title: Multifractality in postural sway supports quiet eye training in aiming tasks: A study of golf putting

abstract: The  quiet eye (QE) approach to visually-guided aiming behavior invests fully in perceptual informations potential to organize coordinated action. Sports psychologists refer to QE as the stillness of the eyes during aiming tasks and increasingly into self- and externally-paced tasks. Amidst the  noisy fluctuations of the athletes body, quiet eyes might leave fewer saccadic interruptions to the coupling between postural sway and optic flow. Postural sway exhibits fluctuations whose multifractal structure serves as a robust predictor of visual and haptic perceptual responses. Postural sway generates optic flow centered on an individuals eye height. We predicted that perturbing the eye height by attaching wooden blocks below the feet would perturb the putting more so in QE-trained participants than participants trained technically. We also predicted that QEs efficacy and responses to perturbation would depend on multifractality in postural sway. Specifically, we predicted that less multifractality would predict more adaptive responses to the perturbation and higher putting accuracy. Results showed that lower multifractality led to more accurate putts, and the perturbation of eye height led to less accurate putts, particularly for QE-trained participants. Models of radial error (i.e., the distance between the balls final position and the hole) indicated that lower estimates of multifractality due to nonlinearity coincided with a more adaptive response to the perturbation. These results suggest that reduced multifractality may act in a context-sensitive manner to restrain motoric degrees of freedom to achieve the task goal.


title: Multi-Omic Profiling of the Liver Across Diets and Age in a Diverse Mouse Population

abstract: Systems biology approaches often use inferred networks of gene expression and metabolite data to identify regulatory factors and pathways connected with phenotypic variance. Generally, study-specific multi-layer "Omics" datasets are used to contextualize generic molecular networks. In this regard separating upstream causal mechanisms, downstream biomarkers, and incidental correlations remains a significant challenge, yet it is essential for designing mechanistic experiments. To address this, we designed a study following a population of 2157 individuals from 89 isogenic BXD mouse strains across their lifespan to identify molecular interactions among genotype, environment, age (GxExA) and metabolic fitness. Each strain was separated into two cohorts, one fed low fat (6% cal/fat) and the other high fat (60% cal/fat) diets. Tissues were collected for 662 individuals (309 cohorts) diverging across age (7, 12, 18, and 24 months), diet, sex, and strain. Transcriptome, proteome, and metabolome data were generated for liver. Of these we identified linear relations among these molecular data with lifespan for the same genomes of mice (Roy et al. 2020), and we defined [~]1100 novel protein-coding genes associated with longevity. We knocked down the ortholog of Ctsd in C. elegans. The treatment reduced longevity both in wildtype and in mutant long-lived strains, thus validating the prediction. Next, to assess the molecular impact of GxExA on gene expression, the multi-omics data was parsed into metabolic networks where connectivity varied due to the independent variables. Differences in edge strengths connecting nodes in these molecular networks according to each variable enabled causal inference by using stability selection, with roughly 21% of novel gene-pathway connections being causally affected by diet and/or age. For instance, Chchd2 is activated by aging and drives changes in the proteasome, oxidative phosphorylation, and mitochondrial translation transcriptional networks. Together, we have developed a large multi-omics resource for studying aging in the liver, and a resource for turning standard associations into causal networks.

category: systems biology


title: Evolving Changes in Cortical and Sub-Cortical Excitability during Movement Preparation: A Study of Brain Potentials and Eye-Blink Reflexes during Loud Acoustic Stimulation.

abstract:
The presentation of Loud Acoustic Stimuli (LAS) during preparation can trigger motor actions at very short latencies in a phenomenon called the StartReact effect. It was initially proposed that a special, separate sub-cortical mechanism which by-passes slower cortical processes could be involved. We sought to examine the evidence for a separate mechanism against the alternative that responses to LAS can be explained by a combination of stimulus intensity effects and preparatory-states - as proposed by activation models of motor control.

To investigate whether cortically mediated preparatory processes are involved in shaping reactions to LAS, we used an auditory reaction task where we manipulated preparation-level within each trial. We contrasted responses to non-intense tones and LAS and examined whether cortical activation, sub-cortical excitability (measured by pre-stimulus EEG and eye-blink startle reflexes, respectively) and the motor response were influenced by preparation-level.

As predicted by the activation model, increases in preparation-level were marked by gradual reductions in RT coupled with increases in cortical activation and sub-cortical excitability - at both condition- and trial-levels. Changes in cortical activation influenced motor and auditory but not visual areas - highlighting the wide-spread yet selective nature of preparation. RTs were shorter to LAS than tones, but the overall pattern of preparation-level effects were the same for both stimuli. These results demonstrate that LAS responses are indeed shaped by cortically mediated preparatory processes. The concurrent changes observed in brain and behaviour with increasing preparation reinforces the notion that preparation is marked by evolving brain states which shape the motor response.

Key PointsO_LIReactions to Loud Acoustic Stimuli can be explained by stimulus intensity and preparation state
C_LIO_LIWe manipulated movement preparation by altering the temporal position of the imperative stimulus
C_LIO_LIPreparation was marked by reductions in RT, and increased cortical and sub-cortical excitability
C_LIO_LIPreparation had the same effect on reactions to Loud Acoustic Stimuli and non-intense tones
C_LIO_LIThe results highlight the widespread, evolving, and strategic nature of movement preparation
C_LI

category: neuroscience


title: Mice learn multi-step routes by memorizing subgoal locations

abstract: The behavioral strategies that mammals use to learn multi-step routes in natural settings are unknown. Here we show that mice spontaneously adopt a subgoal memory strategy. We first investigated how mice navigate to shelter in response to threats when the direct path is blocked. Initially, they fled toward the shelter and negotiated obstacles using sensory cues. Within twenty minutes, they adopted a subgoal strategy, initiating escapes by running directly to the obstacles edge. Mice continued to target this subgoal location after the obstacle was removed, indicating use of spatial memory. However, standard models of spatial learning - egocentric-movement repetition and internal-map building - did not explain how subgoal memories formed. Instead, mice used a hybrid approach: memorizing salient locations encountered during spontaneous  practice runs. This strategy was also used during geometrically identical reward-seeking behavior. These results suggest that subgoal memorization is a fundamental strategy by which rodents learn efficient multi-step routes in new environments.


title: A Human-Immune-System (HIS) humanized mouse model for COVID-19 research (DRAGA: HLA-A2.HLA-DR4.Rag1KO.IL-2Rγc KO.NOD)

abstract: We report the first Human Immune System (HIS)-humanized mouse model ("DRAGA": HLA-A2.HLA-DR4.Rag1KO.IL-2R{gamma}cKO.NOD) for COVID-19 research. This mouse is reconstituted with human cord blood-derived, HLA-matched hematopoietic stem cells. It engrafts human epi/endothelial cells expressing the human ACE2 receptor for SARS-CoV-2 and TMPRSS2 serine protease co-localized on lung epithelia. HIS-DRAGA mice sustained SARS-CoV-2 infection, showing deteriorated clinical condition, replicating virus in the lungs, and human-like lung immunopathology including T-cell infiltrates, microthrombi and pulmonary sequelae. Among T-cell infiltrates, lung-resident (CD103+) CD8+ T cells were sequestered in epithelial (CD326+) lung niches and secreted granzyme B and perforin, indicating cytotoxic potential. Infected mice also developed antibodies against the SARS-CoV-2 viral proteins. Hence, HIS-DRAGA mice showed unique advantages as a surrogate in vivo human model for studying SARS-CoV-2 immunopathology and for testing the safety and efficacy of candidate vaccines and therapeutics.

category: immunology


title: A Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO.IL-2Rγc KO. NOD) for COVID-19


title: Social-like responses are inducible in asocial Mexican cavefish despite the exhibition of strong repetitive behaviour

abstract: Social behaviour is a hallmark of complex animal systems; however, some species appear to have secondarily lost this social ability. In these non-social species, whether social abilities are permanently lost or suppressed is unclear. The blind cavefish Astyanax mexicanus is known to be asocial. Here, we reveal that cavefish exhibited social-like interactions in familiar environments but suppressed these interactions in stress-associated unfamiliar environments. Furthermore, the level of suppression in sociality was positively correlated with that of stereotypic repetitive behaviour, as seen in mammals. Treatment with a human antipsychotic drug targeting the dopaminergic system induced social-like interactions in cavefish, even in unfamiliar environments, while reducing repetitive behaviour. Overall, these results suggest that the antagonistic relationship between repetitive and social-like behaviours is deeply shared from teleosts through mammals.

category: evolutionary biology


title: Exposing distinct subcortical components of the auditory brainstem response evoked by continuous naturalistic speech

abstract: Speech processing is built upon encoding by the auditory nerve and brainstem, yet we know very little about how these processes unfold in specific subcortical structures. These structures are deep and respond quickly, making them difficult to study during ongoing speech. Recent techniques begin to address this problem, but yield temporally broad responses with consequently ambiguous neural origins. Here we describe a method that pairs re-synthesized "peaky" speech with deconvolution analysis of EEG recordings. We show that in adults with normal hearing, the method quickly yields robust responses whose component waves reflect activity from distinct subcortical structures spanning auditory nerve to rostral brainstem. We further demonstrate the versatility of peaky speech by simultaneously measuring bilateral and ear-specific responses across different frequency bands, and discuss important practical considerations such as talker choice. The peaky speech method holds promise as a tool for investigating speech encoding and processing, and for clinical applications.


title: Laboratory evolution of anticipatory gene regulation in Escherichia coli.

abstract: Environmental cues in an ecological niche are often temporal in nature. For instance, in temperate climates, temperature is higher in daytime compared to during night. In response to these temporal cues, bacteria have been known to exhibit anticipatory regulation, whereby they trigger response to a yet to appear cue, anticipating its actual arrival in the near future. Such an anticipatory response in known to enhance Darwinian fitness, and hence, is likely an important feature of regulatory networks in microorganisms. However, the conditions under which an anticipatory response evolves as an adaptive response are not known. In this work, we develop a quantitative model to study response of a population to two temporal environmental cues, and predict variables which are likely important for evolution of anticipatory regulatory response. We follow this with experimental evolution of E. coli in alternating environments of a pentose sugar, rhamnose, and an oxidative stress molecule, paraquat for more than 800 generations. We demonstrate that growth in this cyclical environment leads to evolution of anticipatory regulation, whereby, exposure to rhamnose leads to partial induction of the oxidative stress response regulon. As a result, pre-exposure to rhamnose leads to a greater fitness in paraquat environment. Overall, we show that in niches where environmental stimuli have a cyclical nature, anticipatory regulation can evolve as an adaptive strategy, in a time course of a few hundred generations. This contributes to our understanding of how environment shapes the topology of regulatory networks in an organism.

category: microbiology


title: Patterning the embryonic pulmonary mesenchyme

abstract: Smooth muscle guides morphogenesis of several epithelia during organogenesis, including the mammalian airways. However, it remains unclear how airway smooth muscle differentiation is spatiotemporally patterned and whether it originates from distinct mesenchymal progenitors. Using single-cell RNA-sequencing of embryonic mouse lungs, we show that the pulmonary mesenchyme contains a continuum of cell identities, but no distinct progenitors. Transcriptional variability correlates with sub-epithelial and sub-mesothelial mesenchymal compartments that are regulated by Wnt signaling. Live-imaging and tension-sensors reveal compartment-specific migratory behaviors and cortical forces, and show that sub-epithelial mesenchyme contributes to airway smooth muscle. Cytoskeletal and Wnt signaling pathways are activated early in reconstructed differentiation trajectories. Consistently, Wnt activation stimulates the earliest stages of smooth muscle differentiation and induces local accumulation of mesenchymal F-actin, which influences epithelial morphology. Our single-cell approach uncovers the principles of pulmonary mesenchymal patterning during branching morphogenesis and identifies a morphogenetically active mesenchymal layer that sculpts the airway epithelium.

category: developmental biology


title: Phenotypic reconstruction of the last universal common ancestor reveals a complex cell

abstract: A fundamental concept in evolutionary theory is the last universal common ancestor (LUCA) from which all living organisms originated. While hypotheses regarding the phenotype of LUCA do exist, most are at best based on gene presence or absence. However, despite recent attempts to link genes and phenotypic traits in prokaryotes, it is still inherently difficult to predict phenotype based on the presence or absence of genes alone. Here, we apply a novel approach based on phylogenetically informed ancestral character reconstruction with 22 fundamental descriptors of prokaryotic cells to reconstruct LUCAs phenotype. We infer that the last universal common ancestor of all living organisms was likely an ovoid cell with a large genome, which probably had a cell wall and was actively motile. Potentially obtaining energy from the oxidation of inorganic compounds, tolerant of saltwater and thriving at temperatures above 70{degrees}C, LUCA was probably found living freely in anaerobic conditions in water of neutral pH. Our results depict LUCA as likely to be a far more complex cell than has previously been proposed, challenging the evolutionary model of increased complexity through time in prokaryotes. Given current estimates for the emergence of LUCA we suggest that early life very rapidly evolved considerable cellular complexity.


title: Effects of fine-scale population structure on inbreeding in a long-term study of snapdragons (Antirrhinum majus)

abstract: Many studies have quantified the distribution of heterozygosity and relatedness in natural populations, but surprisingly few have examined the demographic processes driving these patterns. In this study we take a novel approach by studying how population structure affects both pairwise identity and the distribution of heterozygosity in a natural population of a self-incompatible plant Antirrhinum majus. We look at a measure of the variance in heterozygosity within a population, identity disequilibrium (g2), together with FST using a panel of 91 SNPs in 22,353 individuals collected over 11 years. We find that pairwise relatedness (FST) declines rapidly over short spatial scales, and the excess variance in heterozygosity between individuals (g2) reflects significant variation in inbreeding. Additionally, we detect an excess of individuals with around half the average heterozygosity, indicating that some are due to selfing or matings between close relatives. We use two types of simulation to ask whether variation in heterozygosity is consistent with fine-scale spatial population structure. First, by simulating offspring using parents drawn from a range of spatial scales, we show that the known pollen dispersal kernel explains g2. Second, we simulate a 1000-generation pedigree using the known dispersal and spatial distribution and find that the resulting g2 is consistent with that observed from the field data. In contrast, a simulated population with uniform density underestimates g2, indicating that heterogeneous density promotes identity disequilibrium. Our study shows that heterogeneous density and leptokurtic dispersal can together explain the distribution of heterozygosity. Furthermore, our study highlights the limitations of making theoretical predictions from simulations that only assume simple density and dispersal distributions.


title: Effects of fine-scale population structure on the distribution of heterozygosity in a long-term study of (Antirrhinum majus)


title: Object manifold geometry across the mouse cortical visual hierarchy

abstract: Despite variations in appearance we robustly recognize objects. Neuronal populations responding to objects presented under varying conditions form object manifolds and hierarchically organized visual areas untangle pixel intensities into linearly decodable object representations. However, the associated changes in the geometry of object manifolds along the cortex remain unknown. Using home cage training we showed that mice are capable of invariant object recognition. We simultaneously recorded the responses of thousands of neurons to measure the information about object identity across the visual cortex and found that lateral areas LM, LI and AL carry more linearly decodable object information compared to other visual areas. We applied the theory of linear separability of manifolds, and found that the increase in classification capacity is associated with a decrease in the dimension and radius of the object manifold, identifying the key features in the geometry of the population neural code that enable invariant object coding.


title: Global prevalence of potentially pathogenic short-tandem repeats in an epilepsy cohort

abstract: This study aims to decipher the role of short tandem repeats (STRs) in epilepsy patients. Whole genome short-read sequencing data of 734 epileptic patients was used to look for known STR expansions associated with increased risk of neurodevelopmental diseases or epilepsy using three different software. Results show one hit of particular interest on ARX gene associated with Early Infantile Encephalopathic Epilepsy that could be causal for one patient with developmental and epileptic encephalopathy. However, we show that the different software do not agree on most of the calls above the threshold and that experimental validation is still needed for diagnostic, although these algorithms could prove useful for pre-selection of samples to be validated.

category: genetics


title: Virucidal and antiviral activity of astodrimer sodium against SARS-CoV-2 in vitro

abstract: An effective response to the ongoing coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will involve a range of complementary preventive modalities. The current studies were conducted to evaluate the in vitro SARS-CoV-2 antiviral and virucidal activity of astodrimer sodium, a dendrimer with broad spectrum antimicrobial activity, including against enveloped viruses in in vitro and in vivo models, that is marketed for antiviral and antibacterial applications. We report that astodrimer sodium inhibits replication of SARS-CoV-2 in Vero E6 and Calu-3 cells, with 50% effective concentrations (EC50) for i) reducing virus-induced cytopathic effect of 0.002 to 0.012 mg/mL in Vero E6 cells, and ii) infectious virus release by plaque assay of 0.019 to 0.031 mg/mL in Vero E6 cells and 0.031 to 0.045 mg/mL in Calu-3 cells. The selectivity index (SI) in these assays was as high as 2197. Astodrimer sodium was also virucidal, reducing SARS-CoV-2 infectivity by >99.9% (>3 log10) within 1 minute of exposure, and up to >99.999% (>5 log10) shown at astodrimer sodium concentrations of 10 to 30 mg/mL in Vero E6 and Calu-3 cell lines. Astodrimer sodium also inhibited infection in a primary human airway epithelial cell line. The data were similar for all investigations and were consistent with the potent antiviral and virucidal activity of astodrimer sodium being due to inhibition of virus-host cell interactions, as previously demonstrated for other viruses. Further studies will confirm if astodrimer sodium binds to SARS-CoV-2 spike protein and physically blocks initial attachment of the virus to the host cell. Given the in vitro effectiveness and significantly high SI, astodrimer sodium warrants further investigation for potential as a nasally administered or inhaled antiviral agent for SARS-CoV-2 prevention and treatment applications.


title: Extensive age-dependent loss of antibody diversity in naturally short-lived turquoise killifish

abstract:
Aging individuals exhibit a pervasive decline in adaptive immune function, with important implications for health and lifespan. Previous studies have found a pervasive loss of immune-repertoire diversity in human peripheral blood during aging; however, little is known about repertoire aging in other immune compartments, or in species other than humans. Here, we perform the first study of immune-repertoire aging in an emerging model of vertebrate aging, the African turquoise killifish (Nothobranchius furzeri). Despite their extremely short lifespans, these killifish exhibit complex and individualized heavy-chain repertoires, with a generative process capable of producing millions of distinct productive sequences. Whole-body killifish repertoires decline rapidly in within-individual diversity with age, while between-individual variability increases. Large, expanded B-cell clones exhibit far greater diversity loss with age than small clones, suggesting important differences in how age affects different B cell populations. The immune repertoires of isolated intestinal samples exhibit especially dramatic age-related diversity loss, related to an elevated prevalence of expanded clones. Lower intestinal repertoire diversity was also associated with transcriptomic signatures of reduced B-cell activity, supporting a functional role for diversity changes in killifish immunosenescence. Our results highlight important differences in systemic vs. organ-specific aging dynamics in the adaptive immune system.

Significance StatementImmune function declines dramatically with age, with important consequences for health in the elderly. Changes in the diversity and composition of the bodys antibody repertoire are an important contributor to this age-related dysfunction. However, studying repertoire aging at high spatial and temporal resolution is hampered by the long lifespans of humans and other vertebrate models, and the difficulty of acquiring human samples other than peripheral blood. Here, we characterize the aging of the antibody repertoire in a naturally short-lived vertebrate, the turquoise killifish (Nothobranchius furzeri). We found that aging killifish undergo rapid loss of repertoire diversity in both whole-body and intestinal samples within four months of hatching. Loss of repertoire diversity in the gut was also associated with broader gene-expression changes indicative of reduced B-cell activity. As such, killifish represent a powerful laboratory model to study adaptive immune aging.


title: CASP microdomain formation requires cross cell wall stabilization of domains and non-cell autonomous action of LOTR1

abstract: Efficient uptake of nutrients in both animal and plant cells requires tissue-spanning diffusion barriers separating inner tissues from the outer lumen/soil. However, we poorly understand how such contiguous three-dimensional superstructures are formed in plants. Here, we show that correct establishment of the plant Casparian Strip (CS) network relies on local neighbor communication. We show that positioning of Casparian Strip membrane domains (CSDs) is tightly coordinated between neighbors in wild-type and that restriction of domain formation involves the putative extracellular protease LOTR1. Impaired domain restriction in lotr1 leads to fully functional CSDs at ectopic positions, forming "half strips" LOTR1 action in the endodermis requires its expression in the stele. LOTR1 endodermal expression cannot complement, while cortex expression causes a dominant-negative phenotype. Our findings establish LOTR1 as a crucial player in CSD positioning acting in a directional, non-cell-autonomous manner to restrict and coordinate CS positioning.

category: plant biology


title: Mechanisms of signalling-memory governing progression through the eukaryotic cell cycle

abstract: As cells pass through each replication-division cycle, they must be able to postpone further progression if they detect any threats to genome integrity, such as DNA damage or misaligned chromosomes. Once a  decision is made to proceed, the cell unequivocally enters into a qualitatively different biochemical state, which makes the transitions from one cell cycle phase to the next switch-like and irreversible. Each transition is governed by a unique signalling network; nonetheless, they share a common characteristic of bistable behaviour, a hallmark of molecular memory devices. Comparing the cell cycle signalling mechanisms acting at the Restriction Point, G1/S, G2/M and meta-to-anaphase transitions, we deduce a generic network motif of coupled positive and negative feedback loops underlying each transition.


title: Effects of scent enrichment on behavioural andphysiological indicators of stress in zoo primates

abstract: Captive breeding is vital for primate conservation, with modern zoos serving a crucial role in breeding populations of threatened species and educating the general public. However, captive populations can experience welfare issues that may also undermine their reproductive success. In order to enhance the well-being of endangered zoo primates, we conducted a study to assess the effects of a new scent enrichment programme on captive red-ruffed lemurs (Varecia rubra), black howler monkeys (Alouatta caraya), siamangs (Symphalangus syndactylus), Lar gibbons (Hylobates lar) and orangutans (Pongo pygmaeus pygmaeus). We combined behavioural observations and faecal endocrinology analyses to evaluate the effects of a series of essential oils (benzoin, lavender, lemongrass) on five captive troops (N = 19) housed at Dudley Zoo & Castle and Twycross Zoo (UK). We recorded observations of natural species-specific and abnormal stress-related behaviours for 480 hr using instantaneous scan sampling. We collected 189 faecal samples and measured the faecal cortisol concentrations using radioimmunoassay. We found a significant effect of the scent enrichment on behaviours, with red-ruffed lemurs and black howler monkeys reducing their social interactions, as well as red-ruffed lemurs and Lar gibbons decreasing their stress-related behaviours, after they were exposed to the series of essential oils. We also found that red-ruffed lemurs displayed a significant increase in faecal glucocorticoids following the exposure to essential oils. Our contradictory findings suggest that the effects of this series of essential oils may change depending on the species-specific social lives and olfactory repertoires of primates. In conclusion, we cannot recommend using these essential oils widely with zoo primates without additional evaluation.

category: animal behavior and cognition


title: Theta and alpha power across fast and slow timescales in cognitive control

abstract: Theta and alpha frequency neural oscillations are important for learning and cognitive control, but their exact role has remained obscure. In particular, it is unknown whether they operate at similar timescales, and whether they support different cognitive processes. We recorded EEG in 30 healthy human participants while they performed a learning task containing both novel (block-unique) and repeating stimuli. We investigated behavior and electrophysiology at both fast (i.e., within blocks) and slow (i.e., between blocks) time scales. Behaviorally, both response time and accuracy improved (resp. decrease and increase) over both fast and slow timescales. However, on the spectral level, theta power significantly decreased along the slow timescale, whereas alpha power instead significantly increased along the fast timescale. We thus demonstrate that theta and alpha both play a role during learning, but operate at different timescales. This result poses important empirical constraints for theories on learning, cognitive control, and neural oscillations.


title: Value shapes the structure of schematic representations in the medial prefrontal cortex

abstract: Adaptive cognition is fostered by knowledge about the structure and value of our environment. Here, we hypothesize that these two kinds of information are inherently intertwined as value-weighted schemas in the medial prefrontal cortex (mPFC). Schemas (e.g., of a social network) emerge by extracting commonalities across experiences and can be understood as graphs comprising nodes (e.g., people) and edges (e.g., their relationships). We sampled information about unique real-life environments (i.e., about personally familiar people and places) and probed the neural representations of their schemas with fMRI. Using representational similarity analysis, we show that the mPFC encodes indeed both, the nodes and edges of the schemas. Critically, as hypothesized, the strength of the edges is not only determined by experience and centrality of a node but also by value. We thus account for the involvement of the mPFC in disparate functions and suggest that valuation emerges naturally from encoded memory representations.


title: Compartment-Specific Neurexin Nanodomains Orchestrate Tripartite Synapse Assembly

abstract: At tripartite synapses, astrocytes enmesh synaptic contacts, but how astrocytes contribute to the formation, maturation and plasticity of synapses remains elusive. Here we show that both astrocytes and neurons abundantly express neurexin-1, a presynaptic adhesion molecule that controls synaptic properties. Using super-resolution imaging, we demonstrate that presynaptic neuronal and astrocytic neurexin-1 form discrete nanoclusters at excitatory synapses. We find that distinct patterns of heparan sulfate modification and alternative splicing confer onto astrocytic and neuronal neurexin-1 different ligand specificities, thereby enabling compartment-specific signaling by neurexin-1. At hippocampal Schaffer-collateral synapses, deletion of neurexin-1 from either astrocytes or neurons did not alter synapse numbers, but differentially impaired synapse function. Neuronal neurexin-1 was essential for NMDA-receptor-mediated synaptic responses, whereas astrocytic neurexin-1 was required for maturation of silent synapses, AMPA-receptor recruitment, and long-term potentiation. Thus, astrocytes and neurons surprisingly use the same synaptic adhesion molecule to control distinct synapse properties.


title: Combining repetition suppression and pattern analysis provides new insights into the role of M1 and parietal areas in skilled sequential actions

abstract:
How does the brain change during learning? In functional magnetic resonance imaging studies, both multivariate pattern analysis and repetition suppression (RS) have been used to detect changes in neuronal representations. In the context of motor sequence learning, the two techniques have provided discrepant findings: pattern analysis showed that only premotor and parietal regions, but not primary motor cortex (M1), develop a representation of trained sequences. In contrast, RS suggested trained sequence representations in all these regions. Here we applied both analysis techniques to a 5-week finger sequence training study, in which participants executed each sequence twice before switching to a different sequence. Both RS and pattern analysis indicated learning-related changes for parietal areas, but only RS showed a difference between trained and untrained sequences in M1. A more fine-grained analysis, however, revealed that the RS effect in M1 reflects a fundamentally different process than in parietal areas. On the first execution, M1 represents especially the first finger of each sequence, likely reflecting preparatory processes. This effect dramatically reduces during the second execution. In contrast, parietal areas represent the identity of a sequence, and this representation stays relatively stable on the second execution. These results suggest that the RS effect does not reflect a trained sequence representation in M1, but rather a preparatory signal for movement initiation. More generally, our study demonstrates that across regions RS can reflect different representational changes in the neuronal population code, emphasizing the importance of combining pattern analysis and RS techniques.

Significance statementPrevious studies using pattern analysis have suggested that primary motor cortex (M1) does not represent learnt sequential actions. However, a study using repetition suppression (RS) has reported M1 changes during motor sequence learning. Combining both techniques, we first replicate the discrepancy between them - with learning-related changes in M1 in RS, but not pattern dissimilarities. We further analysed the representational changes with repetition, and found that the RS effects differ across regions. M1s activity represents the starting finger of the sequence, an effect that vanishes with repetition. In contrast, activity patterns in parietal areas exhibit sequence dependency, which persists with repetition. These results demonstrate the importance of combining RS and pattern analysis to understand the function of brain regions.


title: Evidence for a role of phenotypic mutations in virus adaptation

abstract: Viruses interact extensively with the host molecular machinery, but the underlying mechanisms are poorly understood. Bacteriophage T7 recruits the small protein thioredoxin of the E. coli host as an essential processivity factor for the viral DNA polymerase. We challenged the phage to propagate in a host in which thioredoxin had been extensively modified to hamper its recruitment. The virus adapted to the engineered host without losing the capability to propagate in the original host, but no genetic mutations were fixed in the thioredoxin binding domain of the viral DNA polymerase. Virus adaptation correlated with mutations in the viral RNA polymerase, supporting that promiscuous thioredoxin recruitment was enabled by phenotypic mutations caused by transcription errors. These results point to a hitherto unknown mechanism of virus adaptation that may play a role in crossspecies transmission. We propose that phenotypic mutations may generally contribute to the capability of viruses to evade antiviral strategies.


title: Variable branching characteristics of peripheral taste neurons indicates differential convergence.

abstract:
Taste neurons are functionally and molecularly diverse, but their morphological diversity remains completely unexplored. Using sparse cell genetic labeling, we provide the first reconstructions of peripheral taste neurons. The branching characteristics across 96 taste neurons show surprising diversity in their complexities. Individual neurons had 1 to 17 separate arbors entering between 1 to 7 taste buds, 18 of these neurons also innervated non-taste epithelia. Axon branching characteristics are similar in gustatory neurons from male and female mice. Cluster analysis separated the neurons into four groups according to branch complexity. The primary difference between clusters was the amount of the nerve fiber within the taste bud available to contact taste-transducing cells. Consistently, we found that the maximum number of taste-transducing cells capable of providing convergent input onto individual gustatory neurons varied with a range of 1-22 taste-transducing cells. Differences in branching characteristics across neurons indicate that some neurons likely receive input from a larger number of taste-transducing cells than other neurons (differential convergence). By dividing neurons into two groups based on the type of taste-transducing cell most contacted, we found that neurons contacting primarily sour transducing cells were more heavily branched than those contacting primarily sweet/bitter/umami transducing cells. This suggests that neuron morphologies may differ across functional taste quality. However, the considerable remaining variability within each group also suggests differential convergence within each functional taste quality. Each possibility has functional implications for the system.

Significance statementTaste neurons are considered relay cells, communicating information from taste-transducing cells to the brain, without variation in morphology. By reconstructing peripheral taste neuron morphologies for the first time, we found that some peripheral gustatory neurons are simply branched, and can receive input from only a few taste-transducing cells. Other taste neurons are heavily branched, contacting many more taste-transducing cells than simply branched neurons. Based on the type of taste-transducing cell contacted, branching characteristics are predicted to differ across (and within) quality types (sweet/bitter/umami vs sour). Therefore, functional differences between neurons likely depends on the number of taste-transducing cells providing input and not just the type of cell providing input.


title: Defining the early stages of intestinal colonisation by whipworms

abstract: Whipworms are large metazoan parasites that inhabit distinct multi-intracellular epithelial burrows described as syncytial tunnels, in the large intestine of their hosts. How first-stage larvae invade host epithelia and establish infection remains unclear. Here, we investigate early infection events both using Trichuris muris infections of mice and murine caecaloids, the first in-vitro system for whipworm infection. We show that larvae degrade the mucus layers to access epithelial cells. In early syncytial tunnels, larvae are completely intracellular but woven through multiple live enterocytes and goblet cells. We also use single cell RNA sequencing for the first time to describe the mouse caecum. From infected caeca, the transcriptome data reveal the progression of infection results in cell damage and an expansion of enterocytes with a type-I interferon (IFN) signature, characterised by the expression of Isg15, instigating the host immune response to the whipworm and tissue repair. Our results unravel intestinal epithelium invasion by whipworms and reveal new specific interactions between the host and the parasite that allow the whipworm to establish its multi-intracellular niche.


title: Leading edge stability in motile cells is an emergent property of branched actin network growth

abstract:
Animal cell migration is predominantly driven by the coordinated, yet stochastic, polymerization of thousands of nanometer-scale actin filaments across micron-scale cell leading edges. It remains unclear how such inherently noisy processes generate robust cellular behavior. We employed high-speed imaging of migrating neutrophil-like HL-60 cells to explore the fine-scale shape fluctuations that emerge and relax throughout the process of leading edge maintenance. We then developed a minimal stochastic model of the leading edge that reproduces this stable relaxation behavior. Remarkably, we find lamellipodial stability naturally emerges from the interplay between branched actin network growth and leading edge shape - with no additional feedback required - based on a synergy between membrane-proximal branching and lateral spreading of filaments. These results thus demonstrate a novel biological noise-suppression mechanism based entirely on system geometry. Furthermore, our model suggests that the Arp2/3-mediated ~70-80{degrees} branching angle optimally smooths lamellipodial shape, addressing its long-mysterious conservation from protists to mammals.

Significance StatementAll cellular functions are driven by the stochastic dynamics of macromolecules, and thus are subject to biological noise. Here, as a model system for noise-suppression in the context of cell migration, we investigate lamellipodial maintenance - where thousands of stochastically polymerizing filaments self-organize into a highly-stable, micron-scale leading edge. Combining experiment and computational modeling, we (1) establish lamellipodial stability is an emergent property of dendritically-branched actin network growth, (2) outline a noise-suppression mechanism based on the geometry of lamellipodial actin, and (3) determine the evolutionarily-conserved Arp2/3-mediated ~70-80{degrees} branching angle optimally suppresses stochastic fluctuations. Our results not only explain the essential role of Arp2/3-mediated branching in lamellipodial formation, but also address the decades-old question of why this specific geometry is so well-conserved.

category: cell biology


title: Social immunity and chemical communication in the honeybee: immune-challenged bees enter enforced or self-imposed exile

abstract: Animals living in large colonies are especially vulnerable to infectious pathogens, and may there-fore have evolved additional defences. Eusocial insects supplement their physiological immune systems with  social immunity, a set of adaptations that impedes the entrance, establishment, and spread of pathogens in the colony. We here find that honey bee workers (Apis mellifera) that had been experimentally immune-challenged with lipopolysaccharide (LPS) often exited the hive and subsequently died; some individuals were dragged out by other workers, while others appeared to leave voluntarily. In a second experiment, we found that healthy workers treated with surface chemicals from LPS-treated bees were evicted from the hive more often than controls, indicating that immune-challenged bees produce chemical cues or signals that elicit their eviction. Thirdly, we observed pairs of bees in the lab, and found that pairs spent more time apart when one member of the pair had received LPS, relative to controls. Our findings suggest that immune-challenged bees altruistically banish themselves, and that workers evict sick individuals which they identify using olfactory cues, putatively because of (kin) selection to limit the spread of pathogens within colonies.


title: Single-component multilayered self-assembling nanoparticles presenting rationally designed glycoprotein trimers as Ebola virus vaccines

abstract: Ebola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. Here, we first investigate the contribution of the stalk and heptad repeat 1-C (HR1C) regions to GP metastability. Specific stalk and HR1C modifications in a mucin-deleted form (GP{Delta}muc) increase trimer yield, whereas alterations of HR1C exert a more complex effect on thermostability. Crystal structures are determined to validate two rationally designed GP{Delta}muc trimers in their unliganded state. We then display a modified GP{Delta}muc trimer on reengineered nanoparticles that encapsulate a layer of locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicit cross-ebolavirus NAbs, as well as non-NAbs that enhance pseudovirus infection. Repertoire sequencing reveals quantitative profiles of vaccine-induced B-cell responses. This study demonstrates a promising vaccine strategy for filoviruses, such as EBOV, based on GP stabilization and nanoparticle display.


title: Suppression without inhibition: How retinal computation contributes to saccadic suppression

abstract: Visual perception remains stable across saccadic eye movements, despite the concurrent strongly disruptive visual flow. This stability is partially associated with a reduction in visual sensitivity, known as saccadic suppression, which already starts in the retina with reduced ganglion cell sensitivity. However, the retinal circuit mechanisms giving rise to such suppression remain unknown. Here, we describe these mechanisms using electrophysiology in mouse, pig, and macaque retina, 2-photon calcium imaging, computational modeling, and human psychophysics. We find that sequential stimuli, such as those that naturally occur during saccades, trigger three independent suppressive mechanisms in the retina. The main suppressive mechanism is triggered by contrast-reversing sequential stimuli and originates within the receptive field center of ganglion cells. It does not involve inhibition or other known suppressive mechanisms such as saturation or adaptation. Instead, it relies on temporal filtering of the inherently slow response of cone photoreceptors coupled with downstream nonlinearities. Two further mechanisms of suppression are present predominantly in ON ganglion cells and originate in the receptive field surround, highlighting a novel disparity between ON and OFF ganglion cells. The mechanisms uncovered here likely play a role in shaping the retinal output following eye movements and other natural viewing conditions where sequential stimulation is ubiquitous.


title: Hemodynamic responses link individual differences in informational masking to the vicinity of superior temporal gyrus

abstract: Suppressing unwanted background sound is crucial for aural communication. A particularly disruptive type of background sound, informational masking (IM), often interferes in social settings. However, IM mechanisms are incompletely understood. At present, IM is identified operationally: when a target should be audible, based on suprathreshold target/masker energy ratios, yet cannot be heard because perceptually similar background sound interferes. Here, functional near infrared spectroscopy recordings show that task-evoked blood oxygenation changes near the superior temporal gyrus (STG) covary with behavioral speech detection performance for high-IM but not low-IM background sound, suggesting that the STG is part of an IM-dependent network. Moreover, listeners who are more vulnerable to IM show increased hemodynamic recruitment near STG. In contrast, task-evoked responses near another auditory region of cortex, the caudal inferior frontal sulcus (cIFS), do not predict behavioral sensitivity, suggesting that the cIFS belongs to an IM-independent network. Results are consistent with the idea that cortical gating shapes individual vulnerability to IM.


title: In-situ observation of mitochondrial biogenesis as the early event of apoptosis

abstract:
Mitochondrial biogenesis is a cell response to external stimuli which is generally believed to suppress apoptosis. However, during the process of apoptosis, whether mitochondrial biogenesis occurs in the early stage of the apoptotic cells remains unclear. To address this question, we constructed the COX8-EGFP-ACTIN-mCherry HeLa cells with recombinant fluorescent proteins respectively tagged on the nucleus and mitochondria, and monitored the mitochondrial changes in living cells exposed to gamma-ray radiation. Besides in situ detection of mitochondrial fluorescence changes, we also examined the cell viability, nuclear DNA damage, reactive oxygen species (ROS), Mitochondrial superoxide, citrate synthase activity, ATP, cytoplasmic and mitochondrial calcium, mitochondrial DNA copy number and expression of transcription genes related to mitochondrial biogenesis as well as the apoptosis biomarkers. As a result, we confirmed that significant mitochondrial biogenesis took place preceding the radiation-induced apoptosis, and the change of mitochondrial biogenesis at early time was closely correlated with the apoptotic cells at late stage. The involved mechanism was also discussed.

HighlightsO_LIA dual fluorescence reporter system was successfully constructed for in-situ observation of mitochondrial biogenesis in living cells.
C_LIO_LIThe whole process of radiation-induced mitochondrial biogenesis and apoptosis was scrutinized.
C_LIO_LIThe conception of the relationship between mitochondrial biogenesis and apoptosis was revised.
C_LIO_LIAssessment of the early event of mitochondrial biogenesis is critical for prediction of the late fate of cells.
C_LI

Graphical abstract

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title: MECOM permits pancreatic acinar cell dedifferentiation avoiding cell death under stress conditions

abstract:
Maintenance of the pancreatic acinar cell phenotype suppresses tumor formation. Hence, repetitive acute or chronic pancreatitis, stress conditions in which the acinar cells dedifferentiate, predispose for cancer formation in the pancreas. Dedifferentiated acinar cells acquire a large panel of duct cell-specific markers. However, it remains unclear to what extent dedifferentiated acini differ from native duct cells and which genes are uniquely regulating acinar cell dedifferentiation. Moreover, most studies have been performed in mice since the availability of human cells is scarce.

Here, we applied a non-genetic lineage tracing method of human pancreatic exocrine acinar and duct cells that allowed cell-type-specific gene expression profiling by RNA sequencing. Subsequent to this discovery analysis, one transcription factor that was unique for dedifferentiated acinar cells was functionally characterized.

RNA sequencing analysis showed that human dedifferentiated acinar cells expressed genes in  Pathways of cancer with prominence of MECOM (EVI-1), a transcription factor that was not expressed by duct cells. During mouse embryonic development, pre-acinar cells also transiently expressed MECOM and in the adult mouse pancreas, MECOM was re-expressed when mice were subjected to acute and chronic pancreatitis, conditions in which acinar cells dedifferentiate. In human cells and in mice, MECOM expression correlated with and was directly regulated by SOX9. Mouse acinar cells that, by genetic manipulation, lose the ability to upregulate MECOM showed impaired cell adhesion, more prominent acinar cell death and suppressed acinar cell dedifferentiation by limited ERK signaling.

In conclusion, we transcriptionally profiled the two major human pancreatic exocrine cell types, acinar and duct cells, during experimental stress conditions. We provide insights that in dedifferentiated acinar cells, cancer pathways are upregulated in which MECOM is a critical regulator that suppresses acinar cell death by permitting cellular dedifferentiation.

category: cancer biology


title: DNA extraction and host depletion methods significantly impact and potentially bias bacterial detection in a biological fluid

abstract:
Untargeted sequencing of nucleic acids present in food can inform the detection of food safety and origin, as well as product tampering and mislabeling issues. The application of such technologies to food analysis could reveal valuable insights that are simply unobtainable by targeted testing, leading to the efforts of applying such technologies in the food industry. However, before these approaches can be applied, it is imperative to verify that the most appropriate methods are used at every step of the process: gathering primary material, laboratory methods, data analysis, and interpretation.

The focus of this study is in gathering the primary material, in this case, DNA. We used bovine milk as a model to 1) evaluate commercially available kits for their ability to extract nucleic acids from inoculated bovine milk; 2) evaluate host DNA depletion methods for use with milk, and 3) develop and evaluate a selective lysis-PMA based protocol for host DNA depletion in milk.

Our results suggest that magnetic-based nucleic acid extraction methods are best for nucleic acid isolation of bovine milk. Removal of host DNA remains a challenge for untargeted sequencing of milk, highlighting that the individual matrix characteristics should always be considered in food testing. Some reported methods introduce bias against specific types of microbes, which may be particularly problematic in food safety where the detection of Gram-negative pathogens and indicators is essential. Continuous efforts are needed to develop and validate new approaches for untargeted metagenomics in samples with large amounts of DNA from a single host.

ImportanceTracking the bacterial communities present in our food has the potential to inform food safety and product origin. To do so, the entire genetic material present in a sample is extracted using chemical methods or commercially available kits and sequenced using next-generation platforms to provide a snapshot of what the relative composition looks like. Because the genetic material of higher organisms present in food (e.g., cow in milk or beef, wheat in flour) is around one thousand times larger than the bacterial content, challenges exist in gathering the information of interest. Additionally, specific bacterial characteristics can make them easier or harder to detect, adding another layer of complexity to this issue. In this study, we demonstrate the impact of using different methods in the ability of detecting specific bacteria and highlight the need to ensure that the most appropriate methods are being used for each particular sample.

category: molecular biology


title: Evolution of flowering time in a selfing annual plant: Roles of adaptation and genetic drift

abstract: Resurrection studies are a useful tool to measure how phenotypic traits have changed in populations through time. If these traits modifications correlate with the environmental changes that occurred during the time period, it suggests that the phenotypic changes could be a response to selection. Selfing, through its reduction of effective size, could challenge the ability of a population to adapt to environmental changes. Here, we used a resurrection study to test for adaptation in a selfing population of Medicago truncatula, by comparing the genetic composition and flowering times across 22 generations. We found evidence for evolution towards earlier flowering times by about two days and a peculiar genetic structure, typical of highly selfing populations, where some multilocus genotypes (MLGs) are persistent through time. We used the change in frequency of the MLGs through time as a multilocus fitness measure and built a selection gradient that suggests evolution towards earlier flowering times. Yet, a simulation model revealed that the observed change in flowering time could be explained by drift alone, provided the effective size of the population is small enough (<150). These analyses suffer from the difficulty to estimate the effective size in a highly selfing population, where effective recombination is severely reduced.


title: Analysis of paralogs in target enrichment data pinpoints multiple ancient polyploidy events in Alchemilla s.l. (Rosaceae)

abstract: Target enrichment is becoming increasingly popular for phylogenomic studies. Although baits for enrichment are typically designed to target single-copy genes, paralogs are often recovered with increased sequencing depth, sometimes from a significant proportion of loci, especially in groups experiencing whole-genome duplication (WGD) events. Common approaches for processing paralogs in target enrichment datasets include random selection, manual pruning, and mainly, the removal of entire genes that show any evidence of paralogy. These approaches are prone to errors in orthology inference or removing large numbers of genes. By removing entire genes, valuable information that could be used to detect and place WGD events is discarded. Here we use an automated approach for orthology inference in a target enrichment dataset of 68 species of Alchemilla s.l. (Rosaceae), a widely distributed clade of plants primarily from temperate climate regions. Previous molecular phylogenetic studies and chromosome numbers both suggested ancient WGDs in the group. However, both the phylogenetic location and putative parental lineages of these WGD events remain unknown. By taking paralogs into consideration, we identified four nodes in the backbone of Alchemilla s.l. with an elevated proportion of gene duplication. Furthermore, using a gene-tree reconciliation approach we established the autopolyploid origin of the entire Alchemilla s.l. and the nested allopolyploid origin of four major clades within the group. Here we showed the utility of automated tree-based orthology inference methods, previously designed for genomic or transcriptomic datasets, to study complex scenarios of polyploidy and reticulate evolution from target enrichment datasets.


title: Longitudinal tracking reveals sustained polyclonal repopulation of human-HSPC in humanized mice despite vector integration bias

abstract:
BackgroundCurrent understanding of hematopoiesis is largely derived from mouse models that are physiologically distant from humans. Humanized mice provide the most physiologically relevant small animal model to study human diseases, most notably preclinical gene-therapy studies. However, the clonal repopulation dynamics of human hematopoietic stem and progenitor cells (HSPC) in these animal models is only partially understood. Using a new clonal tracking methodology designed for small sample volumes, we aim to reveal the underlying clonal dynamics of human cell repopulation in a mouse environment.

MethodsHumanized BLT (bone marrow-liver-thymus) mice were generated by transplanting lentiviral vector transduced human fetal liver HSPC (FL-HSPC) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice implanted with a piece of human fetal thymus. We developed a methodology to track vector integration sites (VIS) in a mere 25{micro}l of mouse blood for longitudinal and quantitative clonal analysis of human HSPC repopulation in mouse environment. We explored transcriptional and epigenetic features of human HSPC for possible VIS bias.

Results897 HSPC clones were longitudinally tracked in BLT mice--providing a first-ever demonstration of clonal dynamics and competitive expansion of therapeutic and control vector-modified human cell populations simultaneously repopulating in the same humanized mice. The polyclonal repopulation stabilized at 19 weeks post-transplant and the contribution of the largest clone doubled within 4 weeks. Moreover, 550 ([~]60%) clones persisted over 6 weeks and were highly shared between different organs. The normal clonal profiles confirmed the safety of our gene therapy vectors. Multi-omics analysis of human FL-HSPC revealed that 54% of vector integrations in repopulating clones occurred within {+/-}1kb of H3K36me3-enriched regions.

ConclusionsHuman repopulation in mice is polyclonal and stabilizes more rapidly than that previously observed in humans. VIS preference for H3K36me3 has no apparent negative effects on HSPC repopulation. Our study provides a methodology to longitudinally track clonal repopulation in small animal models extensively used for stem cell and gene-therapy research and with lentiviral vectors designed for clinical applications. Results of this study provide a framework for understanding the clonal behavior of human HPSC repopulating in a mouse environment, critical for translating results from humanized mice models to the human settings.


title: Intra and inter-annual climatic conditions have stronger effect than grazing intensity on root growth of permanent grasslands

abstract:
AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackground and AimsC_ST_ABSUnderstanding how direct and indirect changes in climatic conditions, management, and species composition affect root production and root traits is of prime importance for the delivery of carbon sequestration services of grasslands. This study considers the effects of climatic variability and gradients of herbage utilisation by grazing on root production over the course of two years. The root and leaf traits of the plant communities were determined to detect their capacity to predict above- and below-ground net primary production, ANPP and BNPP, respectively.

MethodsA long-term field experiment was used to compare the effects of abandonment and low (Ca-) and high (Ca+) grazing intensities (resulting in mean residual plant heights of 15.2 cm and 7.7 cm, respectively) induced by grazing rotations on upland fertile grasslands after 10 years of treatment application. Ingrowth cores and exclusion cages were used to measure, respectively, the root and shoot mass production several times each year and at an annual scale. The root and leaf traits of the communities were measured near the vegetations peak growing season.

ResultsWe observed strong seasonal root production across treatments in both a wet and a dry year, but the response to grazing intensity was hardly observable within growing seasons. In the abandonment treatment, the spring and autumn root growth peaks were delayed by approximately one month compared to the two cattle treatments, possibly due to a late plant canopy green-up induced by lower soil temperatures and an accumulation of litter. The BNPP was slightly lower in the abandonment treatment compared to the cattle treatments only during the dry year, whereas a decline of the ANPP in the abandonment treatment compared to the Ca+ treatment was observed during the wet year. In response to drought, which occurred during the second year, the root-to-shoot biomass ratio was stable in the cattle treatments but declined in the abandonment treatment. The higher allocation to root mass could benefit plant communities under drier conditions.

ConclusionsRotational grazing pressures and climatic condition variabilities had limited effects on root growth seasonality, although drought had stronger effects on the BNPP than on the ANPP. The stability of the root-to-shoot biomass ratio during the dry year evidenced a higher resistance to drought by grazed versus abandoned grassland communities.

category: ecology


title: Variation in adaptation across geographic ranges is consistent with strong selection in marginal climates and legacies of range expansion

abstract: Every species experiences limits to its geographic distribution. Some evolutionary models predict that populations at range edges are less well-adapted to their local environments due to drift, expansion load, or swamping gene flow from the range interior. Alternatively, populations near range edges might be uniquely adapted to marginal environments. In this study, we use a database of transplant studies that quantify performance at broad geographic scales to test how local adaptation, site quality, and population quality change from spatial and climatic range centers towards edges. We find that populations from poleward edges perform relatively poorly, both on average across all sites (15% lower population quality) and when compared to other populations at home (31% relative fitness disadvantage), consistent with these populations harboring high genetic load. Populations from equatorial edges also perform poorly on average (18% lower population quality) but, in contrast, outperform foreign populations (16% relative fitness advantage), suggesting that populations from equatorial edges have strongly adapted to unique environments. Finally, we find that populations from sites that are thermally extreme relative to the species niche demonstrate strong local adaptation, regardless of their geographic position. Our findings indicate that both nonadaptive processes and adaptive evolution contribute to variation in adaptation across species ranges.


title: Relabeling metabolic pathway data with groups to improve prediction outcomes

abstract: Metabolic pathway inference from genomic sequence information is an integral scientific problem with wide ranging applications in the life sciences. As sequencing throughput increases, scalable and performative methods for pathway prediction at different levels of genome complexity and completion become compulsory. In this paper, we present reMap (relabeling metabolic pathway data with groups) a simple, and yet, generic framework, that performs relabeling examples to a different set of labels, characterized as groups. A pathway group is comprised of a subset of statistically correlated pathways that can be further distributed between multiple pathway groups. This has important implications for pathway prediction, where a learning algorithm can revisit a pathway multiple times across groups to improve sensitivity. The relabeling process in reMap is achieved through an alternating feedback process. In the first feed-forward phase, a minimal subset of pathway groups is picked to label each example. In the second feed-backward phase, reMaps internal parameters are updated to increase the accuracy of mapping examples to pathway groups. The resulting pathway group dataset is then be used to train a multi-label learning algorithm. reMaps effectiveness was evaluated on metabolic pathway prediction where resulting performance metrics equaled or exceeded other prediction methods on organismal genomes with improved predictive performance.

category: bioinformatics


title: Theoretical basis for stabilizing messenger RNA through secondary structure design

abstract:
RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery, and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. These computational tests were carried out on both model mRNAs and COVID-19 mRNA vaccine candidates. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term  superfolder mRNAs. These designs exhibit wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity, and their folding is robust to temperature, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1, and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable.

Significance statementMessenger RNA (mRNA) medicines that encode and promote translation of a target protein have shown promising use as vaccines in the current SARS-CoV-2 pandemic as well as infectious diseases due to their speed of design and manufacturing. However, these molecules are intrinsically prone to hydrolysis, leading to poor stability in aqueous buffer and major challenges in distribution. Here, we present a principled biophysical model for predicting RNA degradation, and demonstrate that the stability of any mRNA can be increased at least two-fold over conventional design techniques. Furthermore, the predicted stabilization is robust to post-design modifications. This conceptual framework and accompanying algorithm can be immediately deployed to guide re-design of mRNA vaccines and therapeutics to increase in vitro stability.

category: biophysics


title: Evolution and antiviral activity of a human protein of retroviral origin

abstract:
Endogenous retroviruses are abundant components of mammalian genomes descended from ancient germline infections. In several mammals, the envelope proteins encoded by these elements protect against exogenous viruses, but this activity has not been documented in human. We report that our genome harbors a large pool of envelope-derived sequences with the potential to restrict retroviral infection. To further test this, we characterize in detail the envelope-derived protein, Suppressyn. We found that Suppressyn is expressed in preimplantation embryos and developing placenta using its ancestral retroviral promoter. Restriction assays in cell culture show that Suppressyn, and its hominoid orthologs, can restrict infection by extant mammalian type D retroviruses. Our data support a generalizable model of retroviral envelope cooption for host immunity and genome defense.

SummaryWe found that the human genome expresses a vast pool of envelope sequences of retroviral origin and provide proof of principle that such proteins can restrict zoonotic viruses.


title: Calling small variants with universality and Bayesian-frequentist hybridism

abstract:
The accuracy of variant calling is crucially important in clinical settings, as the misdiagnosis of a genetic disease such as cancer can compromise patient survival. Although many variant callers were developed, variant-calling accuracy is still insufficient for clinical applications.

Here we describe UVC, a method for calling small variants of germline or somatic origin. By combining contrary assumptions with sublation, we found two principles to improve variant calling. First, we discovered the following power-law universality: allele fraction is inversely proportional to the cubic root of variant-calling error rate. Second, we found that zero inflation can combine Bayesian and frequentist models of sequencing bias.

We evaluated UVC with other state-of-the-art variant callers by considering a variety of calling modes (germline, somatic, tumor-only, and cell-free DNA with unique molecular identifiers (UMIs)), sequencing platforms (Illumina, BGI, and IonTorrent), sequencing types (whole-genome, whole-exome, and PCR-amplicon), human reference genomes (hg19, hs37d5, and GRCh38), aligners (BWA and NovoAlign), and representative sequencing depths and purities for both tumor and normal. UVC generally outperformed other germline variant callers on the GIAB germline truth sets. UVC strongly outperformed other somatic variant callers on 192 scenarios of in silico mixtures simulating 192 combinations of tumor/normal sequencing depths and tumor/normal purities. UVC strongly outperformed other somatic variant callers on the GIAB somatic truth sets derived from physical mixture and on the SEQC2 somatic reference sets derived from the breast-cancer cell-line HCC1395. UVC achieved 100% concordance with the manual review conducted by multiple independent researchers on a Qiagen 71-gene-panel dataset derived from 16 patients with colon adenoma. Additionally, UVC outperformed Mageri and smCounter2, the state-of-the-art UMI-aware variant callers, on the tumor-only datasets used for publishing these two variant callers. Performance is measured by using sensitivity-specificity trade off for all called variants. The improved variant calls generated by UVC from previously published UMI-based sequencing data are able to provide additional biological insight about DNA damage repair.

UVC enables highly accurate calling of small variants from a variety of sequencing data, which can directly benefit patients in clinical settings. UVC is open-sourced under the BSD 3-Clause license at https://github.com/genetronhealth/uvc and quay.io/genetronhealth/gcc-6-3-0-uvc-0-6-0-441a694.


title: Single-cell analysis of patient-derived PDAC organoids reveals cell state heterogeneity and a conserved developmental hierarchy

abstract: Pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer mortality by 2030. Bulk transcriptomic analyses have distinguished  classical pancreatic tumors from  basal-like tumors with more aggressive clinical behaviour. We derived PDAC organoids from primary tumors of 18 patients, together with two matched samples from liver metastases. By single-cell RNA sequencing, we show that PDAC organoids consist of ductal cells with patient-specific expression of several gene groups, including genes which encode cell surface proteins. We report  classical and  basal-like cells coexisting within single primary tumors or metastases, with greater intratumor subtype heterogeneity linked to higher tumor grade. Single-cell transcriptome analysis of PDAC organoids and primary PDAC identified distinct tumor cell states shared across patients, including a cycling progenitor cell state and a differentiated secretory state. We show that these cell states are connected by a differentiation hierarchy, with  classical subtype cells concentrated at the endpoint of this hierarchy. In an imaging-based drug screen, expression of  classical subtype genes also correlates with better response to clinical drugs. Our results thus uncover a functional hierarchy of PDAC cell states linked to transcriptional tumor subtypes, and support the use of PDAC organoids as a clinically relevant model for in vitro studies of tumor heterogeneity.


title: Computational Modeling: Human Dynamic Model

abstract: Improvements in quantitative measurements of human physical activity are proving extraordinarily useful for studying the underlying musculoskeletal system. Dynamic models of human movement support clinical efforts to analyze, rehabilitate injuries. They are also used in biomechanics to understand and diagnose motor pathologies, find new motor strategies that decrease the risk of injury, and predict potential problems from a particular procedure. In addition, they provide useful constraints for underlying neural circuits. This paper describes a physics-based movement analysis method for analyzing and simulating bipedal humanoid movements. A 48 degree of freedom dynamic model of humans has been developed to report humanoid movements energetic components. It has sufficient speed and accuracy to analyze and synthesize real-time interactive applications, such as psychophysics experiments using virtual reality or human-in-the-loop teleoperation of a simulated robotic system. The dynamic model is fast and robust while still providing results sufficiently accurate to be used to believably animate a humanoid character or estimate internal joint forces used during a movement for creating effort-contingent experimental stimuli. A virtual reality environment developed as part of this research supports controlled experiments for systematically recording human behaviors.

category: physiology


title: Focal Electrical Stimulation of Human Retinal Ganglion Cells for Vision Restoration

abstract: Vision restoration with retinal implants that electrically stimulate retinal ganglion cells (RGCs), which transmit visual information to the brain, is limited by indiscriminate activation of many cells and cell types. Recent work in isolated macaque retina has demonstrated that direct electrical stimulation of RGCs can be performed with single-cell, single-spike resolution. However, the fidelity of epiretinal stimulation has not been examined in the human retina. Here, electrical activation of the major RGC types was examined using large-scale, multi-electrode recording and stimulation in the human retina ex vivo and compared directly to results from macaque. Targeted activation with single-cell, single-spike resolution was often possible without activating overlying axon bundles, at low stimulation current levels similar to those in macaque. Distinct cell types could be identified and targeted based on their distinct electrical signatures. Simulation based on these measurements revealed that a novel, dynamic stimulation approach would produce a nearly optimal evoked visual signal. These results indicate that high-fidelity control of spiking in human RGCs is achievable with extracellular stimulation and that the macaque retina is an accurate model for vision restoration with epiretinal implants.


title: Value Certainty in Drift-Diffusion Models of Preferential Choice

abstract: The drift-diffusion model (DDM) is widely used and broadly accepted for its ability to account for binary choices (in both the perceptual and preferential domains) and response times (RT), as a function of the stimulus or the choice alternative (or option) values. The DDM is built on an evidence accumulation-to-bound concept, where, in the value domain, a decision maker repeatedly samples the mental representations of the values of the available options until satisfied that there is enough evidence (or support) in favor of one option over the other. As the signals that drive the evidence are derived from value estimates that are not known with certainty, repeated sequential samples are necessary to average out noise. The classic DDM does not allow for different options to have different levels of precision in their value representations. However, recent studies have shown that decision makers often report levels of certainty regarding value estimates that vary across choice options. There is therefore a need to extend the DDM to include an option-specific value certainty component. We present several such DDM extensions and validate them against empirical data from four previous studies. The data support best a DDM version in which the drift of the accumulation is based on a sort of signal-to-noise ratio of value for each option (rather than a mere accumulation of samples from the corresponding value distributions). This DDM variant accounts for the impact of value certainty on both choice consistency and response time present in the empirical data.


title: Dissociable Roles of Pallidal Neuron Subtypes in Regulating Motor Patterns

abstract:
We have previously established that PV+ neurons and Npas1+ neurons are distinct neuron classes in the GPe-- they have different topographical, electrophysiological, circuit, and functional properties. Aside from Foxp2+ neurons, which are a unique subclass within the Npas1+ class, we lack driver lines that effectively capture other GPe neuron subclasses. In this study, we examined the utility of Kcng4-Cre, Npr3-Cre, and Npy2r-Cre mouse lines (both males and females) for the delineation of GPe neuron subtypes. By using these novel driver lines, we have provided the most exhaustive investigation of electrophysiological studies of GPe neuron subtypes to date. Corroborating our prior studies, GPe neurons can be divided into two statistically distinct clusters that map onto PV+ and Npas1+ classes. By combining optogenetics and machine learning-based tracking, we showed that optogenetic perturbation of GPe neuron subtypes generated unique behavioral structures. Our findings further highlighted the dissociable roles of GPe neurons in regulating movement and anxiety-like behavior. We concluded that Npr3+ neurons and Kcng4+ neurons are distinct subclasses of Npas1+ neurons and PV+ neurons, respectively. Finally, by examining local collateral connectivity, we inferred the circuit mechanisms involved in the motor patterns observed with optogenetic perturbations. In summary, by identifying mouse lines that allow for manipulations of GPe neuron subtypes, we created new opportunities for interrogations of cellular and circuit substrates that can be important for motor function and dysfunction.

Significance statementWithin the basal ganglia, the external globus pallidus (GPe) has long been recognized for its involvement in motor control. However, we lacked an understanding of precisely how movement is controlled at the GPe level as a result of its cellular complexity. In this study, by using transgenic and cell-specific approaches, we showed that genetically-defined GPe neuron subtypes have distinct roles in regulating motor patterns. In addition, the in vivo contributions of these neuron subtypes are in part shaped by the local, inhibitory connections within the GPe. In sum, we have established the foundation for future investigations of motor function and disease pathophysiology.


title: Thymidine starvation promotes c-di-AMP dependent inflammation during infection

abstract: Antibiotics remain one of the most effective methods for controlling bacterial infection. However, the diverse impacts of antimicrobials on bacterial physiology and host immunity remain unclear. A comprehensive antibiotic screen revealed that disruption of thymidine synthesis in Firmicutes with anti-folate antibiotics promoted elevated levels of the bacterial second messenger cyclic di-AMP, and consequently induced host STING activation during infection. Extensive exposure to antibiotics targeting folate synthesis drives the emergence of thymidine-dependent Staphylococcus aureus SCVs (TD-SCVs). Respiratory infections with TD-SCVs are common among children with cystic fibrosis and are associated with worse clinical outcomes, although the underlying pathophysiological mechanisms remain to be defined. Our study reveals that TD-SCV isolates exhibited excessive c-di-AMP production and STING activation in a thymidine-dependent manner. Murine lung infection with TD-SCVs revealed STING-dependent elevation of proinflammatory cytokines, leading to higher airway neutrophil infiltration and activation comparing to normal colony S. aureus and hemin-dependent SCV. Our results suggest the elevated inflammatory capacity of TD-SCVs contribute to their pathogenesis and revealed a new aspect of STING signaling in the airway by characterizing its role in neutrophil recruitment.


title: A CAR RNA FISH assay to study functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue

abstract:
Chimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer. Here, we describe a CAR RNA FISH histo-cytometry platform combined with a random reaction seed image analysis algorithm to quantitate spatial distribution and in vivo functional activity of a CAR T cell population at a single cell resolution for preclinical models. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions of genetically engineered cells in situ with their target cells in tissues with high statistical power and thus, can serve as an important tool for preclinical assessment of CAR T cell effectiveness.

Brief summaryWe developed an imaging platform and analysis pipeline to study large populations of engineered cells at a single cell level in situ.

One Sentence SummaryWe developed a CAR RNA FISH assay to study chimeric antigen receptor T cell trafficking and function in mouse tissue.


title: Gradual evolution of allopolyploidy in Arabidopsis suecica

abstract: The majority of diploid organisms have polyploid ancestors. The evolutionary process of polyploidization (and subsequent re-diploidization) is poorly understood, but has frequently been conjectured to involve some form of "genome shock" -- partly inspired by studies in crops, where polyploidy has been linked to major genomic changes such as genome reorganization and subgenome expression dominance. It is unclear, however, whether such dramatic changes would be characteristic of natural polyploidization, or whether they are a product of domestication. Here, we study polyploidization in Arabidopsis suecica (n = 13), a post-glacial allopolyploid species formed via hybridization of A. thaliana (n = 5) and A. arenosa (n = 8). We generated a chromosome-level genome assembly of A. suecica and complemented it with polymorphism and transcriptome data from multiple individuals of all species. Despite a divergence of [~]6 Mya between the two ancestral species and appreciable differences in their genome composition, we see no evidence of a genome shock: the A. suecica genome is highly colinear with the ancestral genomes, there is no subgenome dominance in expression, and transposable element dynamics appear to be stable. We do, however, find strong evidence for changes suggesting gradual adaptation to polyploidy. In particular, the A. thaliana subgenome shows upregulation of meiosis-related genes, possibly in order to prevent aneuploidy and undesirable homeologous exchanges that are frequently observed in experimentally generated A. suecica, and the A. arenosa subgenome shows upregulation of cyto-nuclear related processes, possibly in response to the new cytoplasmic environment of A. suecica, with plastids maternally inherited from A. thaliana.


title: The spatial landscape of gene expression isoforms in tissue sections

abstract: In situ capturing technologies add tissue context to gene expression data, with the potential of providing a greater understanding of complex biological systems. However, splicing variants and fulllength sequence heterogeneity cannot be characterized at spatial resolution with current transcriptome profiling methods. To that end, we introduce Spatial Isoform Transcriptomics (SiT), an explorative method for characterizing spatial isoform variation and sequence heterogeneity. We show in mouse brain how SIT can be used to profile isoform expression and sequence heterogeneity in different areas of the tissue. SiT reveals regional isoform switching of Plp1 gene between different layers of the olfactory bulb, and use of external single cell data allowed to nominate cell types expressing each isoform. Furthermore, SiT identifies differential isoform usage for several major genes implicated in brain function (Snap25, Bin1, Gnas) that we independently validated by in situ sequencing. SiT also provides for the first time an in-depth A-to-I RNA editing map of the adult mouse brain. Data exploration can be performed through an online resource (https://www.isomics.eu), where isoform expression and RNA editing can be visualized in a spatial context.

category: genomics


title: TimTrack: A drift-free algorithm for estimating geometric muscle features from ultrasound images

abstract: Ultrasound imaging is valuable for non-invasively estimating fascicle lengths and other features of pennate muscle, especially when performed computationally. Effective analysis techniques to date typically use optic flow to track displacements from image sequences, but are sensitive to integration drift for longer sequences. We here present an alternative algorithm that objectively estimates geometric features of pennate muscle from ultrasound images, without drift sensitivity. The algorithm identifies aponeuroses and estimates fascicle angles to derive fascicle lengths. Length estimates of human vastus lateralis and lateral gastrocnemius in healthy subjects (N = 9 and N = 1 respectively) compared well (root-mean-square error, RMSE < 0.80 cm) to manual estimates by independent observers (n = 3). The coefficient of multiple correlation (CMC) with manual estimates of fascicle length was comparable to previously reported for state-of-the-art optic flow algorithm (0.93-0.99), suggesting similar accuracy. The algorithm requires minimal manual intervention and can optionally extrapolate fascicle lengths that extend beyond the image frame. It facilitates automated analysis of ultrasound images without drift.


title: Bromodomain and Extraterminal Inhibition Blocks Inflammation-Induced Cardiac Dysfunction and SARS-CoV-2 Infection (Pre-Clinical)

abstract: Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined, but could be direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory  cytokine-storm, a cocktail of interferon gamma, interleukin 1{beta} and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids and hearts of SARS-CoV-2 infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCO and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the FDA breakthrough designated drug apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage.


title: Frequency shapes the quality of tactile percepts evoked through electrical stimulation of the nerves

abstract: AO_SCPLOWBSTRACTC_SCPLOWTouch is critical for our ability to manipulate objects, as evidenced by the deficits incurred when touch is absent. To restore the sense of touch via electrical stimulation of the peripheral nerves requires that we understand how the parameters of stimulation shape the evoked sensation. To this end, we investigated the sensory consequences of changing the frequency of pulse trains (PF) delivered to the peripheral nerves of humans chronically implanted with multi-channel nerve cuff electrodes. We found that increases in PF led to systematic increases in perceived frequency, up to about 50 Hz, at which point further changes in PF had little to no impact on sensory quality. Above this transition frequency, ratings of perceived frequency levelled off, the ability to discriminate changes in PF was abolished, and verbal descriptors selected to characterize the sensation changed abruptly. We conclude that the quality of electrically evoked tactile sensations can be shaped by imposing temporal patterns on a fixed neural population, but this temporal patterning can only be resolved up to about 50 Hz. These findings highlight the importance of spike timing in shaping the quality of a sensation and will contribute to the development of encoding strategies for conveying touch feedback through bionic hands and feet.

category: bioengineering


title: Using temperature to analyse the neural basis of a latent temporal decision

abstract: The basal ganglia (BG) are thought to contribute to decision-making and motor control by learning to select actions based on consequences. These functions are critically dependent on timing information that can be extracted from the evolving state of neural populations in the striatum, the major input area of the BG. However, it is debated whether striatal activity underlies latent, dynamic decision processes or kinematics of overt movement. Here, we measured the impact of temperature on striatal population activity and the behavior of rats and compared the observed effects to neural activity and behavior collected in multiple versions of a temporal categorization task. Cooler temperatures caused dilation, and warmer temperatures contraction, of both neural activity and patterns of judgment in time, mimicking endogenous decision-related variability in striatal activity. However, temperature did not similarly affect movement kinematics. These data demonstrate that the time-course of evolving striatal population activity dictates the speed of a latent process that is used to guide decision-making, but not finer temporal aspects of movement. More broadly, they establish temporal scaling of population activity as a cause and not simply a correlate of timing in the brain.

category: neuroscience


title: BDNF/TrkB signaling endosomes in axons coordinate CREB/mTOR activation and protein synthesis in the cell body to induce dendritic growth in cortical neurons.

abstract: Brain-derived neurotrophic factor (BDNF) and its receptors tyrosine kinase receptor B (TrkB) and the p75 neurotrophin receptor (p75) are the primary regulators of dendritic growth in the central nervous system (CNS). After being bound by BDNF, TrkB and p75 are endocytosed into endosomes and continue signaling within the cell soma, dendrites, and axons. We studied the functional role of BDNF axonal signaling in cortical neurons derived from different transgenic mice using compartmentalized cultures in microfluidic devices. We found that axonal BDNF increased dendritic growth from the neuronal cell body in a cAMP response element-binding protein (CREB)-dependent manner. These effects were dependent on axonal TrkB but not p75 activity. Dynein-dependent BDNF-TrkB-containing endosome transport was required for long-distance induction of dendritic growth. Axonal signaling endosomes increased CREB and mTOR kinase activity in the cell body, and this increase in the activity of both proteins was required for general protein translation and the expression of Arc, a plasticity-associated gene, indicating a role for BDNF-TrkB axonal signaling endosomes in coordinating the transcription and translation of genes whose products contribute to learning and memory regulation.


title: Genome-Wide Mapping of Human DNA Replication by Optical Replication Mapping Supports a Stochastic Model of Eukaryotic Replication

abstract: DNA replication is regulated by the location and timing of replication initiation. Therefore, much effort has been invested in identifying and analyzing the sites of human replication initiation. However, the heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation site utilization in metazoans has made mapping the location and timing of replication initiation in human cells difficult. A potential solution to the problem of human replication mapping is single-molecule analysis. However, current approaches do not provide the throughput required for genome-wide experiments. To address this challenge, we have developed Optical Replication Mapping (ORM), a high-throughput single-molecule approach to map newly replicated DNA, and used it to map early initiation events in human cells. The single-molecule nature of our data, and a total of more than 2000-fold coverage of the human genome on 27 million fibers averaging ~300 kb in length, allow us to identify initiation sites and their firing probability with high confidence. In particular, for the first time, we are able to measure genome-wide the absolute efficiency of human replication initiation. We find that the distribution of human replication initiation is consistent with inefficient, stochastic initiation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. In particular, we find sites of human replication initiation are not confined to well-defined replication origins but are instead distributed across broad initiation zones consisting of many initiation sites. Furthermore, we find no correlation of initiation events between neighboring initiation zones. Although most early initiation events occur in early-replicating regions of the genome, a significant number occur in late-replicating regions. The fact that initiation sites in typically late-replicating regions have some probability of firing in early S phase suggests that the major difference between initiation events in early and late replicating regions is their intrinsic probability of firing, as opposed to a qualitative difference in their firing-time distributions. Moreover, modeling of replication kinetics demonstrates that measuring the efficiency of initiation-zone firing in early S phase suffices to predict the average firing time of such initiation zones throughout S phase, further suggesting that the differences between the firing times of early and late initiation zones are quantitative, rather than qualitative. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.