related metabolites (those in the same biological pathway), based on a fragment-based vectorial representation of their chemical structures. We use this representation to predict the biological pathway of a chemical compound with good overall accuracy (AUC 0.74-0.90 depending on the database tested), and analyzed some factors that affect performance. The approach, which is compared with equivalent methods, can in addition detect those molecular fragments characteristic of a pathway. The method is available as a graphical interactive web server http://csbg.cnb.csic.es/iFragMent

category: systems biology


title: On the generalizability of diffusion MRI signal representations across acquisition parameters, sequences and tissue types: chronicles of the MEMENTO challenge.

abstract: Diffusion MRI (dMRI) has become an invaluable tool to assess the microstructural organization of brain tissue. Depending on the specific acquisition settings, the dMRI signal encodes specific properties of the underlying diffusion process. In the last two decades, several signal representations have been proposed to fit the dMRI signal and decode such properties. Most methods, however, are tested and developed on a limited amount of data, and their applicability to other acquisition schemes remains unknown. With this work, we aimed to shed light on the generalizability of existing dMRI signal representations to different diffusion encoding parameters and brain tissue types. To this end, we organized a community challenge - named MEMENTO, making available the same datasets for fair comparisons across algorithms and techniques. We considered two state-of-the-art diffusion datasets, including single-diffusion-encoding (SDE) spin-echo data from a human brain with over 3820 unique diffusion weightings (the MASSIVE dataset), and double (oscillating) diffusion encoding data (DDE/DODE) of a mouse brain including over 2520 unique data points. A subset of the data sampled in 5 different voxels was openly distributed, and the challenge participants were asked to predict the remaining part of the data. After one year, eight participant teams submitted a total of 80 signal fits. For each submission, we evaluated the mean squared error, the variance of the prediction error and the Bayesian information criteria. Most predictions predicted either multi-shell SDE data (37%) or DODE data (22%), followed by cartesian SDE data (19%) and DDE (18%). Most submissions predicted the signals measured with SDE remarkably well, with the exception of low and very strong diffusion weightings. The prediction of DDE and DODE data seemed more challenging, likely because none of the submissions explicitly accounted for diffusion time and frequency. Next to the choice of the model, decisions on fit procedure and hyperparameters play a major role in the prediction performance, highlighting the importance of optimizing and reporting such choices. This work is a community effort to highlight strength and limitations of the field at representing dMRI acquired with trending encoding schemes, gaining insights into how different models generalize to different tissue types and fiber configurations over a large range of diffusion encodings.

category: neuroscience


title: Preparation of large-scale digitization samples for automated electron microscopy of tissue and cell ultrastructure

abstract: Manual selection of targets in experimental or diagnostic samples by transmission electron microscopy (TEM), based on single overview and detail micrographs, has been time- consuming and susceptible to bias. Substantial information and throughput gain may now be achieved by automated acquisition of virtually all structures in a given EM section. Resulting datasets allow convenient pan-and-zoom examination of tissue ultrastructure with preserved microanatomical orientation. The technique is, however, critically sensitive to artifacts in sample preparation. We therefore established a methodology to prepare large-scale digitization samples (LDS) designed to acquire entire sections free of obscuring flaws. For evaluation, we highlight the supreme performance of scanning EM in transmission mode compared to other EM technology. The use of LDS will substantially facilitate access to EM data for a broad range of applications.


title: Multitaper estimates of phase-amplitude coupling

abstract: Phase-amplitude coupling (PAC) is the association of the amplitude of a high-frequency oscillation with the phase of a low-frequency oscillation. In neuroscience, this relationship provides a mechanism by which neural activity might be coordinated between distant regions. The dangers and pitfalls of assessing phase-amplitude coupling with existing statistical measures have been well-documented. The limitations of these measures include: (i) response to non-oscillatory, high-frequency, broad-band activity, (ii) response to high-frequency components of the low-frequency oscillation, (iii) adhoc selection of analysis frequency-intervals, and (iv) reliance upon data shuffling to assess statistical significance. In this work, a multitaper phase-amplitude coupling estimator is proposed that addresses issues (i)-(iv) above. Specifically, issue (i) is addressed by replacing the analytic signal envelope estimator computed using the Hilbert transform with a multitaper estimator that down-weights non-sinusoidal activity using a classical, multitaper super-resolution technique. Issue (ii) is addressed by replacing coherence between the low-frequency and high-frequency components in a standard PAC estimator with multitaper partial coherence, while issue (iii) is addressed with a physical argument regarding meaningful neural oscillation. Finally, asymptotic statistical assessment of the multitaper estimator is introduced to address issue (iv).


title: Transient beta activity and connectivity during sustained motor behaviour

abstract:
Neural oscillations are thought to play a central role in orchestrating activity states between distant neural populations. In humans, long-range neural connectivity has been particularly well characterised for 13-30 Hz beta activity which becomes phase coupled between the motor cortex and the contralateral muscle during isometric contraction. Based on this and related observations, beta activity and connectivity have been linked to sustaining stable cognitive and motor states - or the  status quo - in the brain. Recently, however, beta activity has been shown to be short-lived, as opposed to sustained - though so far this has been reported for regional beta activity in tasks without sustained motor demands. Here, we measured magnetoencephalography (MEG) and electromyography (EMG) in 18 human participants performing an isometric-contraction (gripping) task designed to yield sustained behavioural output. If cortico-muscular beta connectivity is directly responsible for sustaining a stable motor state, then beta activity should be (or become) sustained in this context. In contrast, we found that beta activity and connectivity with the downstream muscle were transient, even when participants engaged in sustained gripping. Moreover, we found that sustained motor requirements did not prolong beta-event duration in comparison to rest. These findings suggest that long-range neural synchronisation may entail short  bursts of frequency-specific connectivity, even when task demands - and behaviour - are sustained.

HighlightsO_LITrial-average 13-30 Hz beta activity and connectivity with the muscle appear sustained during stable motor behaviour
C_LIO_LISingle-trial beta activity and connectivity are short-lived, even when motor behaviour is sustained
C_LIO_LISustained task demands do not prolong beta-event duration in comparison to resting state
C_LI


title: Cooperative Stabilization of Close-Contact Zones Leads to Sensitivity and Selectivity in T-Cell Recognition

abstract: T cells are sensitive to 1 to 10 foreign-peptide-MHC complexes among a vast majority of self-peptide-MHC complexes, and discriminate selectively between peptide-MHC complexes that differ not much in their binding affinity to T-cell receptors (TCRs). Quantitative models that aim to explain this sensitivity and selectivity largely focus on single TCR/peptide-MHC complexes, but T cell adhesion involves a multitude of different complexes. In this article, we demonstrate in a three-dimensional computational model of T-cell adhesion that the cooperative stabilization of close-contact zones is sensitive to 1 to 3 foreign-peptide-MHC complexes and occurs at a rather sharp threshold affinity of these complexes, which implies selectivity. In these close-contact zones with lateral extensions of hundred to several hundred nanometers, few TCR/foreign-peptide-MHC complexes and many TCR/self-peptide-MHC complexes are segregated from LFA-1/ICAM-1 complexes that form at larger membrane separations. Previous high-resolution microscopy experiments indicate that the sensitivity and selectivity in the formation of closed-contact zones reported here is relevant for T-cell recognition, because the stabilization of close-contact zones by foreign, agonist peptide-MHC complexes precedes T-cell signaling and activation in the experiments.

category: immunology


title: Genome-wide screens identify calcium signaling as a key regulator of IgE+ plasma celldifferentiation and survival

abstract: IgE antibodies protect against toxins and parasites, however, they also mediate allergic reactions. In contrast to other antibody isotypes, B cells switched to IgE respond transiently and do not give rise to long-lived plasma cells (PCs) or memory B cells. Although the intrinsic differences of IgE+ B cells have been linked to signaling by the IgE-B cell receptor (BCR), the molecular pathways controlling their behavior remain poorly understood. Here we employ whole-genome CRISPR screening to identify genes regulating IgE+ B cell proliferation, survival and differentiation into PCs. We show that IgE+ B cells are selectively suppressed by the IgE-BCR signaling to intracellular calcium, which inhibits PC differentiation and limits their lifespan after differentiation. Consequently, manipulation of calcium signaling in vivo enhances IgE+ PC responses. Insights from this pathway shed new light on the self-limiting character of IgE responses and open new avenues to eliminate IgE+ PCs in allergy.


title: High-quality complete genome resource of plant pathogenic bacterium Dickeya solani strain IPO 2019 isolated from Hyacinthus orientalis

abstract: Dickeya solani is an emerging plant pathogenic bacterium, causing disease symptoms in a variety of agriculturally relevant crop species worldwide. To date a number of D. solani genomes have been sequenced and characterized, the great majority of these genomes have however come from D. solani strains isolated from potato (Solanum tuberosum L.) and not from other plant hosts. Herewith, we present the first complete, high-quality genome of D. solani strain IPO 2019 (LMG 25990) isolated from ornamental plant Hyacinthus orientalis. The genome of D. solani strain IPO 2019 consists of one chromosome of 4,919,542 bp., with a GC content of 56.2% and no plasmids. The genome contains 4502 annotated features, 22 rRNA genes, 73 tRNA genes and 1 CRISPRS. We believe that the information of this high-quality, complete, closed genome of D. solani strain isolated from host plant different than potato (i.e. hyacinth) will provide resources for comparative genomic studies as well as for analyses targeting adaptation and ecological fitness mechanisms present in Dickeya solani species.


title: Comparative analysis of RNA enrichment methods for preparation of Cryptococcus neoformans RNA sequencing libraries

abstract:
Ribosomal RNA (rRNA) is the major RNA constituent of cells, therefore most RNA sequencing (RNA-Seq) experiments involve removal of rRNA. This process, called RNA enrichment, is done primarily to reduce cost: without rRNA removal, deeper sequencing would need to be performed to balance the sequencing reads wasted on rRNA. The ideal RNA enrichment method would remove all rRNA without affecting other RNA in the sample. We have tested the performance of three RNA enrichment methods on RNA isolated from Cryptococcus neoformans, a fungal pathogen of humans. We show that the RNase H depletion method unambiguously outperforms the commonly used Poly(A) isolation method: the RNase H method more efficiently depletes rRNA while more accurately recapitulating the expression levels of other RNA observed in an unenriched "gold standard". The RNase H depletion method is also superior to the Ribo-Zero depletion method as measured by rRNA depletion efficiency and recapitulation of protein-coding gene expression levels, while the Ribo-Zero depletion method performs moderately better in preserving non-coding RNA (ncRNA). Finally, we have leveraged this dataset to identify novel long non-coding RNA (lncRNA) genes and to accurately map the C. neoformans mitochondrial rRNA genes.

ARTICLE SUMMARYWe compare the efficacy of three different RNA enrichment methods for RNA-Seq in Cryptococcus neoformans: RNase H depletion, Ribo-Zero depletion, and Poly(A) isolation. We show that the RNase H depletion method, which is evaluated in C. neoformans samples for the first time here, is highly efficient and specific in removing rRNA. Additionally, using data generated through these analyses, we identify novel long non-coding RNA genes in C. neoformans. We conclude that RNase H depletion is an effective and reliable method for preparation of C. neoformans RNA-Seq libraries.


title: Targeted Next Generation Sequencing of environmental DNA improves detection and quantification of invasive European green crab (Carcinus maenas)

abstract: In the northeast Pacific Ocean there is high interest in developing eDNA-based survey methods to aid management of invasive populations of European green crab (Carcinus maenas). Expected benefits are improved sensitivity for early detection of secondary spread and quantification of abundances to assess the outcome of eradication efforts. A new eDNA-based approach we term  Targeted Next Generation Sequencing (tNGS) is introduced here and shown to improve detection relative to qPCR at low eDNA concentrations, as is characteristic of founding or spreading populations. tNGS is based on the premise that the number of NGS reads from non-normalized (i.e. equal volumes) targeted PCR amplicons will approximate the starting DNA amount. Standard DNA concentrations that were 10-to 100-times lower than the qPCR limit of detection returned significant numbers of sequencing reads, which in our field assessments translated to a 7% - 10% increase in crab detection probability from tNGS relative to qPCR at low abundances. We also found that eDNA concentration was highly correlated with crab abundance, as measured from traditional trapping methods, for both assays; however, tNGS data had greater precision and less error than qPCR. When partitioning the sources of variation in each assay we identified greater between-site variability for tNGS relative to qPCR, suggesting the former may offer more power for detecting spatial variation in eDNA concentration. When applying this assay in management programs, we suggest including a panel of eDNA samples from sites with trapping data as standards to estimate relative abundance at sites with no a priori information. Results presented here indicate the tNGS approach has great promise for surveillance of green crab and could easily be adopted for surveillance of any species of high interest to management, including endangered species, new incursions of invasive species, and species with low eDNA shedding rates. Pros and cons of this approach compared to qPCR are discussed.

category: molecular biology


title: Modelling the impact of decidual senescence on embryo implantation in human endometrial assembloids

abstract: Decidual remodelling of midluteal endometrium leads to a short implantation window after which the uterine mucosa either breaks down or is transformed into a robust matrix that accommodates the placenta throughout pregnancy. To gain insights into the underlying mechanisms, we established and characterised endometrial assembloids, consisting of gland organoids and primary stromal cells. Single-cell transcriptomics revealed that decidualized assembloids closely resemble midluteal endometrium, harbouring differentiated and senescent subpopulations in both glands and stroma. We show that acute senescence in glandular epithelium drives secretion of multiple canonical implantation factors, whereas in the stroma it calibrates the emergence of anti-inflammatory decidual cells and pro-inflammatory senescent decidual cells. Pharmacological inhibition of stress responses in pre-decidual cells accelerated decidualization by inhibiting senescence and mesenchymal-epithelial transition, processes involved in endometrial breakdown and regeneration, respectively. Accelerated decidualization resulted in entrapment of co-cultured human blastocysts in a largely static decidual matrix. By contrast, the presence of senescent decidual cells created a dynamic implantation environment, enabling embryo expansion and attachment, although their persistence led to gradual disintegration of assembloids. Our findings demonstrate that senescence controls endometrial fate decisions at implantation and highlight how endometrial assembloids may accelerate the discovery of new treatments to prevent reproductive failure.

category: developmental biology


title: Uncovered genetic diversity in Hemidactylus mabouia (Reptilia: Gekkonidae) from Madeira Island reveals uncertain sources of introduction

abstract: Hemidactylus mabouia is one of the most widely distributed species within its genus. It was first reported to Madeira in 2002 and the first individuals were considered to have originated from Cape Verde. Almost 20 years later, we found that H. mabouia has substantially expanded its distribution and can now be found >10 km away from Funchal, where it was first reported. Based on a 12S phylogenetic analysis and using 29 individuals from Funchal and Camara de Lobos we found that Madeira actually harbours two distinct lineages of H. mabouia: one exclusively South American and another widespread in America and Africa. However, the lack of genetic diversity typical of this species outside its native range and the obtained phylogenetic pattern prevent us to infer possible introduction routes or sources. Our study emphasizes that authorities should remain vigilant regarding the arrival of other non-natives and act to prevent their establishment as soon as they are detected.


title: Using Graph Convolutional Neural Networks to Learn a Representation for Glycans

abstract: As the only nonlinear and most diverse biological sequence, glycans offer substantial challenges for computational biology. These complex carbohydrates participate in nearly all biological processes - from protein folding to the cellular entry of viruses - yet are still not well understood. There are few computational methods to link glycan sequences to functions and those that do exist do not take full advantage of all the available information of glycans. SweetNet is a graph convolutional neural network model that uses graph representation learning to facilitate a computational understanding of glycobiology. SweetNet explicitly incorporates the nonlinear nature of glycans and establishes a framework to map any glycan sequence to a representation. We show that SweetNet outperforms other computational methods in predicting glycan properties on all reported tasks. More importantly, we show that glycan representations, learned by SweetNet, are predictive of organismal phenotypic and environmental properties. Finally, we present a new application for glycan-focused machine learning, the prediction of viral glycan-binding, that can be used to discover new viral receptors and monitor rapidly mutating viruses.


title: HiCzin: Normalizing metagenomic Hi-C data and detecting spurious contacts using zero-inflated negative binomial regression

abstract: High-throughput chromosome conformation capture (Hi-C) has recently been applied to natural microbial communities and revealed great potential to study multiple genomes simultaneously. Several extraneous factors may influence chromosomal contacts rendering the normalization of Hi-C contact maps essential for downstream analyses. However, the current paucity of metagenomic Hi-C normalization methods and the ignorance for spurious inter-species contacts weaken the interpretability of the data. Here, we report on two types of biases in metagenomic Hi-C experiments: explicit biases and implicit biases, and introduce HiCzin, a parametric model to correct both types of biases and remove spurious inter-species contacts. We demonstrate that the normalized metagenomic Hi-C contact maps by HiCzin result in lower biases, higher capability to detect spurious contacts, and better performance in metagenomic contig clustering. The HiCzin software and Supplementary Material are available at https://github.com/dyxstat/HiCzin.


title: A modular, dynamic, DNA-based platform for regulating cargo distribution and transport between lipid domains

abstract: Cell membranes regulate the distribution of biological machinery between phase-separated lipid domains to facilitate key processes including signalling and transport, which are among the life-like functionalities that bottom-up synthetic biology aims to replicate in artificial-cellular systems. Here, we introduce a modular approach to program partitioning of amphiphilic DNA nanostructures in co-existing lipid domains. Exploiting the tendency of different hydrophobic "anchors" to enrich different phases, we modulate the lateral distribution of our devices by rationally combining hydrophobes, and by changing nanostructure size and its topology. We demonstrate the functionality of our strategy with a bio-inspired DNA architecture, which dynamically undergoes ligand-induced reconfiguration to mediate cargo transport between domains via lateral re-distribution. Our findings pave the way to next-generation biomimetic platforms for sensing, transduction, and communication in synthetic cellular systems.


title: Meeting experiments at the diffraction barrier: an in-silico widefield fluorescence microscopy

abstract: Fluorescence microscopy allows the visualization of live cells and their components, but even with advances in super- resolution microscopy, atomic resolution remains unattainable. On the other hand, molecular simulations (MS) can easily access atomic resolution, but comparison with experimental microscopy images has not been possible. In this work, a novel in-silico widefield fluorescence microscopy is proposed, which reduces the resolution of MS to generate images comparable to experiments. This technique will allow cross-validation and compound the knowledge gained from experiments and MS. We demonstrate that in-silico images can be produced with different optical axis, object focal planes, exposure time, color combinations, resolution, brightness and amount of out-of-focus fluorescence. This allows the generation of images that resemble those obtained from widefield, confocal, light-sheet, two-photon and super-resolution microscopy. This technique not only can be used as a standalone visualization tool for MS, but also lays the foundation for other in-silico microscopy methods.


title: State-Transition Analysis of Time-Sequential microRNA Expression Predicts Development of Acute Myeloid Leukemia

abstract:
MicroRNAs (miRNAs) are small non-coding RNA molecules involved in post-transcriptional regulation of gene expression and have been shown to hold prognostic value in a variety of settings, including acute myeloid leukemia (AML). However, the temporal dynamics of miRNA expression profiles as it relates to AML initiation and progression is poorly understood. Using serial samples from a mouse model of AML, we show that the miRNA transcriptome undergoes state-transition during AML initiation and progression. The AML state-transition was visualized and modeled by constructing an AML state-space from singular value decomposition of the time-series miRNA sequencing data. Within the AML state-space, we identified critical points of AML development characterized by unique differentially expressed miRNAs compared to healthy controls at critical points of leukemogenesis (early, transition, and late). Interestingly, we observed that changes in miRNA expression during leukemogenesis followed two patterns: 1) a monotonic pattern with continuously increasing or decreasing expression; and 2) a non-monotonic pattern with a local maximum or minimum at the transition critical point which was the "point of no-return" from health to AML. We validated the AML state-space and dynamics in an independent cohort of mice and demonstrated the state-transition model accurately predicted time to AML. Of note, we show that the miRNA-derived state-transition model produced a state-space and critical points that were strikingly similar, but not identical to that produced by the coding (i.e., messenger [m]RNA-based) transcriptome. This indicates that while both miRNA and mRNA expression may provide similar information, they also capture independent features of AML state-transition.

SignificanceWe show that the microRNA transcriptome undergoes a global state transition during the initiation and progression of acute myeloid leukemia, and accurately predicts time to disease development.

category: cancer biology


title: Optimal BR signalling is required for adequate cell wall orientation in the Arabidopsis root meristem

abstract: The plant steroid hormones brassinosteroids (BRs) regulate growth in part through altering the properties of the cell wall, the extracellular matrix of plant cells. Conversely, cell wall signalling connects the state of cell wall homeostasis to the BR receptor complex and modulates BR activity. Here we report that both pectin-triggered cell wall signalling and impaired BR signalling result in altered cell wall orientation in the Arabidopsis root meristem. BR-induced defects in the orientation of newly placed walls are associated with aberrant localization of the cortical division zone but with normal specification of its positioning. Tissue- specific perturbations of BR signalling revealed that the cellular malfunction is unrelated to previously described whole organ growth defects. Thus, tissue type separates the pleiotropic effects of cell wall/BR signals and highlights their importance during cell wall placement.


title: Arabidopsis annexin 5 controls plasma membrane properties in mature pollen grains

abstract: In Arabidopsis, a dry stigma surface enables a gradual hydration of pollen grains by a controlled release of water. Occasionally the grains may be exposed to extreme precipitations that cause rapid water influx, swelling and eventually lead to pollen membrane (PM) rupture. In metazoans, calcium- and phospholipids-binding proteins, referred to as annexins participate in repair of the plasma membrane damages. It remains unclear, however, how this process is conducted in plants. Here, we examined whether the plant annexin 5 (ANN5), the most abundant member of the annexin family in pollen, is involved in the restoration of PM integrity. We analyzed a cellular dynamics of ANN5 in the pollen grains undergoing in vitro and in vivo hydration. We observed a transient ANN5 association to PM during the in vitro hydration that did not occur in the pollen grains being hydrated on the stigma. To simulate a rainfall, we performed spraying of the pollinated stigma with deionized water that induced ANN5 accumulation at PM. Similarly, calcium or magnesium application affected PM properties and induced ANN5 recruitment to PM. Our data suggest a model, in which ANN5 is involved in the maintenance of membrane integrity in pollen grains exposed to osmotic or ionic imbalances.


title: Leveraging joint mechanics simplifies the neural control of movement

abstract:
Behaviors we perform every day, such as manipulating an object or walking, require precise control of interaction forces between our bodies and the environment. These forces are generated by active muscle contractions, specified by the nervous system, and by joint mechanics, determined by the intrinsic properties of the musculoskeletal system. Depending on behavioral goals, joint mechanics might simplify or complicate control of movement by the nervous system. However, whether humans can exploit joint mechanics to simplify neural control remains unclear. Here we evaluated if leveraging joint mechanics can simplify neural control by comparing performance in three tasks that required subjects to generate specified torques about the ankle during imposed sinusoidal movements; only one task required torques that could be generated by leveraging the intrinsic mechanics of the joint. We developed a novel approach that used continuous estimates of impedance, a quantitative description of joint mechanics, and measures of muscle activity to determine the mechanical and neural contributions to the net ankle torque generated in each motor task. We found that the torque resulting from changes in neural control was reduced when ankle impedance was consistent with the task being performed, resulting in a task that required less muscular effort. Subjects perceived this task to be easier than those that were not consistent with the impedance of the ankle and were able to perform it with the highest level of consistency. These results demonstrate that leveraging the mechanical properties of a joint can simplify task completion and improve performance.

KEY POINTSO_LIInteracting with our environment requires production of interaction forces, which are generated by muscle contractions, specified by the nervous system, and by joint mechanics, determined by the intrinsic properties of the musculoskeletal system.
C_LIO_LIWe assessed whether leveraging joint mechanics can simplify neural control by having subjects complete 3 tasks, only one of which could be accomplished by leveraging joint mechanics.
C_LIO_LIWe found that subjects reduced their muscular effort, perceived the task to be easier and completed the task more consistently when the mechanics of the ankle were consistent with the task.
C_LIO_LIThese results highlight the importance of considering limb mechanics when interpreting measures of neural control related to movement and may benefit the design of mechanical interfaces that optimize human performance.
C_LI


title: Hypothalamic Overexpression of Neurosecretory Protein GL Leads to Obesity in Mice

abstract:
IntroductionThe mechanisms underlying obesity are not fully understood, necessitating the creation of novel animal models for investigation of metabolic disorders from the cellular-molecular to behavioral levels of analysis. We have previously found that neurosecretory protein GL (NPGL), a newly-identified hypothalamic neuropeptide, is involved in feeding behavior and fat accumulation in rats. Given the broad availability of genetic tools in mice, the present investigation sought to establish a mouse model of NPGL-induced obesity.

MethodsWe overexpressed the NPGL-precursor gene (Npgl) in the hypothalamus using adeno-associated virus in C57BL/6J mice fed normal chow (NC) or a high-calorie diet (HCD). After 9 weeks of Npgl overexpression, we measured adipose tissues, muscle, and several organ masses in addition to food intake and body mass. To assess the effects of Npgl overexpression on peripheral tissues, we analyzed mRNA expression of lipid metabolism-related genes by quantitative RT-PCR.

ResultsNpgl overexpression increased food intake, body mass, adipose tissues and liver masses, food efficiency, and circulating insulin levels under both NC and HCD, resulting in obesity observable within 8 weeks. Furthermore, we observed fat accumulation in adipose tissues and liver. Additionally, mRNA expression of lipid metabolism-related factors was increased in white adipose tissue and the liver after Npgl overexpression.

ConclusionTaken together, the present study suggests that NPGL is an endogenous obesogenic factor that acts within a short period of time in mice. As a result, this animal model can be widely applied to study the etiology of obesity from genes to behavior.


title: Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

abstract: Previously, we showed that cholinergic interneurons of the dorsal striatum lose cilia in mice harboring the Parkinsons disease associated, kinase activating, R1441C LRRK2 mutation (Dhekne et al., 2018). Here we show that this phenotype is also seen in two mouse strains carrying the most common human G2019S LRRK2 mutation. Heterozygous loss of the PPM1H phosphatase that is specific for LRRK2-phosphorylated Rab GTPases (Berndsen et al., 2019) yields the same cilia loss phenotype, strongly supporting a connection between Rab GTPase phosphorylation and cilia loss. In addition, astrocytes throughout the striatum show a ciliation defect in LRRK2 and PPM1H-/+ mutant models. Hedgehog signaling requires cilia, and loss of cilia correlates here with a loss in induction of Hedgehog signaling as monitored by in situ hybridization of Gli1 transcripts. These data support a model in which LRRK2 and PPM1H mutant mice struggle to receive and respond to critical Hedgehog signals in the nigral-striatal pathway.


title: Metabolic dysfunction induced by high-fat diet modulates hematopoietic stem and myeloid progenitor cells in brown adipose tissue of mice

abstract: Brown adipose tissue (BAT) may be an important metabolic regulator of whole-body glucose. While important roles have been ascribed to macrophages in regulating metabolic functions in BAT, little known is known of the roles of other immune cells subsets, particularly dendritic cells (DCs). Eating a high fat diet may compromise the development of hematopoietic stem and progenitor cells (HSPC) - which give rise to DCs - in bone marrow, with less known of its effects in BAT. We have previously demonstrated that ongoing exposure to low-dose ultraviolet radiation (UVR) significantly reduced the  whitening effect of eating a high-fat diet upon interscapular (i)BAT of mice. Here, we examined whether this observation may be linked to changes in the phenotype of HSPC and myeloid-derived immune cells in iBAT and bone marrow of mice using 12-colour flow cytometry. Many HSPC subsets declined in both iBAT and bone marrow with increasing metabolic dysfunction. Conversely, with rising adiposity and metabolic dysfunction, conventional (c)DCs increased in both of these tissues. When compared to low-fat diet, consumption of high-fat diet significantly reduced proportions of myeloid, common myeloid and megakaryocyte-erythrocyte progenitors in iBAT, and short-term hematopoietic stem cells in bone marrow. In mice fed a high-fat diet, exposure to low-dose UVR significantly reduced proportions of cDCs in iBAT, independently of nitric oxide release from irradiated skin (blocked using the scavenger, cPTIO), but did not significantly modify HSPC subsets in either tissue. Further studies are needed to determine whether changes in these cell populations contribute towards metabolic dysfunction.


title: Plant hydraulics coordinated with photosynthetic traits and climate

abstract: The coupling between water loss and carbon dioxide uptake drives the coordination of plant hydraulic and photosynthetic traits. Analysing multi-species measurements on a 3000 m elevation gradient, we found that hydraulic and leaf-economic traits were less plastic, and more closely associated with phylogeny, than photosynthetic traits. The two trait sets are linked by the sapwood-to-leaf area ratio (Huber value, vH), shown here to be codetermined by sapwood hydraulic conductance (KS), leaf mass-per-area (LMA) and photosynthetic capacity (Vcmax). Substantial hydraulic diversity was related to the trade-off between KS and vH. Leaf drought tolerance (inferred from turgor loss point, -{pi}tlp) increased with wood density, but the trade-off between hydraulic efficiency (KS) and -{pi}tlp was weak. The least-cost optimality framework was extended to predict trait (KS-dominated) and environmental (temperature-dominated) effects on vH. These results suggest an approach to include photosynthetic-hydraulic coordination in land-surface models; however, prediction of non-plastic trait distributions remains a challenge.


title: Dynamic neurogenomic responses to social interactions and dominance outcomes in female paper wasps

abstract: Social interactions have large effects on individual physiology and fitness. In the immediate sense, social stimuli are often highly salient and engaging. Over longer time scales, competitive interactions often lead to distinct social ranks and differences in physiology and behavior. Understanding how initial responses lead to longer-term effects of social interactions requires examining the changes in responses over time. Here we examined the effects of social interactions on transcriptomic signatures at two points, at the end of a 45-minute interaction and 4 hours later, in female Polistes fuscatus paper wasp foundresses. Female P. fuscatus have variable facial patterns that are used for visual individual recognition, so we separately examined the transcriptional dynamics in the optic lobe and the central brain. Results demonstrate much stronger transcriptional responses to social interactions in the central brain compared to the optic lobe. Differentially regulated genes in response to social interactions are enriched for memory-related transcripts. Comparisons between winners and losers of the encounters revealed similar overall transcriptional profiles at the end of an interaction, which significantly diverged over the course of 4 hours, with losers showing changes in expression levels of genes associated with aggression and reproduction in paper wasps. On nests, subordinate foundresses are less aggressive, do more foraging and lay fewer eggs compared to dominant foundresses and we find losers shift expression of many genes, including vitellogenin, related to aggression, worker behavior, and reproduction within hours of losing an encounter. These results highlight the early neurogenomic changes that likely contribute to behavioral and physiological effects of social status changes in a social insect.

category: genomics


title: High-content screening of coronavirus genes for innate immune suppression revealsenhanced potency of SARS-CoV-2 proteins

abstract: Suppression of the host intracellular innate immune system is an essential aspect of viral replication. Here, we developed a suite of medium-throughput high-content cell-based assays to reveal the effect of individual coronavirus proteins on antiviral innate immune pathways. Using these assays, we screened the 196 protein products of seven coronaviruses (SARS-CoV-2, SARS-CoV-1, 229E, NL63, OC43, HKU1 and MERS). This includes a previously unidentified gene in SARS-CoV-2 encoded within the Spike gene. We observe immune-suppressing activity in both known host-suppressing genes (e.g., NSP1, Orf6, NSP3, and NSP5) as well as other coronavirus genes, including the newly identified SARS-CoV-2 protein. Moreover, the genes encoded by SARS-CoV-2 are generally more potent immune suppressors than their homologues from the other coronaviruses. This suite of pathway-based and mechanism-agnostic assays could serve as the basis for rapid in vitro prediction of the pathogenicity of novel viruses based on provision of sequence information alone.


title: FAM57B is a modulator of ceramide synthesis that regulates sphingolipid homeostasis and synaptic composition in the developing brain

abstract: The complex 16p11.2 Deletion Syndrome (16pdel) is accompanied by neurological disorders, including epilepsy, autism spectrum disorder and intellectual disability. We demonstrate that 16pdel iPSC differentiated neurons showed augmented local field potential activity and altered ceramide-related lipid species relative to unaffected. FAM57B, a poorly characterized gene in the 16p11.2 interval, has emerged as a candidate tied to symptomatology. We found that FAM57B modulates ceramide synthase (CerS) activity, but is not a CerS per se. In FAM57B mutant human neuronal cells and zebrafish brain, composition and levels of sphingolipids and glycerolipids associated with cellular membranes are disrupted. Consistently, we observed aberrant plasma membrane architecture and synaptic protein mislocalization, which were accompanied by depressed brain and behavioral activity. Together, these results suggest that haploinsufficiency of FAM57B contributes to changes in neuronal activity and function in 16pdel Syndrome, through a crucial role for the gene in lipid metabolism.


title: Protection against neonatal respiratory viral infection via maternal treatment during pregnancy with the benign immune training agent OM-85

abstract:
ObjectivesIncomplete maturation of immune regulatory functions at birth are antecedent to the heightened risk for severe respiratory infections during infancy. Our forerunner animal model studies demonstrated that maternal treatment with the benign microbial-derived immune modulating agent OM-85 during pregnancy promotes accelerated maturation of immune regulatory networks in the developing fetal bone marrow. Here, we aimed to establish proof-of-concept that this would enhance resilience to severe early life respiratory viral infection during the neonatal period.

MethodsPregnant BALB/c mice were treated orally with OM-85 during gestation and their offspring infected intranasally with a mouse-adapted rhinovirus (vMC0) at postnatal day 2. We then assessed clinical course, lung viral titres and lung immune parameters to determine whether offspring from OM-85 treated mothers demonstrate enhanced immune protection against neonatal vMC0 infection.

ResultsOffspring from OM-85 treated mothers display enhanced capacity to clear an otherwise lethal respiratory viral infection during the neonatal period, with a concomitant reduction in the exaggerated nature of the ensuing immune response. These treatment effects were associated with accelerated postnatal myeloid cell seeding of neonatal lungs and enhanced expression of microbial sensing receptors in lung tissues, coupled in particular with enhanced capacity to rapidly expand and maintain networks of lung dendritic cells expressing function-associated markers crucial for maintenance of local immune homeostasis in the face of pathogen challenge.

ConclusionMaternal OM-85 treatment may represent a novel therapeutic strategy to reduce the burden, and potential long-term sequlae, of severe neonatal respiratory viral infection by accelerating development of innate immune competence.


title: Intrinsic cardiac adrenergic cell contributes to septic cardiomyopathy

abstract: Occurring independently of cardiac sympathetic nervous system, the intrinsic cardiac adrenergic (ICA) cells have been identified as an important regulator in both of developing and adult cardiac physiological and pathological processes. However, its role in septic cardiomyopathy remains unknown. Herein, we report that lipopolysaccharide (LPS) dose- and time-dependently increased norepinephrine (NE) release from ICA cells, which aggravates myocardial TNF- production and dysfunction. Inhibition of NE synthesis in ICA cells alleviated LPS-elicited cardiac dysfunction as well as TNF- production in Langendorff perfusing hearts. Mechanistically, ICA cell expressed Toll-like receptor 4 (TLR4), activated by LPS, to increase the expression of tyrosine hydroxylase, a key enzyme responsible for NE biosynthesis, via AP-1 binding to its promoter. Surprisingly, LPS-TLR4 signaling triggered no TNF- production in ICA cells due to the elevated Nfkbia and Tnfaip6 expression. In LPS-treated co-culture of ICA cells and cardiomyocytes, the raised NE from ICA cells activated cardiomyocyte {beta}1-adrenergic receptor ({beta}1-AR), driving Ca2+/calmodulin-dependent protein kinase II (CaMKII) to increase the activities of NF-{kappa}B and mitogen-activated protein kinase pathways, which were mimicked by dobutamine. Our findings reveal a cell type-specific TLR4 function triggering NE synthesis, but not TNF- production in inflammatory pathogenesis, and identify ICA cell-derived NE as a paracrine signal in the cross talk among different cardiac cells to enhance myocardial injury during LPS challenge, suggesting that targeting ICA cell-derived NE may be a potential therapeutic strategy for septic cardiomyopathy.


title: Intrinsic cardiac adrenergic cells contribute to septic cardiomyopathy


title: Factors influencing maternal microchimerism throughout infancy and its impact on infant T cell immunity

abstract:
Determinants of the acquisition and maintenance of maternal microchimerism (MMc) during infancy and the impact of MMc on infant immune responses are unknown. We examined factors which influence MMc detection and level across infancy and the effect of MMc on T cell responses to BCG vaccination in a cohort of HIV exposed, uninfected and HIV unexposed infants in South Africa. MMc was measured in whole blood from 58 infants using a panel of quantitative PCR assays at day one and 7, 15, and 36 weeks of life. Infants received BCG at birth, and selected whole blood samples from infancy were stimulated in vitro with BCG and assessed for polyfunctional CD4+ T cell responses. MMc was present in most infants across infancy with levels ranging from 0-1,193/100,000 genomic equivalents and was positively impacted by absence of maternal HIV, maternal-infant HLA compatibility, infant female sex, and exclusive breastfeeding. Initiation of maternal antiretroviral therapy prior to pregnancy partially restored MMc level in HIV exposed, uninfected infants. Birth MMc was associated with an improved polyfunctional CD4+ T cell response to BCG. These data emphasize that both maternal and infant factors influence the level of MMc, which may subsequently impact infant T cell responses.

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: High resolution profiling of MHC-II peptide presentation capacity, by Mammalian Epitope Display, reveals SARS-CoV-2 targets for CD4 T cells and mechanisms of immune-escape

abstract: Understanding the mechanisms of immune evasion is critical for formulating an effective response to global threats like SARS-CoV2. We have fully decoded the immune synapses for multiple TCRs from acute patients, including cognate peptides and the presenting HLA alleles. Furthermore, using a newly developed mammalian epitope display platform (MEDi), we determined that several mutations present in multiple viral isolates currently expanding across the globe, resulted in reduced presentation by multiple HLA class II alleles, while some increased presentation, suggesting immune evasion based on shifting MHC-II peptide presentation landscapes. In support, we found that one of the mutations present in B1.1.7 viral strain could cause escape from CD4 T cell recognition in this way. Given the importance of understanding such mechanisms more broadly, we used MEDi to generate a comprehensive analysis of the presentability of all SARS-CoV-2 peptides in the context of multiple common HLA class II molecules. Unlike other strategies, our approach is sensitive and scalable, providing an unbiased and affordable high-resolution map of peptide presentation capacity for any MHC-II allele. Such information is essential to provide insight into T cell immunity across distinct HLA haplotypes across geographic and ethnic populations. This knowledge is critical for the development of effective T cell therapeutics not just against COVID-19, but any disease.


title: Vaccination with SARS-CoV-2 Spike Protein and AS03 Adjuvant Induces Rapid Anamnestic Antibodies in the Lung and Protects Against Virus Challenge in Nonhuman Primates

abstract: Adjuvanted soluble protein vaccines have been used extensively in humans for protection against various viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized spike trimers (preS dTM) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) were formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHP). Binding and functional neutralization assays and systems serology revealed that NHP developed AS03-dependent multi-functional humoral responses that targeted multiple spike domains and bound to a variety of antibody FC receptors mediating effector functions in vitro. Pseudovirus and live virus neutralizing IC50 titers were on average greater than 1000 and significantly higher than a panel of human convalescent sera. NHP were challenged intranasally and intratracheally with a high dose (3x106 PFU) of SARS-CoV-2 (USA-WA1/2020 isolate). Two days post-challenge, vaccinated NHP showed rapid control of viral replication in both the upper and lower airways. Notably, vaccinated NHP also had increased spike-specific IgG antibody responses in the lung as early as 2 days post challenge. Moreover, vaccine-induced IgG mediated protection from SARS-CoV-2 challenge following passive transfer to hamsters. These data show that antibodies induced by the AS03-adjuvanted preS dTM vaccine are sufficient to mediate protection against SARS-CoV-2 and support the evaluation of this vaccine in human clinical trials.


title: A heterohexameric protein consisting of six linked single-domain antibodies is highly protective for BoNT/A, BoNT/B and BoNT/E exposures

abstract:
Botulinum neurotoxin (BoNT) serotypes A, B and E cause the vast majority of human botulism cases and pose the greatest bioterrorism threats. We previously identified multiple camelid single-domain antibodies (VHHs) that each neutralize BoNT/A, BoNT/B or BoNT/E. We also demonstrated that heterodimers of linked toxin-neutralizing VHHs are much more potent than VHH monomer pools in preventing BoNT intoxication. In this study, we expressed two different heterohexamer proteins (VNA1-ABE and VNA2-ABE) of ~100 kDa secreted from mammalian host cells, each containing the same six linked anti-BoNT VHH components ordered in two different combinations. Each heterohexamer contained two VHHs that neutralize BoNT/A, BoNT/B or BoNT/E. Both heterohexameric antitoxins displayed similar strong binding properties for the three targeted BoNT serotypes by ELISA. One ug of each heterohexameric antitoxin fully protected groups of mice co-administered with 100 LD50 of BoNT/A, BoNT/B or BoNT/E, or a pool containing 100 LD50 of each of the three toxins. The results demonstrate that long chains of at least six different linked VHHs can be expressed such that all component VHHs in the multimer retain their target binding activities. These findings make more feasible the development of a BoNT antitoxin product consisting of a small pool of proteins that, in combination, neutralize all known BoNT serotypes and subtypes.

Key ContributionHeteromultimeric proteins consisting of six linked, VHH antibodies, and including VHHs that neutralize BoNT/A, BoNT/B and BoNT/E, retain high potency to protect mice challenged with high doses of all three of these BoNT serotypes.


title: Compositional analysis of ALS-linked stress granule-like structures reveals factors and cellular pathways dysregulated by mutant FUS under stress

abstract: Formation of cytoplasmic RNA-protein structures called stress granules (SGs) is a highly conserved cellular response to stress. Abnormal metabolism of SGs may contribute to the pathogenesis of (neuro)degenerative diseases such as amyotrophic lateral sclerosis (ALS). Many SG proteins are affected by mutations causative of these conditions, including fused in sarcoma (FUS). Mutant FUS variants have high affinity to SGs and also spontaneously form de novo cytoplasmic RNA granules. Mutant FUS-containing assemblies (mFAs), often called "pathological SGs", are proposed to play a role in ALS-FUS pathogenesis. However, global structural differences between mFAs and physiological SGs remain largely unknown, therefore it is unclear whether and how mFAs may affect cellular stress responses. Here we used affinity purification to characterise the protein and RNA composition of normal SGs and mFAs purified from stressed cells. Comparison of the SG and mFA proteomes revealed that proteasome subunits and certain nucleocytoplasmic transport factors are depleted from mFAs, whereas translation elongation, mRNA surveillance and splicing factors as well as mitochondrial proteins are enriched in mFAs, as compared to SGs. Validation experiments for a hit from our analysis, a splicing factor hnRNPA3, confirmed its RNA-dependent sequestration into mFAs in cells and into pathological FUS inclusions in a FUS transgenic mouse model. Furthermore, silencing of the Drosophila hnRNPA3 ortholog dramatically enhanced FUS toxicity in transgenic flies. Comparative transcriptomic analysis of SGs and mFAs revealed that mFAs recruit a significantly less diverse spectrum of RNAs, including reduced recruitment of transcripts encoding proteins involved in protein translation, DNA damage response, and apoptotic signalling. However mFAs abnormally sequester certain mRNAs encoding proteins involved in stress signalling cascades. Overall, our study establishes molecular differences between physiological SGs and mFAs and identifies the spectrum of proteins, RNAs and respective cellular pathways affected by mFAs in stressed cells. In conclusion, we show that mFAs are compositionally distinct from SGs and that they cannot fully substitute for SG functions while gaining novel, potentially toxic functions in cellular stress response. Results of our study support a pathogenic role for stress-induced cytoplasmic FUS assemblies in ALS-FUS.