title: Analysis of community connectivity in spatial transcriptomics data

abstract:
The advent of high throughput spatial transcriptomics (HST) has allowed for unprecedented characterization of spatially distinct cell communities within a tissue sample. While a wide range of computational tools exist for detecting cell communities in HST data, none allow for characterization of community connectivity, i.e., the relative similarity of cells within and between found communities - an analysis task that can elucidate cellular dynamics in important settings such as the tumor microenvironment. To address this gap, we introduce the concept of analysis of community connectivity (ACC), which entails not only labeling distinct cell communities within a tissue sample, but understanding the relative similarity of cells within and between communities. We develop a Bayesian multi-layer network model called BANYAN for integration of spatial and gene expression information to achieve ACC. We use BANYAN to implement ACC in invasive ductal carcinoma, and uncover distinct community structure relevant to the interaction of cell types within the tumor microenvironment. Next, we show how ACC can help clarify ambiguous annotations in a human white adipose tissue sample. Finally, we demonstrate BANYANs ability to recover community connectivity structure via a simulation study based on real sagittal mouse brain HST data.

AvailabilityAn R package banyan is available at https://github.com/carter-allen/banyan.

Contactchung.911@osu.edu

Supplementary informationSupplementary data are available online.

Author SummaryThe proliferation of spatial transcriptomics technologies have prompted the development of numerous statistical models for characterizing the makeup of a tissue sample in terms of distinct cell sub-populations. However, existing methods regard inferred sub-populations as static entities and do not offer any ability to discover the relative similarity of cells within and between communities, thereby obfuscating the true interactive nature of cells in a tissue sample. We develop BANYAN: a statistical model for implementing analysis of community connectivity (ACC), i.e., the process of inferring the similarity of cells within and between cell sub-populations. We demonstrate the utility of ACC through the analysis of a publicly available breast cancer data set, which revealed distinct community structure between tumor suppressive and invasive cancer cell sub-populations. We then showed how ACC may help elucidate ambiguous sub-population annotations in a publicly available human white adipose tissue data set. Finally, we implement a simulation study to validate BANYANs ability to recover true community connectivity structure in HST data.


title: Multimodal single cell data integration challenge: results and lessons learned

abstract: Biology has become a data-intensive science. Recent technological advances in single-cell genomics have enabled the measurement of multiple facets of cellular state, producing datasets with millions of single-cell observations. While these data hold great promise for understanding molecular mechanisms in health and disease, analysis challenges arising from sparsity, technical and biological variability, and high dimensionality of the data hinder the derivation of such mechanistic insights. To promote the innovation of algorithms for analysis of multimodal single-cell data, we organized a competition at NeurIPS 2021 applying the Common Task Framework to multimodal single-cell data integration. For this competition we generated the first multimodal benchmarking dataset for single-cell biology and defined three tasks in this domain: prediction of missing modalities, aligning modalities, and learning a joint representation across modalities. We further specified evaluation metrics and developed a cloud-based algorithm evaluation pipeline. Using this setup, 280 competitors submitted over 2600 proposed solutions within a 3 month period, showcasing substantial innovation especially in the modality alignment task. Here, we present the results, describe trends of well performing approaches, and discuss challenges associated with running the competition.

category: bioinformatics


title: Super-Resolution NMR Spectroscopy using the Intersection of Non-Redundant Information on Resonance Groups

abstract: The resolution of spectra is a major limitation in the application of Nuclear Magnetic Resonance (NMR) spectroscopy to large and complex molecular systems. In this report, we introduce a technique to enhance the resolution of NMR spectra beyond the intrinsic limitations of a spectrometer for a single spectrum by using the Intersection of Non-Redundant Information on Resonance Groups (INIR). With INIR, we reconstruct 900-MHz (21.1T) spectra from a 500-MHz (11.7T) NMR spectrometer, which compare favorably to experimental 900-MHz spectra. INIR holds promise in significantly enhancing the resolution of NMR spectra and in extending the size and complexity of molecules studied by NMR.

category: biophysics


title: Long-range morphogen gradient formation by cell-to-cell signal propagation

abstract: Morphogen gradients are a central concept in developmental biology. Their formation often involves the secretion of morphogens from a local source, that spread by diffusion in the cell field, where molecules eventually get degraded. This implies limits to both the time and length scales over which morphogen gradients can form which are set by diffusion coefficients and degradation rates. Towards the goal of identifying plausible mechanisms capable of extending the gradient range, we here use theory to explore properties of a cell-to-cell signaling relay. Inspired by the millimeter-scale Wnt-expression and signaling gradients in flatworms, we consider morphogen-mediated morphogen production in the cell field. We show that such a relay can generate stable morphogen and signaling gradients that emanate from a source of morphogen at a boundary. This gradient formation can be related to an effective diffusion and an effective degradation that result from morphogen production due to signaling relay. If the secretion of morphogen produced in response to the relay is polarized, it further gives rise to an effective drift. We find that signaling relay can generate long-ranged gradients in relevant times without relying on extreme choices of diffusion coefficients or degradation rates, thus exceeding the limits set by physiological diffusion coefficients and degradation rates. A signaling relay is hence an attractive principle to conceptualize long-ranged gradient formation by slowly diffusing morphogens that are relevant for patterning in adult contexts such as regeneration and tissue turn-over.


title: Pharmacological Interrogation of the C. elegans Pharyngeal Cuticle Reveals Small Molecule Amyloid Disruptors

abstract: Understanding how to pharmacologically manipulate amyloids is an essential step towards managing dozens of amyloid-based human diseases. Here, we present a pipeline that yields small molecule antagonists of amyloidogenesis. Screens for small molecules that perturb the development of the nematode C. elegans revealed 24 distinct small molecule scaffolds that crystalize on the non-luminal face of the pharyngeal cuticle. Consistent with the amyloid-like nature of the pharyngeal cuticle, 25% of these scaffolds are known to bind human amyloids with nanomolar affinity. The growing crystals preferentially disrupt the amyloid-like material within the cuticle but leave its chitin-based matrix relatively intact. We screened a collection of drugs and natural products for those that suppress crystal formation and found 45 distinct scaffolds, 33% of which are known amyloid busters. Screening for pharmacological suppressors of crystal formation therefore represents an inexpensive high-throughput in vivo approach that enriches for small molecules with amyloid-busting potential.

category: pharmacology and toxicology


title: Metabolite profiles across populations of Palmer amaranth (Amaranthus palmeri) highlight the specificity and inducibility of phytochemical response to glyphosate stress.

abstract:
Modifications of the phytochemical profile form a vital component of physiological stress adaptation in plants. However, the specificity and uniqueness of phytochemical changes with respect to the identity of stressors is less known. Here, we investigated the commonality and specificity of metabolic perturbations induced by a specific stressor - glyphosate, and a general stressor - drought, across multiple glyphosate-resistant (GR) and -susceptible (GS) biotypes of a dominant agricultural weed, Amaranthus palmeri. In the absence of stress, the native metabolite profile of GS- and GR-biotypes was similar, and amplification of the EPSPS gene in GR-biotypes did not translate to a higher abundance of downstream metabolites. Further, glyphosate treatment initially inhibited the shikimate pathway in both GS- and GR-biotypes, from which the GR-biotypes recovered, indicating inducibility in the functionalization of the EPSPS enzyme. The accumulation of phenylpropanoids produced downstream of the shikimate pathway, was higher in GR-biotypes than GS-biotypes, with a preferential accumulation of compounds with higher antioxidant potential. However, this increase was not observed in response to drought treatment, where the metabolic perturbations were pervasive but limited in magnitude compared to glyphosate stress. Overall, while native phytochemistry of A. palmeri was similar irrespective of the level of glyphosate susceptibility, the specific stressor, glyphosate, imparted metabolic perturbations that were localized but higher in magnitude, while the specificity of phytochemical response to the general stressor, drought, was minimal. Taken together, these results suggest that, at the metabolic level, the glyphosate resistance mechanism in A. palmeri is partly induced and specific to herbicide stress.

SIGNIFICANCE STATEMENTUnderstanding changes in physiology, especially those related to secondary metabolites with adaptogenic functions, is imperative to decipher the basis of stress adaptation in plants. This study provides critical information on native and stress-induced phytochemical differences between multiple glyphosate-resistant and -susceptible weed biotypes, thus, shedding light on the metabolome-level orchestration of gene amplification-mediated glyphosate resistance mechanism in an economically devastating weed, Palmer amaranth (Amaranthus palmeri).

category: plant biology


title: The Temporal Dynamics of Willed Attention in Vision

abstract: Most models of attention distinguish between voluntary and involuntary attention, the latter being driven in a bottom-up fashion by salient sensory signals. Studies of voluntary visual-spatial attention have used informational or instructional cues, such as arrows, to induce or instruct observers to direct selective attention to relevant locations in visual space in order to detect or discriminate subsequent target stimuli. In everyday vision, however, voluntary attention is influenced by a host of factors, most of which are quite different from the laboratory paradigms that utilize attention-directing cues. These factors include priming, experience, reward, meaning, motivations, and high-level behavioral goals. Attention that is endogenously directed in the absence of external cues has been referred to as self-initiated attention, or in our prior work as "willed attention". Such studies typically replace attention-directing cues with a "prompt" that signals the subject when to choose where they will attend in preparation for the upcoming target stimulus. We used a novel paradigm that was designed to minimize external influences (i.e., cues or prompts) as to where, as well as when, spatial attention would be shifted and focused. Participants were asked to view bilateral dynamic dot motion displays, and to shift their covert spatial attention to either the left or right visual field patch at a time of their own choosing, thus allowing the participants to control both when and where they attended on each trial. The task was to discriminate and respond to a pattern in the attended dot motion patch. Our goal was to identify patterns of neural activity in the scalp-recorded EEG that revealed when and where attention was focused. Using machine learning methods to decode attention-related EEG alpha band activity, we were able to identify the onset of voluntary (willed) shifts of visual-spatial attention, and to determine where attention was focused. This work contributes to our understanding of the neural antecedents of voluntary attention, opening the door for improved models of attentional control, and providing steps toward development of brain-computer interfaces using non-invasive electrical recordings of brain activity.

category: neuroscience


title: Evidence for shared resources in visuospatial attention: Endogenous attention misleads the decoding of exogenous attention in alpha rhythms

abstract:
Prevailing accounts of visuospatial attention differentiate exogenous orienting, which corresponds to involuntary shifts of attention, from endogenous orienting, which reflects the voluntary control of attention. While these different forms of attention are functionally separable, evidence shows that they can nevertheless interact with each other. These dynamics have been at the center of ongoing debates for more than two decades. One hypothesis regarding these interactions is that, while both forms of attention orienting map onto independent functional systems, they are capable of mutual interference. This viewpoint implies that exogenous attention, a ballistic and stimulus-driven process, is not impervious to the influence of endogenous attention. Evidence in support of this view remains sparse and the mechanisms that underlie these interactions remain largely unknown. The present study tackles these questions by examining whether endogenous attention influences the neural processes of exogenous attention using multivariate pattern classification to electroencephalography (EEG). We contrasted the effects of exogenous attention across two experimental conditions: Single cueing where exogenous attention is engaged alone, and double cueing where exogenous attention is concurrently engaged with endogenous attention. Our results show that the simultaneous engagement of endogenous attention alters the emergence of exogenous attention across cue-related and target-related neural processes. Exogenous attention is therefore not impervious to the influence of endogenous attention. More importantly, we observed that classifiers trained to decode exogenous attention from the power and phase of alpha band activity during single cueing condition were systematically misled by endogenous attention when tested in the context of double cueing. Hence, both the power and phase of alpha rhythms are central to exogenous and endogenous attention. These findings not only highlight overlapping neural processes between both form of attention orienting, but further demonstrate that the dynamics of exogenous and endogenous attention are characterized by limited processing resources.

Significance StatementVisuospatial attention is often dichotomized into top-down and bottom-up components: Top-down attention reflects slow voluntary shifts while bottom-up attention corresponds to fast involuntary demands. A large body of findings supports the view that these forms of attention orienting are functionally separable. Following longstanding debates about their interactions, the current study examines the relationship between top-down and bottom-up attention. Using electroencephalography (EEG) and multivariate pattern classification techniques, the researchers show that top-down attention modified the brain activity of bottom-up attention. Moreover, machine learning classifiers trained to decode bottom-up attention based on alpha band activity (8-12 Hz), a well-known marker of visuospatial attention, are systematically misled by top-down attention. The researchers conclude that both forms of visuospatial orienting are supported by several overlapping neural processes, which implies shared neural resources between them.


title: Sex-specific differences in rotarod performance and type 1 cannabinoid receptor levels in a rat model of traumatic brain injury treated with {triangleup}9-tetrahydrocannabinol

abstract:
Traumatic brain injuries (TBI) remain one of the leading causes of death and disability world-wide. One emerging area of TBI research is the involvement of the endocannabinoid system (ECS) in response to TBI. Endogenous cannabinoids modulate inflammation, pain, anxiety, and neurotransmitter release through the activation of the cannabinoid receptors CB1R and CB2R. CB1R and CB2R are activated by exogenous cannabinoids such as {Delta}9-tetrahydrocannabinol (THC) found in Cannabis sativa. As public perceptions change in the wake of Cannabis legalization, research into the potential harmful and therapeutic effects of THC following TBI deserve exploration. In this preliminary study, we investigated sex differences in behavioral effects, CB1R abundance, and cytokine profiles in a rat model of moderate TBI treated with 1 mg{middle dot}kg-1 THC (i.p.). Neither TBI nor THC treatment altered catalepsy, body temperature, nociception, or spontaneous alternation as measured in the y-maze. TBI reduced male rotarod performance in both vehicle and THC-treated groups, and THC treatment decreased performance in Sham-TBI rats when compared to vehicle controls. Female rats that received a TBI and THC exhibited lower relative CB1R density when compared to the Sham-TBI+THC group. TBI was associated with reduced interleukin-4 in males; THC increased interleukin-6 in TBI males compared to Sham-TBI. These preliminary results highlight fundamental sex differences in the response of the ECS following TBI. Our results indicate the need for further investigation of the ECS and phytocannabinoids post-TBI in both acute and chronic phases.

Significance StatementThe endogenous cannabinoid system is a potential target in the pathophysiology and treatment of traumatic brain injury (TBI). In this study we observed TBI reduced rotarod performance in male rats only and performance was not affected by THC. Female rats the received THC and TBI displayed lower cortical cannabinoid receptor 1 levels. These early results showcase sex differences in rodent models of TBI and the endogenous cannabinoid system.


title: Electrophysiological normative responses to emotional, neutral, and cigarette-related images.

abstract: To create reproducible emotional probes, affective scientists rely on sets of standardized pictures that are normed using subjective ratings of valence and emotional arousal. Yet, to investigate psychophysiological emotional responses, it might be more appropriate to select pictures using normative neurophysiological responses rather than normative subjective ratings. Here, we provide electrophysiological normative responses for 323 emotional pictures (215 from the IAPS) covering a wide range of categories (erotica, romantic, appetizing foods, landscapes, people engaged in mundane activities, household objects, disgusting objects, accidents, sad people, violence, mutilations, and cigarette-related contents). Event-related potentials (ERPs) and subjective ratings of pleasure and emotional arousal were collected from 763 individuals (52% females, 41% white) aged between 18 and 65 (mean = 43). For each image, the mean amplitude of the late positive potential (LPP, an electrophysiological index of motivational relevance) and the mean subjective ratings of valence and arousal were calculated. We validated our procedure by showing that the subjective ratings of valence and arousal from this sample were highly correlated to the IAPS published norms (Pearson r=.97 for pleasure and r=.82 for emotional arousal). LPP responses and subjective ratings of emotional arousal also were correlated (Pearson r = .61), but some categories that participants reported being significantly more arousing than neutral (i.e., food, landscapes, and unpleasant objects) did not evoke LPPs significantly different from those evoked by neutral pictures. Researchers interested in probing the brains affective systems can use these electrophysiological normative responses to create emotional probes that evoke reliable neuroaffective responses.


title: Olfactory receptor neurons generate multiple response motifs, increasing coding space dimensionality

abstract: Odorants binding to olfactory receptor neurons (ORNs) trigger bursts of action potentials, providing the brain with its only experience of the olfactory environment. Our recordings made in vivo from locust ORNs showed odor-elicited firing patterns comprise four distinct response motifs, each defined by a reliable temporal profile. Different odorants could elicit different response motifs from a given ORN, a property we term motif switching. Further, each motif undergoes its own form of sensory adaptation when activated by repeated plume-like odor pulses. A computational model constrained by our recordings revealed that organizing responses into multiple motifs provides substantial benefits for classifying odors and processing complex odor plumes: each motif contributes uniquely to encode the plumes composition and structure. Multiple motifs and motif switching further improve odor classification by expanding coding dimensionality. Our model demonstrated these response features could provide benefits for olfactory navigation, including determining the distance to an odor source.


title: A discovery pipeline for identification and in vivo validation of drugs that alter T cell/ dendritic cell interaction

abstract: The interaction between T cells and dendritic cells (DC) is central to determining whether or not an adaptive immune response develops. Autoimmune diseases like rheumatoid arthritis result from inappropriate activation of self-antigen specific T cells, therefore targeting the interaction between T cells and DCs is of considerable therapeutic interest. Here, we describe the development and validation of a drug development platform that includes a high-throughput image-based screening assay and a complementary in vivo model. The in vitro assay directly measures the ability of compounds to influence the interaction of T cells with DC, both negatively and positively. While the in vivo model allows the validation of hit molecules directly in the lymph nodes of intact animals. Using this pipeline, we identify a new function for the protein kinase inhibitor AS703569 by inhibiting T cell/ DC interactions in vitro and in vivo.

category: immunology


title: Transient Oxygen Exposure Causes Profound and Lasting Changes to a Benzene-Degrading Methanogenic Community

abstract:
We investigated the impact of oxygen on a strictly anaerobic, methanogenic benzene-degrading enrichment culture derived decades ago from oil-contaminated sediment. The culture includes a benzene fermenter from Deltaproteobacteria Candidate clade Sva0485 (referred to as ORM2) and methanogenic archaea. A relatively small one-time injection of air, simulating a small leak into a batch culture bottle, had no measurable impact on benzene degradation rates, although retrospectively, a tiny enrichment of aerobic taxa was detected. A subsequent 100 times larger injection of air stalled methanogenesis and caused drastic perturbation of the microbial community. A benzene-degrading Pseudomonas became highly enriched and consumed benzene and all available oxygen. Anaerobic benzene-degrading ORM2 cell numbers plummeted during this time; re-growth and associated recovery of methanogenic benzene degradation took almost one year. These results highlight the oxygen-sensitivity of this methanogenic culture and confirm that the mechanism for anaerobic biotransformation of benzene is independent of oxygen, fundamentally different from established aerobic pathways, and is carried out by distinct microbial communities. The study further highlights the importance of including microbial decay in characterizing and modelling and mixed microbial communities.

SYNOPSISMethanogenic benzene degradation in a highly enriched anaerobic consortium was inhibited for a year after transient exposure to oxygen, causing mass decay of benzene-fermenting bacteria.

GRAPHIC FOR ABSTRACT ART O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

category: microbiology


title: Differences in the genetic structure between and within two landlocked Ayu groups with different migration patterns in Lake Biwa revealed by environmental DNA analysis

abstract: Ayu, Plecoglossus altivelis, is largely an annual amphidromous fish, although a landlocked population lives in Lake Biwa, the largest lake in Japan. The landlocked population comprises two migrant groups, spring migrants and autumn migrants, which run to inlet rivers from the lake at different seasons. We used environmental DNA (eDNA) analysis, which is reported to be more sensitive and cost-effective than capture surveys, to clarify the genetic structure of this landlocked Ayu population with different migration patterns in Lake Biwa. We took water samples in 11 inlet rivers in the spring and autumn for two years in a row and quantitatively detected a total of 265 haplotypes of the mitochondrial D-loop region. The pairwise fixation index (FST) value and haplotype diversity indicated that there were genetic differences between the two migrant groups in their respective rivers, and the FST values were negatively related to latitude and the presence of artificial fish release. Additionally, the isolation by distance within each migrant group was observed when the lake was divided into the east and west sides. These findings show that the landlocked Ayu population in Lake Biwa has the genetic structure associated with migration patterns and geographical distance. This study demonstrates that the eDNA approach will be effective for conducting a large-scale investigation of genetic structure beyond simple presence/absence tests.

category: ecology


title: On the interaction between Janzen-Connell effects and habitat partitioning in spatially structured environments

abstract:
Janzen-Connell Effects and Habitat Partitioning have both been proposed as potential stabilizing mechanisms in species-rich forest communities. Janzen-Connell Effects describe the process in which specialized predators are attracted to adult trees, which reduce the survivorship of nearby conspecific juveniles. This is thought to generate stabilizing negative frequency dependence. Habitation Partitioning describes when species exhibit different fitness responses to spatially heterogeneous environmental factors, typically causing species to aggregate into favorable habitat types. Despite substantial empirical evidence that both processes occur simultaneously in empirical systems, the theoretical implications of how their interactions shape species diversity remain underdeveloped. In this study, I examine a spatially explicit model that incorporates both processes. I show that Janzen-Connell Effects and Habitat Partitioning can act synergistically to promote coexistence such that their contribution to species coexistence is greater than the sum of their parts when environmental spatial heterogeneity is positively autocorrelated. The results of this study highlight the need to explicitly model the interaction between co-occurring spatially dependent coexistence mechanisms to understand the role they play in the maintenance of species diversity.

Significance StatementA primary goal of ecology is to explain the maintenance of species diversity. However, the mechanisms that underlie the coexistence of highly diverse tropical rainforests remain contentious. Two leading hypotheses are Janzen-Connell Effects and Habitat Partitioning. Both hypotheses are based on space. Janzen-Connell Effects are based on the observation that natural enemies attracted to adult trees reduce the survival of their nearby seeds and seedlings. In contrast, Habitation Partitioning states that diversity can be maintained when species exhibit different responses to spatially varying environmental conditions such that species congregate into their preferred habitat types. In this paper, I demonstrate that these mechanisms can interact synergistiacally to promote high species diversity.


title: Accurate Genome-Wide Phasing from IBD Data

abstract:
As genotype databases increase in size, so too do the number of detectable segments of identity by descent (IBD): segments of the genome where two individuals share an identical copy of one of their two parental haplotypes, due to shared ancestry. We show that given a large enough genotype database, these segments of IBD collectively overlap entire chromosomes and can be used to phase them accurately. Furthermore, primarily using instances of IBD that span multiple chromosomes, we can accurately phase an entire genome.

We are able to separate the DNA inherited from each parent completely, across the entire genome, with 98% median accuracy in a test set of 30,000 individuals. We estimate the IBD data requirements for accurate phasing, and we propose a method for estimating confidence in the resulting phase. We show that our methods do not require the genotypes of close family, and that they are robust to genotype errors and missing data. In fact, our method can impute missing data accurately and correct genotype errors.

Author summaryWe present a method for phasing, separating the DNA inherited from each parent, of an entire genome using short segments of DNA that are shared between the genome of the person we wish to phase and those of distant cousins. Essentially, if we have enough of these distant cousins genotypes available, we can piece together overlapping segments until we have phased the entire genome.

We have developed a method that can phase large numbers of individuals, and that makes special considerations for potential close family and potential genotype errors in data.

We report results on experiments phasing 30,000 individuals. We analyze how many such segments are required to phase accurately, and propose a model-based method to estimate accuracy. We also show that our method can accurately impute missing data and correct genotype errors.

category: genetics


title: Reanalysis shows the extreme decline effect does not exist in fish ocean acidification studies

abstract: A meta-analysis published in PLoS Biology by Clements et al. (2022) claims there is an extreme decline effect in studies published between 2009-2019 on the impacts of ocean acidification (OA) on fish behaviour. Here I show that the extreme decline effect reported by Clements et al. is a statistical artifact caused by the way they corrected for zero values in percentage data, which was more common in the earliest experiments compared with later studies. Furthermore, selective choices for excluding or including data, along with serious errors in the compilation of data and missing studies with strong effects, weakened the effect sizes reported for papers after 2010, further exacerbating the decline effect reported by Clements et al. When the data is reanalyzed using appropriate corrections for zero values in percentage and proportional data, and using a complete, corrected and properly screened data set, the extreme decline effect reported by Clements et al. no longer exists.

category: animal behavior and cognition


title: Beyond bold versus shy: Zebrafish exploratory behavior falls into several behavioral clusters and is influenced by strain and sex

abstract: Consistent individual differences in exploratory behavior have been found across a range of taxa, including zebrafish, and are thought to contribute to evolutionary fitness. Animals that explore more of a novel environment, and visit areas of high predation risk, are considered bold, whereas animals with the opposite pattern of behavior are considered shy. Here, we examined whether this bimodal characterization of bold versus shy adequately captures the breadth of exploratory behavior exhibited by zebrafish or if, instead, behavior falls into multiple distinct behavioral subtypes. To identify the presence of behavioral subtypes, we applied unsupervised machine learning to behaviors extracted from three-dimensional swim traces from over 400 adult zebrafish across four strains (AB, TL, TU, and WIK) and both sexes. We found that behavior stratified into four distinct clusters. These included previously described bold and shy behavior as well as two new behavioral types: wall-huggers and active explorers. Consistent with prior work that found individual differences to be stable across time and influenced by biological factors like genetics and sex, the behavioral subtypes we identified were stable for up to 10 weeks and were influenced by the strain and sex of the animals. Taken together, our work suggests that individual differences in zebrafish exploratory behavior goes beyond bold versus shy and exhibits greater complexity than is typically assumed.


title: Stair Recognition for Robotic Exoskeleton Control using Computer Vision and Deep Learning

abstract: Computer vision can be used in robotic exoskeleton control to improve transitions between different locomotion modes through the prediction of future environmental states. Here we present the development of a large-scale automated stair recognition system powered by convolutional neural networks to recognize indoor and outdoor real-world stair environments. Building on the ExoNet database - the largest and most diverse open-source dataset of wearable camera images of walking environments - we designed a new computer vision dataset, called StairNet, specifically for stair recognition with over 515,000 images. We then developed and optimized an efficient deep learning model for automatic feature engineering and image classification. Our system was able to accurately predict complex stair environments with 98.4% classification accuracy. These promising results present an opportunity to increase the autonomy and safety of human-exoskeleton locomotion for real-world community mobility. Future work will explore the mobile deployment of our automated stair recognition system for onboard real-time inference.

category: bioengineering


title: Reducing peptide sequence bias in quantitative mass spectrometry data with machine learning

abstract: Quantitative mass spectrometry measurements of peptides necessarily incorporate sequence-specific biases that reflect the behavior of the peptide during enzymatic digestion, liquid chromatography, and in the mass spectrometer. These sequence-specific effects impair quantification accuracy, yielding peptide quantities that are systematically under- or over-estimated. We provide empirical evidence for the existence of such biases, and we use a deep neural network, called Pepper, to automatically identify and reduce these biases. The model generalizes to new proteins and new runs within a related set of MS/MS experiments, and the learned coefficients themselves reflect expected physicochemical properties of the corresponding peptide sequences. The resulting adjusted abundance measurements are more correlated with mRNA-based gene expression measurements than the unadjusted measurements. Pepper is suitable for data generated on a variety of mass spectrometry instruments, and can be used with labeled or label-free approaches, and with data-independent or data-dependent acquisition.


title: Thermophoresis of Molecules and Structures of different Sizes in Self-assembled Biomatrices

abstract: Upon subjecting biomolecules to non-equilibrium conditions, many biochemical and biophysical features such as biomolecular diffusion, protein folding, interaction kinetics, as well as enzyme-catalyzed reactions can be characterized in an aqueous solution. However, most assays under non-equilibrium conditions cannot be performed in complex self-assembled biomatrices (e.g. extracellular matrices) due to the limitations associated with sample handling, reaction design, and optical detection. Herein, we report the study of biomolecular thermodiffusion in non-covalently assembled synthetic or naturally derived hydrogels. This approach has been demonstrated with a large variety of analytes, including small molecules, polysaccharides, DNAs, DNA origami, and proteins in various polymer networks. The in-biomatrix method has also shown advantages over in-solution measurements: First, it allows us to analyze biomolecules in 3D matrices in a high-throughput fashion. Second, the aggregation of analytes can be remarkably prevented. Although the underlying physics of thermodiffusion is still not well-understood, we demonstrated that the thermodiffusion of surrounding networks will enhance the thermodiffusion of the analyte, an effect counteracting the hindered movement by the polymer network.


title: Bayesian Parameter Estimation for Dynamical Models in Systems Biology

abstract: Dynamical systems modeling, particularly via systems of ordinary differential equations, has been used to effectively capture the temporal behavior of different biochemical components in signal transduction networks. Despite the recent advances in experimental measurements, including sensor development and  -omics studies that have helped populate protein-protein interaction networks in great detail, systems biology modeling lacks systematic methods to estimate kinetic parameters and quantify associated uncertainties. This is because of multiple reasons, including sparse and noisy experimental measurements, lack of detailed molecular mechanisms underlying the reactions, and missing biochemical interactions. Additionally, the inherent nonlinearities with respect to the states and parameters associated with the system of differential equations further compound the challenges of parameter estimation. In this study, we propose a comprehensive framework for Bayesian parameter estimation and complete quantification of the effects of uncertainties in the data and models. We apply these methods to a series of signaling models of increasing mathematical complexity. Systematic analysis of these dynamical systems showed that parameter estimation depends on data sparsity, noise level, and model structure, including the existence of multiple steady states. These results highlight how focused uncertainty quantification can enrich systems biology modeling and enable additional quantitative analyses for parameter estimation.

category: systems biology


title: Sensory conflict disrupts circadian rhythms in the sea anemone Nematostella vectensis.

abstract: Circadian clocks allow organisms to optimize their phenotypes in relation to rhythmic environmental conditions. Clocks must synchronize, or entrain, to environmental signals called zeitgebers (such as light and temperature) while maintaining internal rhythms that are robust to environmental noise. We know surprisingly little about how clocks function in the presence of multiple zeitgebers, although these are the conditions that exist in nature. Misalignment between zeitgeber cycles (here, "sensory conflict") can disrupt circadian rhythms in behavior and gene expression, but it is not clear whether this is generally true across animals. In order to understand general rules about animal clocks, and how clocks have evolved throughout Metazoa, it is necessary to study non-bilaterian animals such as cnidarians, which include corals, jellies, and sea anemones. The sea anemone Nematostella vectensis has emerged as the principal model system for cnidarian circadian biology. In this study, we show that temperature cycles entrain circadian locomotor rhythms in Nematostella, and then conduct extensive behavioral experiments across a range of light and temperature cycles to show that Nematostellas circadian behavior is disrupted by sensory conflict. Chronic exposure to conflicting light and temperature cycles also causes substantial changes to the rhythmic transcriptome, including the breakdown of rhythmic expression of various metabolic processes. These results have important implications for our understanding of cnidarian circadian clocks at both the molecular and organismal levels, and for how sensory information is processed by clocks in the absence of a central nervous system.

category: zoology


title: Typical hippocampal transcriptional response across estrous is dysregulated by Cnih3 gene deletion

abstract: The hippocampus is a critical brain region for coordinating learning, memory, and behavior. In females, the estrous cycle alters these functions through steroid hormone activity, with well-characterized effects on cellular physiology and behavior. However, the molecular basis of these outcomes has not been systematically explored. Therefore, we profiled the transcriptome of dorsal hippocampi from female mice in each estrous cycle stage, and contrasted it with that of males. We identify only subtle sex differences in gene expression between the sexes on average, yet comparing males to individual estrous stages reveals up to thousands of genes deviating from male expression patterns at specific estrous stages. These estrous-responsive genes are especially enriched in gene markers of oligodendrocytes and the dentate gyrus, and in functional gene sets relating to estrogen response, potassium channels, and synaptic gene splicing. Next we profiled Cnih3 knockouts across estrous to provide insight into their previously reported estrous-dependent phenotypes in hippocampal synaptic plasticity, composition, and learning and memory behaviors. Surprisingly, Cnih3 knockouts showed far broader transcriptomic differences between estrous cycle stages and males. Moreover, Cnih3 knockout drove subtle but extensive expression changes accenting sex differential expresssion at diestrus and estrus. Altogether, our profiling constitutes both a resource characterizing estrous-specific gene expression patterns in the adult hippocampus, which can provide insights into mechanisms of sex differential neuropsychiatric functions and dysfunctions, while also highlighting roles of Cnih3 as a buffer against transcriptional effects of estrous and providing insights into the molecular mechanisms that may underlie estrous-dependent phenotypes with its loss.


title: Interneuronal network model of theta-nested fast oscillations predicts differential effects of heterogeneity, gap junctions and short term depression for hyperpolarizing versus shunting inhibition

abstract:
Theta and gamma oscillations in the hippocampus have been hypothesized to play a role in the encoding and retrieval of memories. Recently, it was shown that an intrinsic fast gamma mechanism in medial entorhinal cortex can be recruited by optogenetic stimulation at theta frequencies, which can persist with fast excitatory synaptic transmission blocked, suggesting a contribution of interneuronal network gamma (ING). We calibrated the passive and active properties of a 100-neuron model network to capture the range of passive properties and frequency/current relationships of experimentally recorded PV+ neurons in the medial entorhinal cortex (mEC). The strength and probabilities of chemical and electrical synapses were also calibrated using paired recordings, as were the kinetics and short-term depression (STD) of the chemical synapses. Gap junctions that contribute a noticeable fraction of the input resistance were required for synchrony with hyperpolarizing inhibition; these networks exhibited theta-nested high frequencies ([~]200 Hz) similar to the putative ING observed experimentally in the optogenetically-driven PV-ChR2 mice. With STD included in the model, fast oscillations were only observed before the peak of the theta drive, whereas without STD, they were observed symmetrically before and after the peak. Because hyperpolarizing synapses provide a synchronizing drive that contributes to robustness in the presence of heterogeneity, synchronization decreases as the hyperpolarizing inhibition becomes weaker. In contrast, networks with shunting inhibition required non-physiological levels of gap junctions to synchronize using conduction delays within the measured range; synaptic depression of shunting synapses facilitated fast oscillations, suggesting that shunting inhibition in mEC is desynchronizing.

Author SummaryFast oscillations nested within slower oscillations have been hypothesized to play a role in the encoding and retrieval of memories by chunking information within each fast cycle; networks of parvalbumin positive inhibitory interneurons contribute to the generation of fast oscillations. We show that, in the entorhinal cortex, the intrinsic dynamical properties of these neurons are sufficiently heterogeneous that electrical synapses are likely required to synchronize fast oscillations. Moreover, synchrony likely requires the chemical synapses to have a reversal potential that is negative relative to the action potential threshold of individual neurons during these oscillations. We show that the range of slow phases that support a fast oscillation is controlled by short term synaptic depression. The precise phase locking of the fast oscillation within the slow oscillations is hypothesized to allow for multiplexing of information.


title: A Causal Role of Human Laryngeal Motor Cortex in Speech Lexical Tone and Voicing Perception

abstract: To achieve categorical speech perception, listeners can actively reconstruct motor gestures of the upcoming speech. This is supported by sensorimotor integration function of the dorsal pathway where the "internal models" of articulations implement top-down constraints on auditory speech analyses in the ventral pathway. Moreover, the motor engagement in speech perception is effector-specific, resembling the motor somatotopy during speech production. However, whether and how the laryngeal motor cortex (LMC), which controls the laryngeal muscles that regulate pitch contours and speech voicing, is also involved in speech perception remains largely unknown. To address this question, this study applied transcranial magnetic stimulation (TMS) upon the left or right LMC when participants performed categorical perception tasks on Mandarin lexical tone (pitch contour contrast) or consonant (voice onset time contrast) presented with or without noise. Results showed that, inhibitory stimulation (repetitive TMS or continuous theta-burst stimulation) on the LMC impaired both tone and consonant perception. Furthermore, TMS effects were especially evident when syllables were masked by noise. These results reveal a causal role of the LMC in speech perception that (1) it mirrors its distinctive functions in controlling pitch and voicing during production and (2) it is upregulated and shifts from a left-lateralized to a bilateral engagement in adverse listening conditions as a means of compensation. This study thus provides new evidence to support the motor theory of speech perception, and more generally, active perception and embodied cognition.


title: Causal contributions of the domain-general (Multiple Demand) and the language-selective brain networks to perceptual and semantic challenges in speech comprehension

abstract: 1.Listening to spoken language engages domain-general Multiple Demand (MD, fronto-parietal) regions of the human brain, in addition to domain-selective (fronto-temporal) language regions, particularly when comprehension is challenging. However, there is limited evidence that the MD network makes a functional contribution to core aspects of comprehension. In a behavioural study of volunteers (n=19) with chronic brain lesions, but without aphasia, we assessed the causal role of these networks in perceiving, comprehending and adapting to challenging spoken sentences. A first task measured word report for acoustically degraded (noise-vocoded) sentences before and after training. Participants with greater damage to MD but not language regions required more vocoder channels to achieve 50% word report indicating impaired perception. Perception improved following training, reflecting adaptation to acoustic degradation, but perceptual learning was unrelated to lesion location or extent. A second task used sentence coherence judgements to measure the speed and accuracy of comprehension of spoken sentences using lower-frequency meanings of semantically ambiguous words. Comprehension accuracy was high and unaffected by lesion location or extent. The availability of the lower-frequency meaning, as measured in a subsequent word association task, increased following comprehension (word-meaning priming). Word-meaning priming was reduced for participants with greater damage to language but not MD regions. We conclude that language and MD networks make dissociable contributions to challenging speech comprehension: using recent experience to update word meaning preferences depends on language specialised regions, whereas the domain-general MD network plays a causal role in reporting words from degraded speech.


title: A deep learning-based automated closed-loop optogenetic system for neuromodulation during seizures

abstract: Closed-loop electrical brain stimulation is becoming a popular technique proposed for use as a treatment alternative to surgical resection of brain tissue for drug-resistant seizures in epilepsy patients. Closed-loop optogenetic stimulation is an experimental alternative to electrical stimulation since it can stimulate or inhibit neurons. The closed-loop part contains an online seizure detection algorithm, which, based on the design, can detect the onset of a seizure or evaluate a running window for seizure membership. Conventional configurations of closed-loop optogenetics have several limitations, ranging from the adaptability of hardware-based implementation to inadequate, customized feature selection of seizures, among others. Here we provide a detailed description of our closed-loop components. We used a sequential, fully convolutional neural network regressor for complex feature selection of seizures against the controls from local field potential recordings. Our modular design kept the local field potential-recording headset and optical probe separate. The seizure detection and execution of light delivery are fast and can be precisely timed. This automated system is robust to noise, modular in design, flexible in use, and simple execution. When applied in vivo, the proposed work shows efficacy over the state of the arts in terms of improved seizure detection and reduction in false positives.


title: Extracellular vesicles secreted by Brugia malayi microfilariae modulate the melanization pathway in the mosquito host

abstract:
Vector-borne, filarial nematode diseases represent a significant and affecting disease burden in humans, domestic animals, and livestock worldwide. Parasitic filarial nematodes require both an intermediate (vector) host and a definitive (mammalian) host during the course of their life cycle. In either host, the nematode must evade the host elicited immune response in order to develop and establish infection. There is direct evidence of parasite-derived immunomodulation in mammals, however, there is less evidence of parasite immunomodulation of the vector host. We have previously reported that all life stages of Brugia malayi, a causative agent of lymphatic filariasis, secrete extracellular vesicles (EVs). Here we investigate the immunomodulatory effects of microfilariae derived EVs on the vector host Aedes aegypti. RNA-seq analysis of an A. aegypti cell line treated with B. malayi microfilariae EVs showed differential expression of both mRNAs and miRNAs, some with roles in immune regulation. One downregulated gene, AAEL002590, identified as a serine protease, was shown to have direct involvement in the phenoloxidase (PO) cascade through analysis of PO activity. Similarly, injection of adult female mosquitoes with B. malayi microfilariae EVs validated these results in vivo, eliciting a downregulation of the AAEL002590 transcript and a significant reduction in PO activity. Our data indicates that parasite-derived EVs are capable of interfering with critical immune responses in the vector host, particularly immune responses such as melanization that target extracellular parasites. In addition, this data provides novel targets for transmission control strategies for LF and other parasitic diseases.

Author SummaryVector-borne, filarial nematode diseases represent a significant and affecting disease burden in humans, domestic animals and livestock worldwide. Parasitic nematodes must evade the elicited immune response of their hosts in order to develop and establish infection. While there is evidence for immunomodulation of the mammalian host, the mechanism of this immunomodulation is not fully clear and there is limited evidence for immunomodulation of the vector host. Here we have shown that parasite-derived extracellular vesicles are effector structures for immunomodulation of the vector host. In particular, we have identified that parasite-derived extracellular vesicles can interfere with critical mosquito immune responses against parasites. This data provides insight into parasite biology and novel targets for transmission control strategies for parasitic diseases.


title: Multi-trait genomic prediction improves selection accuracy for enhancing seed mineral concentrations in pea (Pisum sativum L.)

abstract: The superiority of multi-trait genomic selection (MT-GS) over univariate genomic selection (UNI-GS) can be improved by redesigning the phenotyping strategy. In this study, we used about 300 advanced breeding lines from North Dakota State University (NDSU) pulse breeding program and about 200 USDA accessions evaluated for ten nutritional traits to assess the efficiency of sparse testing in MT-GS. Our results showed that sparse phenotyping using MT-GS consistently outperformed UNI-GS when compared to partially balanced phenotyping using MT-GS. This strategy can be further extended to multi-environment multi-trait GS to improve prediction performance and reduce the cost of phenotyping and time-consuming data collection process. Given that MT-GS relies on borrowing information from genetically correlated traits and relatives, consideration should be given to trait combinations in the training and prediction sets to improve model parameters estimate and ultimately prediction performance. Our results point to heritability and genetic correlation between traits as possible parameters to achieve this objective.


title: DNA methylome combined with chromosome cluster-oriented analysis provides an early signature for cutaneous melanoma aggressiveness

abstract:
Aberrant DNA methylation is a well-known feature of tumours and has been associated with metastatic melanoma. However, since melanoma cells are highly heterogeneous, it has been challenging to use affected genes to predict tumour aggressiveness, metastatic evolution, and patients outcomes. We hypothesized that common aggressive hypermethylation signatures should emerge early in tumorigenesis and should be shared in aggressive cells, independent of the physiological context under which this trait arises.

We compared paired melanoma cell lines with the following properties: (i) each pair comprises one aggressive counterpart and its parental cell line, and (ii) the aggressive cell lines were each obtained from different host and their environment (human, rat, and mouse), though starting from the same parent cell line. Next, we developed a multi-step genomic pipeline that combines the DNA methylome profile with a chromosome cluster-oriented analysis.

A total of 229 differentially hypermethylated genes were commonly found in the aggressive cell lines. Genome localization analysis revealed hypermethylation peaks and clusters, identifying eight hypermethylated gene promoters for validation in tissues from melanoma patients.

Five CpG identified in primary melanoma tissues were transformed into a DNA methylation score that can predict survival (Log-rank test, p=0.0008). This strategy is potentially universally applicable to other diseases involving DNA methylation alterations.

category: cancer biology


title: The ADAM17 sheddase complex regulator iTAP modulates inflammation, epithelial repair, and tumor growth

abstract: The metalloprotease ADAM17 catalyzes the shedding of key signalling molecules from the cell surface, including the inflammatory cytokine TNF (tumour necrosis factor) and activating ligands of the EGFR (epidermal growth factor receptor). ADAM17 exists within an assemblage called the "sheddase complex" containing a rhomboid pseudoprotease (iRhom1 or iRhom2). iRhoms control multiple aspects of ADAM17 biology, including its vesicular trafficking, maturation from its precursor pro-form, activation on the cell surface and specificity for subsets of proteolytic targets. Previous studies from our laboratory and others identified the FERM domain-containing protein Frmd8/iTAP as an iRhom-binding protein. iTAP is required to maintain the cell surface stability of the sheddase complex, thereby preventing the precocious shunting of ADAM17 and iRhom2 to lysosomes and their consequent degradation. As pathophysiological role(s) of iTAP have not been addressed, here we sought to characterize the impact of loss of iTAP on ADAM17-associated phenotypes in mice. Our data show that iTAP KO mice exhibit defects in ADAM17 activity in inflammatory and intestinal epithelial barrier repair functions, but do not exhibit the collateral effects associated with global loss of ADAM17. Furthermore, we show that iTAP promotes cancer cell growth in a cell-autonomous manner, and by modulating the tumor microenvironment. Our work suggests that pharmacological intervention at the level of iTAP may be beneficial to target ADAM17 activity in specific compartments during chronic inflammatory diseases or cancer, avoiding the deleterious impact on vital functions associated with the widespread inhibition of ADAM17 in normal tissues.

category: molecular biology


title: Influence of circular RNA topology on microRNA stability

abstract: Circular RNAs (circRNAs) have been proposed to "sponge" or block microRNAs, a property shared with linear RNAs. Alternatively, certain RNAs induce microRNA destruction through the process of Target RNA-Directed MicroRNA Degradation (TDMD). Whether both linear and circular transcripts are equivalent in driving TDMD is unknown. Here we show that RNA topology is critical for TDMD. Through a novel system that expresses a circRNA while reducing the co-expressed cognate linear RNA, we demonstrate that circRNAs cannot induce TDMD. Interestingly, this is attributed to the circular RNA topology and not to its sequence, which is TDMD-competent in its linear form. Similarly, based on the previous knowledge that CDR1as/ciRS-7 circular RNA protects miR-7 from Cyrano-mediated TDMD, we demonstrate that depletion of CDR1as/CirS-7 reduces mir-7 levels, while overexpression of an artificial linear version of CDR1as/ciRS-7 drives TDMD. By analyzing RNA sequencing data of a neuron differentiation system, we suggest that circRNA-mediated microRNA stabilization is widespread. Our results support a model in which circRNAs, unlike linear mRNAs, lead to a topology-dependent TDMD evasion, aiding in the stabilization of specific microRNAs.


title: Evolution of nasal and olfactory infection characteristics of SARS-CoV-2 variants

abstract: SARS-CoV-2 infection of the upper airway and the subsequent immune response are early, critical factors in COVID-19 pathogenesis. By studying infection of human biopsies in vitro and in a hamster model in vivo, we demonstrated a transition in tropism from olfactory to respiratory epithelium as the virus evolved. Analyzing each variants revealed that SARS-CoV-2 WA1 or Delta infects a proportion of olfactory neurons in addition to the primary target sustentacular cells. The Delta variant possesses broader cellular invasion capacity into the submucosa, while Omicron displays longer retention in the sinonasal epithelium. The olfactory neuronal infection by WA1 and the subsequent olfactory bulb transport via axon is more pronounced in younger hosts. In addition, the observed viral clearance delay and phagocytic dysfunction in aged olfactory mucosa is accompanied by a decline of phagocytosis related genes. Furthermore, robust basal stem cell activation contributes to neuroepithelial regeneration and restores ACE2 expression post-infection. Together, our study characterized the nasal tropism of SARS-CoV-2 strains, immune clearance, and regeneration post infection. The shifting characteristics of viral infection at the airway portal provides insight into the variability of COVID-19 clinical features and may suggest differing strategies for early local intervention.

category: pathology


title: An ACAT inhibitor regulates SARS-CoV-2 replication and antiviral T cell activity

abstract: The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies have uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 entry and fusion independent of transmembrane protease serine 2 expression in multiple cell types. We also demonstrate a role for ACAT in regulating SARS-CoV-2 RNA replication in primary bronchial epithelial cells. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled in the acute phase of infection. Thus, re-purposing of available ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects.


title: Strong and consistent associations of waterbird community composition with HPAI H5 occurrence in European wild birds

abstract: Since 2014, highly pathogenic avian influenza (HPAI) H5 viruses of clade 2.3.4.4 have been dominating the outbreaks across Europe, causing massive deaths among poultry and wild birds. However, the factors shaping its broad-scale outbreak patterns remain unclear. With extensive waterbird survey datasets of about 7,000 sites across Europe, we here demonstrated that H5N8 occurrence in wild birds in the 2016/17 and 2020/21 epidemics as well as H5N1 occurrence in 2005/06 epidemic were strongly associated with very similar waterbird community attributes, pointing to the possibility of similar interspecific transmission processes between different epidemics. A simple extrapolation of the model constructed from the 2016/17 epidemic can well predict the H5N8 pattern in wild birds in 2020/21 epidemic. We also found a dilution effect of phylogenetic diversity that was always negatively correlated with H5 occurrence in wild birds. In contrast, H5N8 occurrence in poultry was subject to different risk factors between the two epidemics. In general, waterbird community composition play a much more important role in determining the spatial pattern of H5N8 in wild birds than in poultry. Our work contributes to reveal the factors driving H5N8 patterns, and highlights the value of waterbird community factors in future HPAI surveillance and prediction.


title: A high potent synthetic nanobody with broad-spectrum activity neutralizes SARS-Cov-2 virus and Omicron variant through a unique binding mode

abstract: COVID-19 continues to be a severe public health thread worldwide. The major challenge to control this pandemic is the rapid mutation rate of the SARS-Cov-2 virus, leading to the escape of the protection of vaccines and most of the neutralizing antibodies to date. Thus, it is pivotal to develop neutralizing antibodies with broad-spectrum activity targeting multiple SARS-Cov-2 variants. Here, we first got a synthetic nanobody(named C5) which could interfere with ACE2 binding to SARS-Cov-2 spike protein and its RBD domain. The affinity matured format of C5 clone, named C5G2, has a single digit nanomolar affinity to RBD domain and inhibit its binding to ACE2 with an IC50 of 3.7 nM. Pseudovirus assay indicated that the monovalent C5G2 could protect the cells from the infection of SARS-Cov-2 wild type virus and most of the virus of concern, i.e. Alpha, Beta, Gamma and Omicron variants. Strikingly, C5G2 has the highest potency against omicron among all the variants with the IC50 of 4.9ng/ml(0.3n M). We further solved the Cryo-EM structure of C5G2 in complex with the Spike trimer. The structure data showed that C5G2 bind to RBD mainly through its CDR3 at a vast region that not overlapping with the ACE2 binding surface. Surprisingly, C5G2 also bind to a distinct epitope residing in the NTD domain of spike protein through the same CDR3 loop, which may further increase its potency against the virus infection. Thus, this bi-paratopic nanobody may be served as an effective drug for the prophylaxis and therapy of the Omicron infection.

category: biochemistry


title: Hierarchical Computational Modeling and Dynamic Network Analysis of Allosteric Regulation in the SARS-CoV-2 Spike Omicron Trimer Structures: Omicron Mutations Cooperate to Allosterically Control Balance of Protein Stability and Conformational Adaptability

abstract: Structural and computational studies of the Omicron spike protein in various functional states and complexes provided important insights into molecular mechanisms underlying binding, high transmissibility, and escaping immune defense. However, the regulatory roles and functional coordination of the Omicron mutations are poorly understood and often ignored in the proposed mechanisms. In this work, we explored the hypothesis that the SARS-CoV-2 spike protein can function as a robust allosterically regulated machinery in which Omicron mutational sites are dynamically coupled and form a central engine of the allosteric network that regulates the balance between conformational plasticity, protein stability, and functional adaptability. In this study, we employed coarse-grained dynamics simulations of multiple full-length SARS-CoV-2 spike Omicron trimers structures in the closed and open states with the local energetic frustration analysis and collective dynamics mapping to understand the determinants and key hotspots driving the balance of protein stability and conformational adaptability. We have found that the Omicron mutational sites at the inter-protomer regions form regulatory clusters that control functional transitions between the closed and open states. Through perturbation-based modeling of allosteric interaction networks and diffusion analysis of communications in the closed and open spike states, we quantify the allosterically regulated activation mechanism and uncover specific regulatory roles of the Omicron mutations. The network modeling demonstrated that Omicron mutations form the inter-protomer electrostatic bridges that connect local stable communities and function as allosteric switches of signal transmission. The results of this study are consistent with the experiments, revealing distinct and yet complementary role of the Omicron mutational sites as a network of hotspots that enable allosteric modulation of structural stability and conformational changes which are central for spike activation and virus transmissibility.


title: Broadly neutralizing antibodies target the coronavirus fusion peptide

abstract:
The potential for future coronavirus outbreaks highlights the need to develop strategies and tools to broadly target this group of pathogens. Here, using an epitope-agnostic approach, we identified six monoclonal antibodies that bound to spike proteins from all seven human-infecting coronaviruses. Epitope mapping revealed that all six antibodies target the conserved fusion peptide region adjacent to the S2 cleavage site. Two antibodies, COV44-62 and COV44-79, broadly neutralize a range of alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.1 and BA.2, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2 cleavage site. Importantly, COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings identify the fusion peptide as the target of the broadest neutralizing antibodies in an epitope-agnostic screen, highlighting this site as a candidate for next-generation coronavirus vaccine development.

One-Sentence SummaryRare monoclonal antibodies from COVID-19 convalescent individuals broadly neutralize coronaviruses by targeting the fusion peptide.


title: The SARS-CoV-2 receptor-binding domain facilitates neutrophil transepithelial migration and nanoparticle uptake in the mice airways

abstract:
SARS-CoV-2-induced infection is still dangerous. Mouse models are convenient to the investigation of virus-activated immune response mechanisms. However, mice are not proper model organisms to study COVID-19 due to decreased interaction affinity between the SARS-CoV-2 receptor-binding domain (RBD) and mouse angiotensin-converting enzyme 2 (ACE2) compared with human ACE2. In the present study, we propose a mouse model that allows estimating the influence of SARS-CoV-2 on the immune system. To mimic the effects of RBD- ACE2 high-affinity interaction, mice received the ACE2 inhibitor MLN-4760. To simulate virus loading, we applied 100 nm particles suspended in the solution of RBD via the oropharyngeal route to mice. In this model, MLN-4760 application enhanced neutrophil egress from the bone marrow to the bloodstream and RBD attracted neutrophils to the luminal side of the conducting airway epithelium. By contrast, inert 100 nm particles were not potent to stimulate neutrophil recruitment to the conducting airway mucosa. Using this model, and by altering the dosage of the ACE2 inhibitor, nanoparticles, and RBD, one can adapt it to investigate different COVID-19 states characterized with mild or severe airway inflammation.

StatementThis study presents a mouse model that allows estimating the influence of SARS-CoV-2 on the immune system and investigates immune cell-model virus particle interactions in the conducting airway mucosa.


title: Discrimination of SARS-CoV-2 Omicron sub-lineages BA.1 and BA.2 using a high-resolution melting-based assay: A pilot study

abstract: The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. As of March 2022, Omicron variant BA.2 is rapidly replacing variant BA.1. As variant BA.2 may cause more severe disease than variant BA.1, variant BA.2 requires continuous monitoring. The current study aimed to develop a novel high-resolution melting (HRM) assay for variants BA.1 and BA.2 and to determine the sensitivity and specificity of our method using clinical samples. Here, we focused on the mutational spectra at three regions in the spike receptor-binding domain (RBD; R408, G446/L452, and S477/T478) for the variant-selective HRM analysis. Each variant was identified based on the mutational spectra as follows: no mutations (Alpha variant); L452R and T478K (Delta variant); G446S and S477N/T478K (Omicron variant BA.1); and R408S and S477N/T478K (Omicron variant BA.2). Upon analysis of mutation-coding RNA fragments, the melting peaks of the wild-type fragments were distinct from those of the mutant fragments. The sensitivity and specificity of this method were determined as 100% and more than 97.5%, respectively, based on 128 clinical samples (40 Alpha, 40 Delta, 40 Omicron variants BA.1/BA.1.1, and 8 Omicron BA.2). These results suggest that this HRM-based assay is a promising screening method for monitoring the transmission of Omicron variants BA.1 and BA.2.


title: Genome wide screen of RNAi molecules against SARS-CoV-2 creates a broadly potent prophylaxis

abstract: Expanding the arsenal of prophylactic approaches against SARS-CoV-2 is of utmost importance, specifically those strategies that are resistant to antigenic drift in Spike. Here, we conducted a screen with over 16,000 RNAi triggers against the SARS-CoV-2 genome using a massively parallel assay to identify hyper-potent siRNAs. We selected 10 candidates for in vitro validation and found five siRNAs that exhibited hyper-potent activity with IC50<20pM and strong neutralisation in live virus experiments. We further enhanced the activity by combinatorial pairing of the siRNA candidates to develop siRNA cocktails and found that these cocktails are active against multiple types of variants of concern (VOC). We examined over 2,000 possible mutations to the siRNA target sites using saturation mutagenesis and identified broad protection against future variants. Finally, we demonstrated that intranasal administration of the siRNA cocktail effectively attenuates clinical signs and viral measures of disease in the Syrian hamster model. Our results pave the way to development of an additional layer of antiviral prophylaxis that is orthogonal to vaccines and monoclonal antibodies.

category: genomics


title: Single sample pathway analysis in metabolomics: performance evaluation and application

abstract:
Single sample pathway analysis (ssPA) transforms molecular level omics data to the pathway level, enabling the discovery of patient-specific pathway signatures. Compared to conventional pathway analysis, ssPA overcomes the limitations by enabling multi-group comparisons, alongside facilitating numerous downstream analyses such as pathway-based machine learning. While in transcriptomics ssPA is a widely used technique, there is little literature evaluating its suitability for metabolomics. Here we provide a thorough benchmark of established ssPA methods (ssGSEA, GSVA, SVD (PLAGE), and z-score) using semi-synthetic metabolomics data, alongside the evaluation of two novel methods we propose: ssClustPA and kPCA. While GSEA-based and z-score methods outperformed the others in terms of recall, clustering/dimensionality reduction-based methods provided higher precision at moderate-to-high effect sizes. A case study applying ssPA to inflammatory bowel disease demonstrates how these methods yield a much richer depth of interpretation than conventional approaches, for example by clustering pathway scores to visualise a pathway-based patient subtype-specific correlation network. We also developed the sspa python package (freely available at https://pypi.org/project/sspa/), providing implementations of all the methods benchmarked in this study. This work underscores the value ssPA methods can add to metabolomic studies and provides a useful reference for those wishing to apply ssPA methods to metabolomics data.

Author summaryPathway analysis is a computational method used to draw insights from omics data by identifying groups of molecules (biological pathways) which are important in a study. Single-sample pathway analysis is based on the same principles as conventional pathway analysis but allows researchers to compute the enrichment of pathways at an individual-sample level. This enables pathway-based patient stratification and facilitates a multitude of downstream analyses based on pathways, such as machine learning, or pathway-based visualization which would not be possible using conventional approaches. In this work we investigated the application of single-sample pathway analysis to metabolomics data, a field in which it has not been widely used to date. We first evaluated the most popular methods for single-sample pathway analysis using simulated metabolomics data, as well as two novel methods we developed. Following these tests, we used metabolomics data from inflammatory bowel disease patients to demonstrate how single-sample pathway analysis methods can be used to infer novel pathway-based insights with the advantage of discriminating between disease subtypes. Overall, our analysis provides readers with information on the performance of single-sample pathway analysis methods, whilst highlighting the potential of such methods in metabolomics research.

category: bioinformatics


title: Highly selective transgene expression through double-floxed inverted orientation system by using a unilateral spacer sequence

abstract: The double-floxed inverted orientation (DIO) system with adeno-associated viral (AAV) vector provides a beneficial approach to express transgenes in specific cell populations having Cre recombinase. A significant issue with this system is the protection against non-specific expression of transgenes in tissues after vector injection. We here show that Cre-independent recombination in AAV genome carrying the DIO sequence occurs during the production of viral vectors in packaging cells, which results in transgene expression in off-target populations. Introduction of a relatively longer nucleotide sequence between two recognition sites at the unilateral side of the transgene cassette, termed a unilateral spacer sequence (USS), is useful to suppress recombination during the vector production, leading to the protection of non-specific transgene expression with enhanced gene expression selectivity. Our DIO/USS system offers a powerful strategy for highly specific Cre-dependent transgene expression, aiming at various applications for structural and functional analyses of target cell populations.


title: Housekeeping gene gyrA, a potential molecular marker for Bacillus ecology study

abstract: Bacillus is a ubiquitous microorganism and is one of the most commercially important species widely used in industry, agriculture and healthcare. Bacillus is relatively well understood at the single-cell level; however, molecular tools that determine diversity and ecology of Bacillus community are limited, which limits our understanding of how the Bacillus community works. In the present study, we investigated the potential of the housekeeping gene gyrA as a molecular marker for determining the diversity of Bacillus species. The amplification efficiency for Bacillus species diversity could be greatly improved by primer design. Therefore, we designed a novel primer pair gyrA3 that can detect at least 92 Bacillus species and related species. For Bacillus amyloliquefaciens, Bacillus pumilus, and Bacillus megaterium, we observed that the high variability of the gyrA gene allows for more detailed clustering at the subspecies level that cannot be achieved by the 16S rRNA gene. Since gyrA provides better phylogenetic resolution than 16S rRNA and informs on the diversity of the Bacillus community, we propose that gyrA gene may have broad application prospects in the study of Bacillus ecology.


title: Outside-in: intracellular vesicles in giant sulfur bacteria contain peptidoglycan

abstract: Until recently, the cellular envelopes of bacteria were regarded as static and rigid relative to those of eukaryotes. While investigating peptidoglycan synthesis in populations of giant sulfur bacteria, Candidatus Thiomargarita spp., we observed internal vesicle-like features (VLFs). VLFs, as imaged following the active incorporation of D-amino acids, appear to begin as invaginations and delaminations of the cellular envelope. Staining with wheat germ agglutinin confirmed the presence of peptidoglycan in VLFs, while polymyxin B revealed that the outer membrane is present in some VLFs. Transmission electron microscopy revealed a complex network of interconnected VLFs. Genomes of Ca. Thiomargarita nelsonii lack a canonical divisome, while possessing homologs to genes such as actin, membrane scaffolding proteins, and dynamins that are associated with phagocytosis in eukaryotes. The physiological role of VLFs remains unclear, but the presence of sulfur globules in some suggests compartmentalization of metabolism and energy production. This is the first report of peptidoglycan and outer membrane bound intracellular vesicles within prokaryotic cells. These findings transform the canonical view of the inflexible bacterial cell envelope and further narrow the divide between prokaryotes and eukaryotes.


title: Metabolic-imaging of human glioblastoma explants: a new precision-medicine model to predict tumor treatment response early

abstract:
BackgroundGlioblastoma (GB) is the most severe form of brain cancer, with a 12-15 month median survival. Surgical resection, temozolomide (TMZ) and radiotherapy (RT) remain the primary therapeutic options for GB, and no new therapies have been introduced in recent years. This therapeutic standstill is primarily due to preclinical models that do not reflect the complexity of GB cell biology and fail to test anti-cancer treatments valuably. Better preclinical models for compound screening are therefore needed.

MethodsIn-vitro 3D glioblastoma live explants (GB-EXPs) were derived from patients resected tumors. We then applied metabolic imaging by fluorescence lifetime imaging microscopy (FLIM) on to GB-EXPs to assess drug response, using TMZ as our benchmark drug. Whole-transcriptome and whole-exome analyses were performed.

ResultsUsing FLIM we were able to stratify samples in Responder and Non-Responder tumors. Our functional precision medicine approach was successfully completed within 1 week of surgery. FLIM-based tumor samples stratification was well reflected at the molecular level, highlighting new targets associated with TMZ treatment and identifying a molecular signature associated with survival.

ConclusionsTo the best of our knowledge, this is the first time that FLIM-based metabolic imaging is used on live glioblastoma 3D explants to test anti-neoplastic drugs rapidly. FLIM-based readouts of drug response in GB explants could improve precision treatment of patients with GB and the identification of new anti-GB drugs.

O_LIPatients tumors can be tested for several individual anti-cancer treatments early after surgery
C_LIO_LIFLIM metabolic imaging is an accurate and rapid in vitro tumor treatment response assessment tool
C_LIO_LIOur model can be exploited to identify new drug molecular targets and applied to other cancers
C_LI

Importance of the studyIn this article, we propose an accurate glioblastoma anti-cancer treatment response 3D-approach consisting of FLIM-based metabolic imaging applied directly to in-vitro vital patient-derived glioblastoma explants. Unlike other models, drug response of live explants is assessed within 1 week from patients surgery, therefore with minimal tampering of natural tumor cyto-architecture, preventing the conventional structural and molecular divergence that the tumor undergoes after long time of in vitro culturing and with no animal involvement. This novel in vitro approach for glioblastoma treatment assessment could be exploited as a tool, in a range of clinical and laboratory settings to improve patients clinical management. Selection of the optimal drug regimen for glioblastoma patients remains a clinical challenge and a method to assess the tumor specific response to several therapies at an early stage could be transformative for glioblastoma patients management. Furthermore, this model can be easily used for large-scale screening of new pharmaceutical compounds.


title: Systematic generation and imaging of tandem repeats reveal multivalent base-pairing as a major determinant of RNA aggregation

abstract: Expansion of short tandem repeats (STRs) in the human genome underlies over fifty genetic disorders. A common pathological feature of repeat RNAs is their propensity to aggregate in cells. While these RNA aggregates have been shown to cause toxicity by sequestering RNA-binding proteins, the molecular mechanism of repeat RNA aggregation remains unclear. Here we devised a generalizable method to efficiently generate long tandem repeat DNAs de novo and applied it to systematically determine the sequence features underlying RNA aggregation. Live-cell imaging of repeat RNAs indicated that aggregation was mainly driven by multivalent RNA-RNA interactions via either canonical or noncanonical base pairs. While multiple short runs of two consecutive base pairs were sufficient, longer runs of consecutive base pairs such as those formed by a neurodegeneration-associated hexanucleotide repeat further enhanced aggregation. In summary, our study provides a unifying model for the molecular basis of repeat RNA aggregation and a generalizable approach for identifying the sequence and structural determinants of repeat RNA properties.


title: Ribo-macs derived from nucleoli:big ribosome clusters in the cytoplasm of naive stem cells

abstract: Primed stem cells and naive stem cells are important for understanding early development, but their ribosomes have not been focused on. In this study, we find that big ribosome clusters named Ribo-macs exist in the cytoplasm of naive stem cells. Then, we prove that Ribo-macs are dynamic and physiological in the cytoplasm, and can synthesize proteins associated with biogenesis of endoplasmic reticulum and mitochondria. We also discover and demonstrate that Ribo-macs are the nucleoli, which of significance is promoting us to rethink our understanding of nucleoli. Besides, we reveal that Ribo-macs have a compatible relation with P-bodies and stress granules. In a word, all the results about Ribo-macs provide us with a new insight to understand how cells adapt quickly to environment.

category: developmental biology


title: The admixture histories of Cabo Verde

abstract: From the 15th to the 19th century, the Trans-Atlantic Slave-Trade influenced the genetic and cultural diversity of numerous populations. We explore genomic and linguistic data from the nine islands of Cabo Verde, the earliest European colony of the era in Africa, a major Slave-Trade platform between the 16th and 19th centuries, and a previously uninhabited location ideal for investigating early admixture events between Europeans and Africans. We find that genetic admixture in Cabo Verde occurred primarily between Iberian and Senegambian populations, although forced and voluntary migrations to the archipelago involved numerous other populations. Inter-individual genetic and linguistic variation recapitulate geographic distribution of individuals birth-places across Cabo Verdean islands, suggesting that Kriolu language variants have developed together with genetic divergences. Furthermore, we find that admixture occurred both early in each island, long before the 18th-century massive TAST deportations triggered by the plantation economy, and after this era. Our results illustrate how shifting socio-cultural relationships between enslaved and nonenslaved communities shaped enslaved-African descendants descent on both sides of the Atlantic.


title: Elucidating Mechanisms of Tolerance to Salmonella Typhimurium Across Long-Term Infections Using the Collaborative Cross

abstract:
Understanding the molecular mechanisms underlying resistance and tolerance to pathogen infection may present the opportunity to develop novel interventions. Resistance is the absence of clinical disease with low pathogen burden, while tolerance is minimal clinical disease in the face of high pathogen burden. Salmonella is a worldwide health concern. We studied 18 strains of Collaborative Cross mice that survive acute Salmonella Typhimurium (STm) infections. We infected these strains orally and monitored them for three weeks post-infection. Five strains cleared STm by the end of the experiment (resistant), while 6 strains maintained a bacterial load and survived to the end of the experiment (tolerant). The remaining 7 strains survived longer than 7 days but succumbed to infection before the end of the study period and were called "delayed susceptible" to differentiate them from strains that do not survive to day 7 (susceptible). Tolerant strains were colonized in Peyers patches, mesenteric lymph node, spleen and liver, while resistant strains had significantly reduced bacterial colonization. Tolerant strains had lower pre-infection core body temperatures than both delayed susceptible and resistant strains and had disrupted circadian patterns of body temperature post-infection sooner than resistant strains. Tolerant strains had higher circulating total white blood cells than resistant strains, driven by increased numbers of neutrophils. Tolerant strains had more severe tissue damage and higher circulating levels of MCP-1 and IFN-{gamma}, but lower levels of ENA-78 than resistant strains. QTL analysis revealed 1 significant association and 6 suggestive associations. RNA-seq analysis identified 22 genes that are differentially regulated in tolerant versus resistant animals that overlapped with the QTLs we identified and allowed us to identify the top 5 canonical pathways. Fibrinogen genes (Fga, Fgb, and Fgg) were found across the QTL, RNA, and top canonical pathways making them the best candidate genes for differentiating tolerance and resistance.

Author SummaryAn infected host can respond in multiple ways to bacterial infection including resistance and tolerance. Resistance is a decrease in pathogen load, while in tolerance mild clinical signs are present despite high pathogen load. We infected a collection of 18 strains of genetically diverse mice with Salmonella Typhimurium for up to three weeks. Five strains were resistant, 6 strains were tolerant, and the remaining 7 strains survived an intermediate amount of time ("delayed susceptible"). Tolerant strains maintained bacterial load across several organs, while resistant strains reduced bacterial load. Tolerant strains had the lowest pre-infection core body temperatures and the most rapid disruption in circadian patterns of body temperature post-infection. Tolerant strains had higher circulating neutrophils, higher circulating levels of MCP-1 and IFN-{gamma}, but lower levels of ENA-78 than resistant strains, in addition to more severe tissue damage than resistant strains. QTL analysis revealed multiple associated regions, and gene expression analysis identified 22 genes that are differentially regulated in tolerant versus resistant animals in these regions. Fibrinogen genes (Fga, Fgb, and Fgg) were found across the QTL, RNA, and top canonical pathways making them the best candidate genes for differentiating tolerance and resistance.


title: Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probes

abstract: We introduce a working pipeline for expression, purification and validation of disulfide-bond rich three-finger proteins using E. coli as the expression host. With this pipeline, we have successfully obtained highly purified and bioactive recombinant -Bungarotoxin, k-Bungarotoxin, Hannalgesin, Mambalgin-1, -Cobratoxin, MT, Slurp1, Pate B etc. Milligrams to hundreds of milligrams of recombinant three finger proteins were obtained within weeks in the lab. The recombinant proteins showed specificity in binding assay and six of them were crystallized and structurally validated using X-ray diffraction protein crystallography. As many three finger proteins have attractive therapeutic or research interests and due to the extremely high quality of the recombinant three finger proteins we obtained, our method provides a competitive alternative to either their native counterparts or chemically synthetic ones, and should facilitate related research and applications.


title: Investigating the Consistency of Extracellular Vesicle Production from Breast Cancer Subtypes Using CELLine Adherent Bioreactors

abstract: Extracellular vesicle (EV) research has grown rapidly in recent years, largely due to the potential use of EVs as liquid biopsy biomarkers or therapeutics. However, in-depth characterisation and validation of EVs produced using conventional in vitro cultures can be challenging due to the large area of cell monolayers and volumes of culture media required. To overcome this obstacle, multiple bioreactor designs have been tested for EV production with varying success, but the consistency of EVs produced over time in these systems has not been reported previously. In this study, we demonstrate that several breast cancer cell lines of different subtypes can be cultured simultaneously in space, resource, and time efficient manner using CELLine AD 1000 systems, allowing the consistent production of vast amounts of EVs for downstream experimentation. We report an improved workflow used for inoculating, maintaining, and monitoring the bioreactors, their EV production, and the characterisation of the EVs produced. Lastly, our proteomic analyses of the EVs produced throughout the lifetime of the bioreactors show that core EV-associated proteins are relatively consistent, with few minor variations over time, and that tracking the production of EVs may be a convenient method for indirectly monitoring the bioreactors health. These findings will aid future studies requiring the simultaneous production of large amounts of EVs from several cell lines of different subtypes of a disease and other EV biomanufacturing applications.


title: Spatial Models of Pattern Formation During Phagocytosis

abstract:
Phagocytosis, the biological process in which cells ingest large particles such as bacteria, is a key component of the innate immune response. Fc{gamma} receptor (Fc{gamma}R)-mediated phagocytosis is initiated when these receptors are activated after binding immunoglobulin G (IgG). Receptor activation initiates a signaling cascade that leads to the formation of the phagocytic cup and culminates with ingestion of the foreign particle. In the experimental system termed "frustrated phagocytosis", cells attempt to internalize micropatterned disks of IgG. Cells that engage in frustrated phagocytosis form "rosettes" of actin-enriched structures called podosomes around the IgG disk. The mechanism that generates the rosette pattern is unknown. We present data that supports the involvement of Cdc42, a member of the Rho family of GTPases, in pattern formation. Cdc42 acts downstream of receptor activation, upstream of actin polymerization, and is known to play a role in polarity establishment. Reaction-diffusion models for GTPase spatiotemporal dynamics exist. We demonstrate how the addition of negative feedback and minor changes to these models can generate the experimentally observed rosette pattern of podosomes. We show that this pattern formation can occur through two general mechanisms. In the first mechanism, an intermediate species forms a ring of high activity around the IgG disk, which then promotes rosette organization. The second mechanism does not require initial ring formation but relies on spatial gradients of intermediate chemical species that are selectively activated over the IgG patch. Finally, we analyze the models to suggest experiments to test their validity.

Author SummaryPhagocytosis, the process by which cells ingest foreign bodies, plays an important role in innate immunity. Phagocytosis is initiated when antibodies coating the surface of a foreign body are recognized by immune cells, such as macrophages. To study early events in phagocytosis, we used "frustrated phagocytosis", an experimental system in which antibodies are micropatterned in disks on a cover slip. The cytoskeleton of cells attempting to phagocytose these disks organizes into "rosette" patterns around the disks. To investigate mechanisms that underlie rosette formation we turned to mathematical modeling based on reaction-diffusion equations. Building on existing models for polarity establishment, our analysis revealed two mechanisms for rosette formation. In the first scenario an initial ring of an intermediate signaling molecule forms around the disk, while in the second scenario rosette formation is driven by gradients of positive and negative pathway regulators that are activated over the disk. Finally, we analyze our models to suggest experiments for testing these mechanisms.


title: Modeling tactile pleasantness across skin types at the individual level reveals a reliable and stable basic function

abstract: Touch is perceived most pleasant when delivered at velocities known to optimally activate C Tactile afferents. At the group level, pleasantness ratings of touch delivered at velocities in the range 0.3-30 cm/s follows an inverted-U shape curve, with maximum pleasantness between 1 and 10 cm/s. However, the prevalence, reliability, and stability of this function at the individual level and across skin types remains unknown. Here, we tested a range of seven velocities delivered with a soft brush, on both hairy and non-hairy skin in 123 participants. We showed that the relationship between pleasantness and velocity of touch is significantly best described by a negative quadratic model at the individual level in the majority of participants both on hairy (67.1%) and non-hairy skin (62.6%). Higher interoceptive accuracy and self-reported depression were related to a better fit of the quadratic model and to the steepness of the curve, respectively. The prevalence of the quadratic model was stable across body sites (62.6%), across two experimental sessions (73-78%,), and regardless of the number of trials. Thus, the individual perception of tactile pleasantness follows a characteristic velocity-dependent function across skin types and shows trait characteristics, making it a possible biomarker for mental health disorders.


title: Immotile cilia of the mouse node sense a fluid flow-induced mechanical force for left-right symmetry breaking

abstract: Immotile cilia of crown cells at the node of mouse embryos are required for sensing of a leftward fluid flow1 that gives rise to the breaking of left-right (L-R) symmetry2. The flow-sensing mechanism has long remained elusive, however, with both mechanosensing and chemosensing models having been proposed1, 3-5. Here we show that immotile cilia at the mouse node respond to mechanical force. In the presence of a leftward flow, immotile cilia on the left side of the node bend toward the ventral side whereas those on the right side bend toward the dorsal side. Application of mechanical stimuli to immotile cilia along the dorsoventral axis by optical tweezers induced Ca2+ transients and degradation of Dand5 mRNA--the first known L-R asymmetric molecular events--in the targeted cells. The Pkd2 channel protein was found to be preferentially localized to the dorsal side of immotile cilia on both left and right sides of the node, and the observed induction of Ca2+ transients preferentially by mechanical stimuli directed toward the ventral side could explain the differential response of immotile cilia to the directional flow. Our results thus suggest that immotile cilia at the node sense the direction of fluid flow in a manner dependent on a flow-generated mechanical force.