title: Top-down contextual requirement to suppress self-movement influences grasp-specific modulation of corticospinal excitability during action observation

abstract:
Action observation modulates corticospinal excitability (CSE) measured via transcranial magnetic stimulation (TMS) in humans, which presumably exposes the effect of mirror neuron activation on corticospinal pathways. These responses can consist of both facilitation and suppression, and the balance of these two may restrict the outflow of activity into movement. Evidence also suggests that task context can considerably influence CSE changes during action observation.

Here, we assessed whether embedding action observation within a Go-NoGo paradigm, emphasizing movement withholding on observation and NoGo trials, influenced CSE modulation. Fourteen healthy subjects received single pulse TMS over left primary motor cortex (M1) during a baseline period, grasp observation onset, or after a NoGo cue, while performing, observing, or withholding two distinct reach-to-grasp actions. We assessed modulation of motor evoked potentials (MEPs) in three intrinsic hand muscles, which were recruited in a grasp-specific manner during action execution. CSE modulation was limited, and predominantly suppressive in nature during grasp observation. Seven subjects performed the same task without the NoGo condition ("Go-only" block) immediately before the "Go&NoGo" block. We found evidence for grasp-specific modulation of CSE, which matched the recruitment pattern of the muscles during action execution. Within these subjects, modulation was attenuated when the NoGo condition was introduced, but was still distinct from modulation in the first group.

These results suggest that bottom-up grasp-specific modulation of MEPs during action observation is attenuated by the top-down contextual requirement to suppress self-movement, and facilitation and suppression effects are determined by the balance between these two processes.

SIGNIFICANCE STATEMENTAction observation can activate specific pathways in the motor system of the observer, which are also used to perform the same action. This motor resonance, measured via changes in corticospinal excitability using transcranial magnetic stimulation, is susceptible to task context. In this study, we show that observing grasping actions results in grasp-specific changes in excitability, consistent with mirror neuron activation, but this effect is masked when observation is interleaved within a Go-NoGo paradigm, which emphasises suppression of ones own movement. Top-down task requirements to withhold movement within and across trials, which are present in most action observation studies, likely influence the extent of motor resonance, urging caution in the design and interpretation of results in TMS action observation experiments.


title: Pupil dilation and the slow wave ERP reflect surprise about choice outcome resulting from intrinsic variability in decision confidence

abstract: Central to human and animal cognition is the ability to learn from feedback in order to optimize future rewards. Such a learning signal might be encoded and broadcasted by the brains arousal systems, including the noradrenergic locus coeruleus. Pupil responses and the positive slow wave component of event-related potentials reflect rapid changes in the arousal level of the brain. Here we ask whether and how these variables may reflect surprise: the mismatch between ones expectation about being correct and the outcome of a decision, when expectations fluctuate due to internal factors (e.g., engagement). We show that during an elementary decision-task in the face of uncertainty both physiological markers of phasic arousal reflect surprise. We further show that pupil responses and slow wave ERP are unrelated to each other, and that prediction error computations depend on feedback awareness. These results further advance our understanding of the role of central arousal systems in decision-making under uncertainty.

category: neuroscience


title: Adult Stem Cells and Niche Cells segregate gradually from common precursors that build the adult Drosophila ovary during pupal development

abstract: Adult stem cell function relies on the prior development of appropriate numbers and spatial organization of stem cells and supportive niche cells. Drosophila Follicle Stem Cells (FSCs) present a favorable paradigm for understanding those developmental processes. About sixteen FSCs in an adult germarium produce transit-amplifying Follicle Cells (FCs) from their posterior face and quiescent Escort Cells (ECs) to their anterior. Both ECs and FCs also act as niche cells. Here we show that adult ECs, FSCs and FCs derive from common precursors intermingled with germline cells during pupal development, with progeny of a single precursor commonly including ECs and FSCs, FSCs and FCs, or all three adult cell types. Precursors posterior to germline cells become the first FCs and engulf a largely naked germline cyst projected out of the germarium to form the first egg chamber and set up a key posterior signaling center for regulating adult FSC behavior.

category: developmental biology


title: Coastal winds and larval fish abundance indicate a recruitment mechanism for southeast Australian estuarine fisheries

abstract: Coastal winds transport water masses and larval fish onshore or offshore which may influence estuarine recruitment, yet our understanding of the mechanism underlying this relationship is limited. Here, we combine datasets from a historical database of larval fish off southeast Australia with a high-resolution atmospheric reanalysis model to show that normalised abundance of coastally spawned larvae increased with weak to moderate upwelling favourable winds 14 days prior to sampling. The increase in abundance may reflect increased nutrient and plankton availability for larval fish. Normalised larval abundance decreased following strong upwelling favourable winds but increased after onshore (downwelling favourable) winds, due to wind-driven transport. By combining a commercial estuarine fisheries catch-rate dataset (4 species, 8 estuaries, 10 years) and the high-resolution atmospheric reanalysis model, we show that negative effects of upwelling favourable winds during the spawning period can be detected in lagged estuarine commercial fisheries catch rates (lagged by 2 - 8 years depending on species growth rates), potentially representing the same mechanism proposed for larval fish. Upwelling favourable winds in the southeast Australian region have increased since 1850 while onshore winds have decreased, which may reduce larval recruitment to estuaries. Coastal winds are likely an important factor for estuarine recruitment in the southeast Australian region and future research on the estuarine recruitment of fish should incorporate coastal winds.

category: ecology


title: The Set Increment with Limited Views Encoding Ratio (SILVER) Method for Optimizing Radial Sampling of Dynamic MRI

abstract:
PurposeTo present and assess a method for choosing a fixed increment between spokes in radially sampled MRI that results in higher SNR than other radial sampling increments.

Theory and MethodsSampling uniformity contributes to image SNR when reconstructed using linear methods. Thus, for a radial trajectory, uniformly spaced sampling is ideal. However, uniform sampling lacks post-acquisition reconstruction flexibility, which is often needed in dynamic imaging. Golden ratio-based increments are often used for this purpose. The method presented here, Set Increment with Limited Views Encoding Ratio (SILVER), optimizes sampling uniformity when a limited number of temporal resolutions are required. With SILVER, an optimization algorithm finds the angular increment that provides the highest uniformity for a pre-defined set of reconstruction window sizes. SILVER was tested over multiple sets and assessed in terms of uniformity, noise amplification, and SNR both in simulations and in acquisitions of a phantom and healthy volunteers.

ResultsThe proposed algorithm produced trajectories that, for the optimized window sizes, had higher uniformity and lower image noise than golden ratio sampling both in a simulated single-coil system and in a multi-coil system, assessed using simulation, phantom, and in vivo experiments. The noise in SILVER optimized trajectories was comparable to uniformly distributed spokes whilst retaining flexibility in reconstruction at multiple temporal resolutions. In a resting state fMRI experiment, tSNR increases at different spatial and temporal resolutions ranged from 21-72%.

ConclusionSILVER is a simple addition to any sequence currently using golden ratio sampling and significantly increases sampling efficiency and SNR.


title: Phagocytosis and self-destruction execute dendrite degeneration of Drosophila sensory neurons at distinct levels of NAD+ reduction

abstract: After injury, severed dendrites and axons expose the "eat-me" signal phosphatidylserine (PS) on their surface and degenerate by disassembly. While axon degeneration is controlled by a conserved "axon-death" pathway that is thought to activate self-destruction, how PS exposure is regulated by this pathway and whether PS-induced phagocytosis contributes to neurite breakdown in vivo remain unknown. Here we show that in Drosophila sensory dendrites, PS exposure and self-destruction are triggered by two distinct levels of NAD+ reduction downstream of Sarm activation. Surprisingly, phagocytosis is the main driver of dendrite degeneration induced by both genetic NAD+ disruptions and injury. Furthermore, the axon-death factor Axed is only partially required for self-destruction of injured dendrites, acting in parallel with PS-induced phagocytosis. Lastly, injured dendrites exhibit a unique rhythmic calcium flashing that correlates with self-destruction. Therefore, a special genetic program coordinates PS exposure and self-destruction in injury-induced dendrite degeneration in vivo.

category: cell biology


title: Nfkbid is required for immunity and antibody responses to Toxoplasma gondii

abstract:
Protective immunity to parasitic infections has been difficult to elicit by vaccines. Among parasites that evade vaccine-induced immunity is Toxoplasma gondii, which causes lethal secondary infections in chronically infected mice. Here we report that unlike susceptible C57BL/6J mice, A/J mice were highly resistant to secondary infection. To identify correlates of immunity, we utilized forward genetics to identify Nfkbid, a nuclear regulator of NF-{kappa}B that is required for B cell activation and B-1 cell development. Nfkbid-null mice (bumble) did not generate parasite-specific IgM and lacked robust parasite-specific IgG, which correlated with defects in B-2 cell maturation and class-switch recombination. Though high-affinity antibodies were B-2 derived, transfer of B-1 cells partially rescued the immunity defects observed in bumble mice and were required for 100% vaccine efficacy in bone marrow chimeric mice. Immunity in resistant mice correlated with robust isotype class-switching in both B cell lineages, which can be fine-tuned by Nfkbid gene expression. We propose a model whereby humoral immunity to T. gondii is regulated by Nfkbid and requires B-1 and B-2 cells for full protection.

AUTHOR SUMMARYEukaryotic parasitic diseases account for approximately one fifth of all childhood deaths, yet no highly protective vaccine exists for any human parasite. More research must be done to discover how to elicit protective vaccine-induced immunity to parasitic pathogens. We used an unbiased genetic screen to find key genes responsible for immunity to the eukaryotic parasite Toxoplasma gondii. Our screen found Nfkbid, a transcription factor regulator, which controls B cell activation and innate-like B-1 cell development. Mice without Nfkbid were not protected against T. gondii and were deficient at making antibodies against the parasite. Our survival studies of vaccinated mice with and without B-1 compartments found that B-1 cells improved survival, suggesting that B-1 cells act in conjunction with B-2 cells to provide vaccine-induced immunity. Nfkbid and other loci identified in our unbiased screen represent potential targets for vaccines to elicit protective immune responses against parasitic pathogens.

category: immunology


title: Cheetah: a computational toolkit for cybergenetic control

abstract: Advances in microscopy, microfluidics and optogenetics enable single-cell monitoring and environmental regulation and offer the means to control cellular phenotypes. The development of such systems is challenging and often results in bespoke setups that hinder reproducibility. To address this, we introduce Cheetah - a flexible computational toolkit that simplifies the integration of real-time microscopy analysis with algorithms for cellular control. Central to the platform is an image segmentation system based on the versatile U-Net convolutional neural network. This is supplemented with functionality to robustly count, characterise and control cells over time. We demonstrate Cheetahs core capabilities by analysing long-term bacterial and mammalian cell growth and by dynamically controlling protein expression in mammalian cells. In all cases, Cheetahs segmentation accuracy exceeds that of a commonly used thresholding-based method, allowing for more accurate control signals to be generated. Availability of this easy-to-use platform will make control engineering techniques more accessible and offer new ways to probe and manipulate living cells.

category: synthetic biology


title: Label-free amino acid identification for de novo protein sequencing via tRNA charging and current blockade in a nanopore

abstract: A label-free procedure to identify single amino acids (AAs) for protein sequencing is developed in theory and simulated in part. A terminal AA cleaved from a protein/peptide, a tRNA, its cognate amino-acyl tRNA synthetase (AARS), and adenosine triphosphate (ATP) are brought together in a container where tRNA, if cognate, gets charged with AA and adenosine monophosphate (AMP) is released. The released AMP (and any free AA and ATP molecules) filters into the cis chamber of an adjoining electrolytic cell (e-cell) from where they pass through a nanopore into the trans chamber. Addition of NaOH to the container deacylates the tRNA if it is charged. The resulting free AA passes into the cis chamber of the e-cell, translocates into trans, and causes a current blockade; AA is immediately known from the identity of the tRNA (the two are cognate). If the tRNA is not charged there is no AA bound to it so AA remains unidentified. In this approach there is no need to distinguish among the 20 AAs by blockade size; it suffices to distinguish blockades from noise: thus a high-precision analog measurement has been transformed into a low-precision binary one. Identification is accurate because of tRNA superspecificity (the tRNA charging error rate is < 1/350); parallel execution with 20 different tRNAs can identify AA in one cycle. This is a de novo method in which no prior information about the protein is used or needed.

category: bioengineering


title: Aging leads to DNA methylation alterations associated with loss of lineage fidelity and breast cancer in mammary luminal epithelial cells

abstract: Age is a significant risk factor for breast cancer and luminal epithelial cells (LEps) are a key cell lineage implicated in age-related luminal breast cancers. The aging process is characterized by alterations to the epigenome. However, the extent of age-associated DNA methylation alterations in LEps and its functional consequences have remained unclear. We report here that aging leads to distinct methylation changes in LEps that may increase susceptibility to breast cancer. Regulatory elements display consistent methylation changes at lineage-specific TF binding sites consistent with loss of lineage fidelity. CpG islands and transposable elements (TEs) showed increased methylation disorder with stochastic methylation gain and loss respectively that were also detected in luminal breast cancer. Each of these classes of methylation changes impact the regulation of genes associated with luminal breast cancer. Altogether, our results indicate that aging leads to DNA methylation changes that could determine breast cancer susceptibility.

category: genomics


title: Urbanization promotes specific bacteria in freshwater microbiomes including potential pathogens

abstract: Freshwater ecosystems are characterized by complex and highly dynamic microbial communities that are strongly structured by their local environment and biota. Growing city populations and the process of urbanization substantially alter freshwater environments. To determine the changes in freshwater microbial communities associated with urbanization, full-length 16S rRNA gene PacBio sequencing was performed from surface water and sediments from a wastewater treatment plant, urban and rural lakes in the Berlin-Brandenburg region, Northeast Germany. Water samples exhibited highly habitat specific bacterial communities with multiple genera showing clear urban signatures. We identified potentially harmful bacterial groups associated with environmental parameters specific to urban habitats such as Alistipes, Escherichia/Shigella, Rickettsia and Streptococcus. We demonstrate that urbanization alters natural microbial communities in lakes and, via simultaneous eutrophication, creates favorable conditions that promote specific bacterial genera including potential pathogens. Our findings are of global relevance highlighting a long-term health risk in urbanized waterbodies, at a time of accelerated global urbanization. The results demonstrate the urgency for undertaking mitigation measures such as targeted lake restoration projects and sustainable water management efforts.

category: microbiology


title: A PCNA-K164R mutation impinges on origin activation and mitotic DNA synthesis

abstract:
Ubiquitination of the replication clamp proliferating cell nuclear antigen (PCNA) at the conserved residue lysine 164 (K164) occurs during normal S phase progression and increases after DNA damage induced replication stress. PCNA-K164 ubiquitination is critical for Okazaki fragment maturation and the activation of DNA damage tolerance pathways. Moreover, ubiquitinated PCNA operates in a fork protection pathway parallel to BRCA-RAD51. Whether PCNA ubiquitination regulates other genome maintenance mechanisms is unclear. Utilizing PCNAK164R cells generated by CRISPR-Cas9, we demonstrate that this mutation causes DNA replication defects that impact origin activation. PCNAK164R cells accumulate single-stranded DNA gaps during replication that persist throughout mitosis due to compromised mitotic DNA synthesis (MiDAS). We uncover a novel role for PCNA-K164 ubiquitination in regulating FANCD2 to initiate MiDAS. Persistent gaps hence interfere with MCM2-7 double hexamer loading in the subsequent G1 phase. Our findings demonstrate that the impact of PCNAK164-Ub is not limited to S/G2 phases but extends to mitosis and G1 phase.

SUMMARYPCNA-K164 ubiquitination promotes DNA gap filling during S/G2 phases of the cell cycle. This study identifies a novel role for K164 ubiquitination in replication dynamics and mitotic DNA synthesis and thus provides new insight into the players involved in counteracting under-replication.

category: molecular biology


title: Dense time-course gene expression profiling of the Drosophila melanogaster innate immune response

abstract:
BackgroundImmune responses need to be initiated rapidly, and maintained as needed, to prevent establishment and growth of infections. At the same time, resources need to be balanced with other physiological processes. On the level of transcription, studies have shown that this balancing act is reflected in tight control of the initiation kinetics and shutdown dynamics of specific immune genes.

ResultsTo investigate genome-wide expression dynamics and trade-offs after infection at a high temporal resolution, we performed an RNA-seq time course on D. melanogaster with 20 time points post Imd stimulation. A combination of methods, including spline fitting, cluster analysis, and Granger causality inference, allowed detailed dissection of expression profiles, lead-lag interactions, and functional annotation of genes through guilt-by-association. We identified Imd-responsive genes and co-expressed, less well characterized genes, with an immediate-early response and sustained up-regulation up to five days after stimulation. In contrast, stress response and Toll-responsive genes, among which were Bomanins, demonstrated early and transient responses. We further observed a strong trade-off with metabolic genes, which strikingly recovered to pre-infection levels before the immune response was fully resolved.

ConclusionsThis high-dimensional dataset enabled the comprehensive study of immune response dynamics through the parallel application of multiple temporal data analysis methods. The well annotated data set should also serve as a useful resource for further investigation of the D. melanogaster innate immune response, and for the development of methods for analysis of a post-stress transcriptional response time-series at whole-genome scale.

category: systems biology


title: Tempo and mode of morphological evolution are decoupled from latitude in birds

abstract: The latitudinal diversity gradient is one of the most striking patterns in nature yet its implications for morphological evolution are poorly understood. In particular, it has been proposed that an increased intensity of species interactions in tropical biota may either promote or constrain trait evolution, but which of these outcomes predominates remains uncertain. Here, we develop tools for fitting phylogenetic models of phenotypic evolution in which the impact of species interactions--namely, competition--can vary across lineages. Deploying these models on a global avian trait dataset to explore differences in trait divergence between tropical and temperate lineages, we find that the effect of latitude on the mode and tempo of morphological evolution is weak and clade- or trait-dependent. Our results indicate that competition does not disproportionately impact morphological evolution in tropical bird families and question the validity of previously reported patterns of slower trait evolution in the tropics.

category: evolutionary biology


title: A neural surveyor to map touch on the body

abstract: Perhaps the most recognizable sensory map in all of neuroscience is the somatosensory homunculus. Though it seems straightforward, this simple representation belies the complex link between an activation in a somatotopic map and the associated touch location on the body. Any isolated activation is spatially ambiguous without a neural decoder that can read its position within the entire map, but how this is computed by neural networks is unknown. We propose that the somatosensory system implements multilateration, a common computation used by surveying and GPS systems to localize objects. Specifically, to decode touch location on the body, multilateration estimates the relative distance between the afferent input and the boundaries of a body part (e.g., the joints of a limb). We show that a simple feedforward neural network, which captures several fundamental receptive field properties of cortical somatosensory neurons, can implement a Bayes-optimal multilateral computation. Simulations demonstrated that this decoder produced a pattern of localization variability between two boundaries that was unique to multilateration. Finally, we identify this computational signature of multilateration in actual psychophysical experiments, suggesting that it is a candidate computational mechanism underlying tactile localization.


title: The neural architecture of language: Integrative modeling converges on predictive processing

abstract:
The neuroscience of perception has recently been revolutionized with an integrative modeling approach in which computation, brain function, and behavior are linked across many datasets and many computational models. By revealing trends across models, this approach yields novel insights into cognitive and neural mechanisms in the target domain. We here present a first systematic study taking this approach to higher-level cognition: human language processing, our species signature cognitive skill. We find that the most powerful  transformer models predict nearly 100% of explainable variance in neural responses to sentences and generalize across different datasets and imaging modalities (fMRI, ECoG). Models neural fits ( brain score) and fits to behavioral responses are both strongly correlated with model accuracy on the next-word prediction task (but not other language tasks). Model architecture appears to substantially contribute to neural fit. These results provide computationally explicit evidence that predictive processing fundamentally shapes the language comprehension mechanisms in the human brain.

SignificanceLanguage is a quintessentially human ability. Research has long probed the functional architecture of language processing in the mind and brain using diverse brain imaging, behavioral, and computational modeling approaches. However, adequate neurally mechanistic accounts of how meaning might be extracted from language are sorely lacking. Here, we report an important first step toward addressing this gap by connecting recent artificial neural networks from machine learning to human recordings during language processing. We find that the most powerful models predict neural and behavioral responses across different datasets up to noise levels. Models that perform better at predicting the next word in a sequence also better predict brain measurements - providing computationally explicit evidence that predictive processing fundamentally shapes the language comprehension mechanisms in the human brain.


title: A two-point IC50 method for evaluating the biochemical potency of irreversible enzyme inhibitors

abstract: Irreversible (covalent) enzyme inhibitors cannot be unambiguously ranked for biochemical potency by using IC50 values determined at a single point in time, because the same IC50 value could originate either from relatively low initial binding affinity accompanied by high chemical reactivity, or the other way around. To disambiguate the potency ranking of covalent inhibitors, here we describe a data-analytic procedure relying on two separate IC50 values, determined at two different reaction times. In the case of covalent inhibitors following the two-step kinetic mechanism E + I {rightleftharpoons} E{middle dot}I [->] EI, the two IC50 values alone can be used to estimate both the inhibition constant (Ki) as a measure of binding affinity and the inactivation rate constant (kinact) as a measure of chemical reactivity. In the case of covalent inhibitors following the one-step kinetic mechanism E + I [->] EI, a simple algebraic formula can be used to estimate the covalent efficiency constant (kinact/Ki) from a single experimental value of IC50. The two simplifying assumptions underlying the method are (1) zero inhibitor depletion, which implies that the inhibitor concentrations are always significantly higher than the enzyme concentration; and (2) constant reaction rate in the uninhibited control assay. The newly proposed method is validated by using a simulation study involving 64 irreversible inhibitors with covalent efficiency constants spanning seven orders of magnitude.

category: biochemistry


title: DeepGANnel: Synthesis of fully annotated single molecule patch-clamp data using generative adversarial networks

abstract:
Development of automated analysis tools for "single ion channel" recording is hampered by the lack of available training data. For machine learning based tools, very large training sets are necessary with sample-by-sample point labelled data (e.g., 1 sample point every 100microsecond). In an experimental context, such data are labelled with human supervision, and whilst this is feasible for simple experimental analysis, it is infeasible to generate the enormous datasets that would be necessary for a big data approach using hand crafting. In this work we aimed to develop methods to generate simulated ion channel data that is free from assumptions and prior knowledge of noise and underlying hidden Markov models.

We successfully leverage generative adversarial networks (GANs) to build an end-to-end pipeline for generating an unlimited amount of labelled training data from a small annotated ion channel "seed" record, and this needs no prior knowledge of theoretical dynamical ion channel properties. Our method utilises 2D CNNs to maintain the synchronised temporal relationship between the raw and idealised record. We demonstrate the applicability of the method with 5 different data sources and show authenticity with t-SNE and UMAP projection comparisons between real and synthetic data. The model would be easily extendable to other time series data requiring parallel labelling, such as labelled ECG signals or raw nanopore sequencing data.

category: physiology


title: Sustained neural rhythms reveal endogenous oscillations supporting speech perception

abstract: Rhythmic sensory or electrical stimulation will produce rhythmic brain responses. These rhythmic responses are often interpreted as endogenous neural oscillations aligned (or "entrained") to the stimulus rhythm. However, stimulus-aligned brain responses can also be explained as a sequence of evoked responses, which only appear regular due to the rhythmicity of the stimulus, without necessarily involving underlying neural oscillations. To distinguish evoked responses from true oscillatory activity, we tested whether rhythmic stimulation produces oscillatory responses which continue after the end of the stimulus. Such sustained effects provide evidence for true involvement of neural oscillations. In Experiment 1, we found that rhythmic intelligible, but not unintelligible speech produces oscillatory responses in magnetoencephalography (MEG) which outlast the stimulus at parietal sensors. In Experiment 2, we found that transcranial alternating current stimulation (tACS) leads to rhythmic fluctuations in speech perception outcomes after the end of electrical stimulation. We further report that the phase relation between electroencephalography (EEG) responses and rhythmic intelligible speech can predict the tACS phase that leads to most accurate speech perception. Together, we provide fundamental results for several lines of research - including neural entrainment and tACS - and reveal endogenous neural oscillations as a key underlying principle for speech perception.


title: Effects of H2A.B incorporation on nucleosome structures and dynamics

abstract:
The H2A.B histone variant is an epigenetic regulator involved in transcriptional upregulation, DNA synthesis, and splicing that functions by replacing the canonical H2A histone in the nucleosome core particle. Introduction of H2A.B results in less compact nucleosome states with increased DNA unwinding and accessibility at the nucleosomal entry and exit sites. Despite being well characterized experimentally, the molecular mechanisms by which H2A.B incorporation alters nucleosome stability and dynamics remain poorly understood. To study the molecular mechanisms of H2A.B, we have performed a series of conventional and enhanced sampling molecular dynamics simulation of H2A.B and canonical H2A containing nucleosomes. Results of conventional simulations show that H2A.B weakens protein/protein and protein/DNA interactions at specific locations throughout the nucleosome. These weakened interactions result in significantly more DNA opening from both the entry and exit sites in enhanced sampling simulations. Furthermore, free energy profiles show that H2A.B containing nucleosomes have significantly broader free wells, and that H2A.B allows for sampling of states with increased DNA breathing, which are shown to be stable on the hundreds of nanoseconds timescale with further conventional simulations. Together, our results show the molecular mechanisms by which H2A.B creates less compacted nucleosome states as a means of increasing genetic accessibility and gene transcription.

SIGNIFICANCENature has evolved a plethora of mechanisms for altering the physical and chemical properties of chromatin fibers as a means of controlling gene expression. These epigenetic processes may serve to increase or decrease DNA accessibility, manage the recruitment of remodeling factors, or tune the stability of the nucleosomes that make up chromatin. Here, we have used both conventional and enhanced sampling molecular dynamics simulations to understand how one of these epigenetic mechanisms, the substitution of canonical H2A proteins with the H2A.B variant, exerts its influence on the structures and dynamics of the nucleosome. Results show at the molecular level how this variant alters inter-molecular interactions to increase DNA accessibility as a means of increasing genetic accessibility and gene transcription.

category: biophysics


title: Pectin induced colony expansion of soil-derived Flavobacterium strains

abstract:
The genus Flavobacterium is characterized by the capacity to metabolize complex organic compounds and a unique gliding motility mechanism. They are often abundant in root microbiomes of various plants, but the factors contributing to this high abundance are currently unknown. In this study, we evaluated the effect of various plant-associated poly- and mono-saccharides on colony expansion of two Flavobacterium strains. Both strains were able to spread on pectin and other polysaccharides such as microcrystalline cellulose. However, only pectin (but not pectin monomers), a component of plant cell walls, enhanced colony expansion on solid surfaces in a dose- and substrate-dependent manner. On pectin, flavobacteria exhibited bi-phasic motility, with an initial phase of rapid expansion, followed by growth within the colonized area. Proteomic and gene expression analyses revealed significant induction of carbohydrate metabolism related proteins when flavobacteria were grown on pectin, including selected SusC/D, TonB-dependent glycan transport operons. Our results show a positive correlation between colony expansion and the upregulation of proteins involved in sugar uptake, suggesting an unknown linkage between specific operons encoding for glycan uptake and metabolism and flavobacterial expansion. Furthermore, within the context of flavobacterial-plant interactions, they suggest that pectin may facilitate flavobacterial expansion on plant surfaces in addition to serving as an essential carbon source.

Contribution to the field statementMembers of the Flavobacterium genus are frequently rhizosphere competent, i.e. abundant and generally enriched on root surfaces relative to surrounding bulk soil, and previous studies suggest that they may play a role in plant health and ecosystem functioning. However, little is known about genetic and physiological factors that facilitate flavobacterial colonization and proliferation in this highly competitive environment.

In this study we found that pectin stimulates flavobacterial colonies in a bi-phasic manner, initially characterized by rapid expansion on agar followed by increased biomass production. This appears to be linked to pectin-stimulated induction of specific TonB-associated proteins evidentially involved in the binding and uptake of complex sugars. Given the fact that pectin and other glycans are a primary component of plant cell walls, we hypothesize that these mechanisms are at least partially responsible for the rhizosphere competence of flavobacteria.


title: Revisiting the Genomic Structure of the Genus Pseudomonas with Whole Genome Data: Insights into Diversity and Host-related Genetic Determinants

abstract: Pseudomonas spp. exhibit considerable differences in host specificity and virulence. Most Pseudomonas species were isolated exclusively from environmental sources, ranging from soil to plants, but some Pseudomonas species have been detected from versatile sources, including both human host and environmental sources. Understanding genome variations that generate the tremendous diversity in Pseudomonas biology is important in controlling the incidence of infections. With a data set of 704 Pseudomonas complete whole genome sequences representing 186 species, Pseudomonas intrageneric structure was investigated by hierarchical clustering based on average nucleotide identity, and by phylogeny analysis based on concatenated core-gene alignment. Further comparative functional analyses indicated that Pseudomonas species only living in natural habitats lack multiple functions that are important in the regulation of bacterial pathogenesis, indicating the possession of these functions might be characteristic of Pseudomonas human pathogens. Moreover, we have performed pangenome based homogeneity analyses, and detected genes with conserved structures but diversified functions across the Pseudomonas genomes, suggesting these genes play a role in driving diversity. In summary, this study provided insights into the dynamics of genome diversity and host-related genetic determinants in Pseudomonas, which might help the development of more targeted antibiotics for the treatment of Pseudomonas infections.


title: Deep ancestral introgression shapes evolutionary history of dragonflies and damselflies

abstract: Introgression is arguably one of the most important biological processes in the evolution of groups of related species, affecting at least 10% of the extant species in the animal kingdom. Introgression reduces genetic divergence between species, and in some cases can be highly beneficial, facilitating rapid adaptation to ever-changing environmental pressures. Introgression also significantly impacts inference of phylogenetic species relationships where a strictly binary tree model cannot adequately explain reticulate net-like species relationships. Here we use phylogenomic approaches to understand patterns of introgression along the evolutionary history of a unique, non-model insect system: dragonflies and damselflies (Odonata). We demonstrate that introgression is a pervasive evolutionary force across various taxonomic levels within Odonata. In particular, we show that the morphologically "intermediate" species of Anisozygoptera (one of the three primary suborders within Odonata besides Zygoptera and Anisoptera), which retain phenotypic characteristics of the other two suborders, experienced high levels of introgression likely coming from zygopteran genomes. Additionally, we found evidence for multiple cases of deep inter-superfamilial ancestral introgression.


title: Full-length isoform transcriptome of developing human brain provides new insights into autism

abstract:
Alternative splicing plays important role in brain development, however its global contribution to human neurodevelopmental diseases (NDD) has not been fully investigated. Here, we examined the relationships between full-length splicing isoforms expression in the brain and de novo loss-of-function mutations identified in the patients with NDDs. We analyzed the full-length isoform transcriptome of the developing human brain and observed differentially expressed isoforms and isoform co-expression modules undetectable by gene-level analyses. These isoforms were enriched in loss-of-function mutations and microexons, co-expressed with a unique set of partners, and had higher prenatal expression. We experimentally tested the impact of splice site mutations in five NDD risk genes, including SCN2A, DYRK1A and BTRC, and demonstrated exon skipping. Furthermore, our results suggest that the splice site mutation in BTRC reduces translational efficiency, likely impacting Wnt signaling through impaired degradation of {beta}-catenin. We propose that functional effect of mutations associated with human diseases should be investigated at the isoform-rather than the gene-level resolution.

HighlightsO_LIDifferential isoform expression analysis of the human brain transcriptome reveals neurodevelopmental processes and pathways undetectable by differential gene expression analyses.
C_LIO_LISplicing isoforms impacted by neurodevelopmental disease (NDD) risk mutations exhibit higher prenatal expression, are enriched in microexons and are involved in neuronal-related functions.
C_LIO_LIIsoform co-expression network analysis identifies modules with splicing and synaptic functions that are enriched in NDD mutations.
C_LIO_LISplice site mutations impacting NDD risk genes cause exon skipping and produce novel isoforms with altered biological properties.
C_LIO_LIFunctional impact of mutations should be investigated at the full-length isoform-level rather than the gene-level resolution
C_LI


title: Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis

abstract:
Single-cell transcriptomics provide a systematic map of gene expression in different human cell types. The next challenge is to systematically understand cell-type specific gene function. The integration of CRISPR-based functional genomics and stem cell technology enables the scalable interrogation of gene function in differentiated human cells. Here, we present the first genomewide CRISPR interference and CRISPR activation screens in human neurons.

We uncover pathways controlling neuronal response to chronic oxidative stress, which is implicated in neurodegenerative diseases. Unexpectedly, knockdown of the lysosomal protein prosaposin strongly sensitizes neurons, but not other cell types, to oxidative stress by triggering the formation of lipofuscin, a hallmark of aging, which traps iron, generating reactive oxygen species and triggering ferroptosis. We also determine transcriptomic changes in neurons following perturbation of genes linked to neurodegenerative diseases. To enable the systematic comparison of gene function across different human cell types, we establish a data commons named CRISPRbrain.


title: When killers become thieves: trogocytosed PD-1 inhibits NK cells in cancer

abstract:
Leucocytes often perform trogocytosis, the process by which cells acquire parts of the plasma membrane from interacting cells. Accumulating evidence indicates that trogocytosis modulates immune responses, but the underlying molecular mechanisms are unclear. Here, using two mouse models of leukemia, we found that cytotoxic lymphocytes perform trogocytosis at high rates with tumor cells. While performing trogocytosis, both Natural Killer and CD8+ T cells acquire the checkpoint receptor PD-1 from leukemia cells. In vitro and in vivo investigation revealed that PD-1 protein found on the surface of Natural Killer cells, rather than being endogenously expressed, was derived entirely from leukemia cells. Mechanistically, SLAM receptors were essential for PD-1 trogocytosis. PD-1 acquired via trogocytosis actively suppressed anti-tumor immunity, as revealed by the positive outcome of PD-1 blockade in PD-1-deficient mice. PD-1 trogocytosis was corroborated in patients with clonal plasma cell disorders, where Natural Killer cells that stained for PD-1 also stained for tumor cell markers. Our results, in addition to shedding light on a previously unappreciated mechanism underlying the presence of PD-1 on Natural Killer and cytotoxic T cells, reveal the immune-regulatory effect of membrane transfer occurring when immune cells contact tumor cells.

Once sentence summaryNatural Killer cells are inhibited by PD-1 acquired from the surface of tumor cells via trogocytosis.


title: FMRP activates the translation of large autism/intellectual disability proteins and stimulates N-end rule E3 ligase Poe/UBR4 production within puromycin-sensitive RNP particles

abstract: Mutations in Fmr1, encoding the RNA binding protein FMRP, are leading causes of intellectual disability, autism, and female infertility, but FMRPs mechanism of action is controversial. In contrast to its previously postulated function as a translation repressor acting by stalling elongation, we recently found that FMRP activates translation initiation of large proteins in Drosophila oocytes up to [~]2-fold. We report here that FMRPs function as a translational activator is conserved in the mammalian brain. Reanalysis of mouse cortex ribosome profiling data shows that translation of large proteins in Fmr1 mutants is down-regulated 2.0-1.2-fold; ribosome stalling appears not to influence FMRP target protein translation in either cortex or oocyte tissue. Consistent with an activator function, most FMRP targets are associated with clinical syndromes when reduced, but not when over-expressed. Fmr1-dependent translation of one target, the N-end rule E3 ligase Poe/UBR4, occurs in microscopically visible ribonucleoprotein particles. These "Poe particles" require FMRP for their formation, are distinct from P bodies, and depend on actively elongating ribosomes, as indicated by their dissolution following a brief puromycin treatment. N-end rule-mediated proteolysis via Poe/UBR4 restrains cell growth and limits MAPK signaling in nervous tissue. Thus, loss of FMRP reduces production of an important growth repressor.


title: Regional gene expression patterns are associated with task-specific brain activationduring reward and emotion processing measured with functional MRI

abstract: The spatial relationship between gene expression profiles and neural response patterns known to be altered in neuropsychiatric disorders, e.g. depression, can guide the development of more targeted therapies. Here, we estimated the correlation between human transcriptome data and two different brain activation maps measured with functional magnetic resonance imaging (fMRI) in healthy subjects. Whole-brain activation patterns evoked during an emotional face recognition task were associated with topological mRNA expression of genes involved in cellular transport. In contrast, fMRI activation patterns related to the acceptance of monetary rewards were associated with genes implicated in neuronal development, synapse regulation, and gene transcription. An overlap of these genes with risk genes from major depressive disorder genome-wide associations studies revealed the involvement of the master regulators TCF4 and MEF2C. These results were further validated by means of meta-analytic fMRI data. Overall, the identification of stable relationships between spatial gene expression profiles and fMRI data may reshape the prospects for imaging transcriptomics studies.


title: Network-aware reaction pattern recognition reveals regulatory signatures of mitochondrial dysfunction

abstract: Metabolism forms a complex, interdependent network, and perturbations can have indirect effects that are pervasive. Identifying these patterns and their consequences is difficult, particularly when the effects occur across canonical pathways, and these difficulties have long acted as a bottleneck in metabolic data analysis. This challenge is compounded by technical limitations in metabolomics approaches that garner incomplete datasets. Current network-based tools generally utilize pathway-level analysis lacking the granular resolution required to provide context into the effects of all perturbations, regardless of magnitude, across the metabolic network. To address these shortcomings, we introduce algorithms that allow for the real-time extraction of regulatory patterns and trends from user data. To minimize the impact of missing measurements within the metabolic network, we introduce methods that enable complex pattern recognition across multiple reactions. These tools are available interactively within the user-friendly Metaboverse app (https://github.com/Metaboverse) to facilitate exploration and hypothesis generation. We demonstrate that expected signatures are accurately captured by Metaboverse. Using public lung adenocarcinoma data, we identify a previously undescribed multi-dimensional signature that correlated with survival outcomes in lung adenocarcinoma patients. Using a model of respiratory deficiency, we identify relevant and previously unreported regulatory patterns that suggest an important compensatory role for citrate during mitochondrial dysfunction. This body of work thus demonstrates that Metaboverse can identify and decipher complex signals from data that have been otherwise difficult to identify with previous approaches.


title: Ambient light and mimicry as drivers of wing transparency in Lepidoptera

abstract: Transparency reduces prey detectability by predators. While transparent aquatic species hold higher transparency levels as the light availability of their habitat increases, less is known about such variation in terrestrial species. Lepidoptera species exhibiting transparent wings display various levels of transparency. Using two complementary approaches, we explore how the evolution of different transparency degrees relates to habitat openness, activity rhythm and mimicry syndrome (bee/wasp versus dead-leaf mimic). First, by exposing artificial moth-like prey to wild avian predators in a range of habitat openness, we show that survival is lower in more open habitats. We also found that less transparent morphs are more attacked than more transparent ones, regardless of habitat openness degree. Second, by analysing the evolution of wing features and ecological traits in 107 clearwing species, we found that diurnal species transmit more light than nocturnal species under certain conditions (when considering only forewings, at smaller clearwing surfaces and at larger wing lengths) and that species flying in open habitats and exhibiting large percentages of clearwing surface transmit slightly more light than those flying in closed habitats, although this is reversed at smaller percentages of clearwing surfaces. Additionally, bee/wasp mimics are more often diurnal and have higher and less variable light transmittances than dead-leaf mimics, which are more often nocturnal. Flying during the day, in open habitats and mimicking insects with transparent wings seem to promote high light transmittance under certain circumstances. Activity rhythm, habitat openness and species interactions play a crucial role in determining transparency design on land.


title: Activation of the Crtc2/Creb1 transcriptional network in skeletal muscle enhances weight loss during intermittent fasting

abstract: The Creb-Regulated Transcriptional Coactivator (Crtc) family of transcriptional coregulators drive Creb1-mediated transcription effects on metabolism in many tissues, but the in vivo effects of Crtc2/Creb1 transcription on skeletal muscle metabolism are not known. Skeletal muscle-specific overexpression of Crtc2 (Crtc 2 mice) induced greater mitochondrial activity, metabolic flux capacity for both carbohydrates and fats, improved glucose tolerance and insulin sensitivity, and increased oxidative capacity, supported by upregulation of key metabolic genes. Crtc2 overexpression led to greater weight loss during alternate day fasting (ADF), selective loss of fat rather than lean mass, supported by maintenance of higher energy expenditure during the fast. ADF downregulated most of the mitochondrial electron transport genes, and other regulators of mitochondrial function, that were substantially reversed by Crtc2-driven transcription. Glucocorticoids acted with AMPK to drive atrophy and mitophagy, which was reversed by Crtc2/Creb1 signaling. Crtc2/Creb1-mediated signaling coordinates metabolic adaptations in skeletal muscle that explain how Crtc2/Creb1 regulates metabolism and weight loss.


title: Coordinate transcriptional and post-transcriptional repression of pro-differentiation genes maintains intestinal stem cell identity

abstract:
The role of Processing bodies (P-bodies), key sites of post-transcriptional control, in adult stem cells remains poorly understood. Here, we report that adult Drosophila intestinal stem cells, but not surrounding differentiated cells such as absorptive Enterocytes (ECs), harbor P-bodies that contain Drosophila orthologs of mammalian P-body components DDX6, EDC3, EDC4 and LSM14A/B. A targeted RNAi screen in intestinal progenitor cells identified 39 previously known and 64 novel P-body regulators, including Patr-1, a gene necessary for P-body assembly. Loss of Patr-1-dependent P-bodies leads to a loss of stem cells that is associated with inappropriate translation and expression of EC-fate gene nubbin. Transcriptomic analysis of progenitor cells identifies a cadre of such weakly transcribed pro-differentiation transcripts that are elevated after P-body loss. Altogether, this study identifies a coordinated P-body dependent, translational and transcriptional repression program that maintains a defined set of in vivo stem cells in a state primed for differentiation.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/175398v2_ufig1.gif" ALT="Figure 1">
View larger version (31K):
org.highwire.dtl.DTLVardef@d57273org.highwire.dtl.DTLVardef@14d057aorg.highwire.dtl.DTLVardef@1a2c4a5org.highwire.dtl.DTLVardef@11c5ba3_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIDrosophila intestinal progenitor cells contain constitutive and ultrastructurally organized P-bodies.
C_LIO_LIA P-body regulator Patr-1 is required for intestinal progenitor cell maintenance.
C_LIO_LIEnterocyte (EC) genes such as nubbin are weakly transcribed but not translated in intestinal progenitors.
C_LIO_LIP-bodies repress EC gene translation to promote stem cell maintenance.
C_LI


title: Early-life nutrition interacts with developmental genes to shape the brain and sleep behavior in Drosophila melanogaster

abstract: The mechanisms by which the genotype interacts with nutrition during development to contribute to the variation of complex behaviors and brain morphology of adults are not well understood. Here we use the Drosophila Genetic Reference Panel to identify genes and pathways underlying these interactions in sleep behavior and mushroom body morphology. We show that early-life nutritional restriction has genotype-specific effects on variation in sleep behavior and brain morphology. We mapped genes associated with sleep sensitivity to early-life nutrition, which were enriched for protein-protein interactions responsible for translation, endocytosis regulation, ubiquitination, lipid metabolism, and neural development. By manipulating the expression of candidate genes in the mushroom bodies and all neurons, we confirm that genes regulating neural development, translation and insulin signaling contribute to the variable response of sleep and brain morphology to early-life nutrition. We show that the interaction between differential expression of candidate genes with nutritional restriction in early life resides in the mushroom bodies or other neurons, and that these effects are sex specific. Natural variation in genes that control the systemic response to nutrition and brain development and function interact with early-life nutrition in different types of neurons to contribute to the variation of brain morphology and adult sleep behavior.

category: genetics


title: An Effector Index to Predict Causal Genes at GWAS Loci

abstract:
Drug development and biological discovery require effective strategies to map existing genetic associations to causal genes. To approach this problem, we began by identifying a set of positive control genes for 12 common diseases and traits that cause a Mendelian form of the disease or are the target of a medicine used for disease treatment. We then identified a widely-available set of genomic features enriching GWAS-associated single nucleotide variants (SNVs) for these positive control genes. Using these features, we trained and validated the Effector Index (Ei), a causal gene mapping algorithm using the 12 common diseases and traits. The area under Eis receiver operator curve to identify positive control genes was 80% and area under the precision recall curve was 29%. Using an enlarged set of independently curated positive control genes for type 2 diabetes which included genes identified by large-scale exome sequencing, these areas increased to 85% and 61%, respectively. The best predictors were coding or transcript altering SNVs, distance to gene and open chromatin-based metrics. We provide the Ei algorithm for its widespread use and have created a web-portal to facilitate understanding of results. This work outlines a simple, understandable approach to prioritize genes at GWAS loci for functional follow-up and drug development.

Author summaryIn order to derive biological insight, or develop drugs based on genome-wide association studies (GWAS) data, causal genes at associated loci need to be identified. GWAS usually identify large genome regions containing many genes, but seldomly identifies specific causal genes. We have developed an algorithm to predict which genes in a region of disease association are likely causal and have named this algorithm the Effector Index. The Effector Index was optimized on diseases that have known causal or drug target genes, and further validated to predict these types of genes in independent datasets. The Effector Index formalizes these predictive features into a tool that can be used by researchers, and results from the traits and diseases studied here are available via the Accelerating Medicine Partnership web-portal at http://hugeamp.org/effectorgenes.html.

category: bioinformatics


title: Tissue context determines the penetrance of regulatory DNA variation

abstract: Assessment of the functional consequences of disease-associated sequence variation at non-coding regulatory elements is complicated by their high degree of context sensitivity to both the local chromatin and nuclear environments. Allelic profiling of DNA accessibility across individuals has shown that only a select minority of sequence variation affects transcription factor (TF) occupancy, yet the low sequence diversity in human populations means that no experimental assessment is available for the majority of disease-associated variants. Here we describe high-resolution in vivo maps of allelic DNA accessibility in liver, kidney, lung and B cells from 5 increasingly diverged strains of F1 hybrid mice. The high density of heterozygous sites in these hybrids enables precise quantification of the effect size and cell-type specificity of hundreds of thousands of variants throughout the mouse genome. We show that chromatin-altering variants delineate characteristic sensitivity profiles for hundreds of TF motifs. We develop a compendium of TF-specific sensitivity profiles accounting for genomic context effects. Finally, we link these maps of allelic accessibility to allelic transcript levels in the same samples. This work provides a foundation for quantitative prediction of cell-type specific effects of non-coding variation on TF activity, which will dramatically facilitate both fine-mapping and systems-level analyses of common disease-associated variation in human genomes.


title: Initial spindle formation at the oocyte center protects against incorrect kinetochore-microtubule attachment and aneuploidy in mice

abstract: Spindle formation and positioning are two critical events that must be regulated tightly to avoid erroneous chromosome segregation. Following nuclear envelope breakdown (NEBD), the spindle is predominantly assembled centrally before migrating towards the cortex to allow the first asymmetric division. The biological significance of primary central positioning of the spindle has not yet been studied. Because the spindle forms where NEBD occurs, nucleus position greatly influences the site of spindle assembly. We find that nucleus positioning is a dynamic process that depends on maternal age. Moreover, we show that preferential initial spindle positioning at the oocyte center is an insurance mechanism to avoid premature exposure to cortical Cdc42 signaling that hinders proper kinetochore-microtubule attachments, thereby protecting against the development of aneuploid gametes. These findings will help in understanding why female gametes are notoriously associated with high rates of aneuploidy, the leading genetic cause of miscarriage and infertility in women.


title: Basal epidermis collective migration and local Sonic hedgehog signaling promote skeletal branching morphogenesis in zebrafish fins

abstract: Teleost fish fins, like all vertebrate limbs, comprise a series of bones laid out in characteristic pattern. Each fins distal bony rays typically branch to elaborate skeletal networks providing form and function. Zebrafish caudal fin regeneration studies suggest basal epidermal-expressed Sonic hedgehog (Shh) promotes ray branching by partitioning pools of adjacent pre-osteoblasts. This Shh role is distinct from its famous Zone of Polarizing Activity role establishing paired limb positional information. Therefore, we investigated if and how Shh signaling similarly functions during developmental ray branching of both paired and unpaired fins while resolving cellular dynamics of branching by live imaging. We found shha is expressed uniquely by basal epidermal cells overlying pre-osteoblast pools at the distal aspect of outgrowing juvenile fins. Lateral splitting of each shha-expressing epidermal domain followed by the pre-osteoblast pools precedes overt ray branching. We use ptch2:Kaede fish and Kaede photoconversion to identify short stretches of shha+ basal epidermis and juxtaposed pre-osteoblasts as the Shh/Smoothened (Smo) active zone. Basal epidermal distal collective movements continuously replenish each shha+ domain with individual cells transiently expressing and responding to Shh. In contrast, pre-osteoblasts maintain Shh/Smo activity until differentiating. Smo inhibition using the small molecule BMS-833923 prevents branching in all fins, paired and unpaired, with surprisingly minimal effects on caudal fin initial skeletal patterning, ray outgrowth or bone differentiation. Staggered BMS-833923 addition indicates Shh/Smo signaling acts throughout the branching process. We use live cell tracking to find Shh/Smo restrains the distal movement of basal epidermal cells by apparent  tethering to pre-osteoblasts. We propose short-range Shh/Smo signaling promotes these heterotypic associations to couple instructive basal epidermal collective movements to pre-osteoblast repositioning as a unique mode of branching morphogenesis.


title: Dynamics of isoflurane-induced vasodilation and blood flow of cerebral vasculature revealed by multi-exposure speckle imaging

abstract:
BackgroundAnesthetized animal models are used extensively during neurophysiological and behavioral studies despite systemic effects from anesthesia that undermine both accurate interpretation and translation to awake human physiology. The majority of work examining the impact of anesthesia on cerebral blood flow (CBF) has been restricted to before and after measurements with limited spatial resolution.

New MethodWe used multi-exposure speckle imaging (MESI), an advanced form of laser speckle contrast imaging (LSCI), to characterize the dynamics of isoflurane anesthesia induction on cerebral vasculature and blood flow in the mouse brain.

ResultsThe large anatomical changes caused by isoflurane are depicted with wide-field imagery and video highlighting the induction of general anesthesia. Within minutes of exposure, both vessel diameter and blood flow increased drastically compared to the awake state and remained elevated for the duration of imaging. An examination of the dynamics of anesthesia induction reveals that blood flow increased faster in arteries than in veins or parenchyma regions.

Comparison with Existing MethodsMESI offers robust hemodynamic measurements across large fields-of-view and high temporal resolutions sufficient for continuous visualization of cerebrovascular events featuring major changes in blood flow.

ConclusionThe large alterations caused by isoflurane anesthesia to the cortical vasculature and CBF are readily characterized using MESI. These changes are unrepresentative of normal physiology and provide further evidence that neuroscience experiments would benefit from transitioning to un-anesthetized awake animal models.

category: neuroscience


title: Colloblasts act as a biomechanical sensor for suitable prey in Pleurobrachia

abstract:
Ctenophores are a group of largely-planktonic, gelatinous carnivores whose most common method of prey capture is nearly a phylum-defining trait. Tentaculate ctenophores release an unknown proteinaceous adhesive from specialised colloblast cells lining their tentacles following prey contact with the tentacles. There exist no extant studies of the mechanical properties of colloblast adhesive. We use live microscopy techniques to visualise adhesion events between Pleurobrachia pileus colloblasts and probes of different surface chemistries in response to probing with varying contact areas. We further define two mechanisms of adhesion termination upon probe retraction. Adapting a technique for measuring surface tension, we examine the adhesive strength of tentacles in the ctenophore Pleurobrachia bachei under varying pH and bonding time conditions, and demonstrate the destructive exhaustion of colloblast adhesive release. We find that colloblast-mediated adhesion is rapid, and that the bonding process is robust against shifts in ambient pH. However, we find that the Pleurobrachia colloblast adhesive system is among the weakest biological adhesive systems yet described. We place this surprising observation into a broader ecophysiological context by modeling prey capture for prey of a range of sizes. We find that limited use of colloblast adhesive with high surface area contact is suitable both for capturing appropriately sized prey and rejecting, by detachment, prey above a certain size threshold. This allows Pleurobrachia, lacking a mechanism to directly "see" potential prey they are interacting with, to invest in capturing only prey of an appropriate size, decreasing the risk of injury.

Summary statementCtenophore colloblast adhesive is found to be strong, but few colloblasts are simultaneously active, producing a weakly-adhering system. A physical model demonstrates how such a system may filter unsuitable prey.

category: zoology


title: Cocaine-induced neuron subtype mitochondrial dynamics through Egr3 transcriptional regulation

abstract: Mitochondrial function is required for brain energy homeostasis and neuroadaptation. Recent studies demonstrate that cocaine affects mitochondrial dynamics and morphological characteristics within the nucleus accumbens (NAc). Further, mitochondria are differentially regulated by cocaine in dopamine receptor-1 containing medium spiny neurons (D1-MSNs) vs dopamine receptor-2 (D2)-MSNs. However, there is little understanding into cocaine-induced transcriptional mechanisms and their role in regulating mitochondrial processes. Here, we demonstrate that cocaine enhances binding of the transcription factor, early growth response factor 3 (Egr3), to nuclear genes involved in mitochondrial function and dynamics. Moreover, cocaine exposure regulates mRNA of these mitochondria-associated nuclear genes in both contingent or noncontingent cocaine administration and in both rodent models and human postmortem tissue. Interestingly, several mitochondrial nuclear genes showed distinct profiles of expression in D1-MSNs vs D2-MSNs, with cocaine exposure generally increasing mitochondrial-associated nuclear gene expression in D1-MSNs vs suppression in D2-MSNs. We further show that blunting Egr3 expression in D1-MSNs blocks cocaine-enhancement of the mitochondrial-associated transcriptional coactivator, peroxisome proliferator-activated receptor gamma coactivator (PGC1), and the mitochondrial fission molecule, dynamin related protein 1 (Drp1). Finally, reduction of D1-MSN Egr3 expression attenuates cocaine-induced enhancement of small-sized mitochondria, causally demonstrating that Egr3 regulates mitochondrial morphological adaptations. Collectively, these studies demonstrate cocaine exposure impacts mitochondrial dynamics and morphology by Egr3 transcriptional regulation of mitochondria-related nuclear gene transcripts; indicating roles for these molecular mechanisms in neuronal function and plasticity occurring with cocaine exposure.


title: Facilitation of neural responses to targets moving against optic flow

abstract:
For the human observer, it can be difficult to follow the motion of small objects, especially when they move against background clutter. In contrast, insects efficiently do this, as evidenced by their ability to capture prey, pursue conspecifics, or defend territories, even in highly textured surrounds. We here recorded from target selective descending neurons (TSDNs) which likely subserve these impressive behaviors. To simulate the type of background optic flow that would be generated by the pursuers own movements through the world, we used the coherent motion of a perspective distorted sparse dot field. We show that hoverfly TSDN responses to target motion are suppressed when such background optic flow moves in the same direction as the target. Indeed, the neural responses are strongly attenuated against both translational sideslip as well as rotational yaw. More strikingly, we show that TSDNs are facilitated by background optic flow in the opposite direction to the target, if the target moves horizontally. Furthermore, we show that a small, frontal spatial window of background optic flow is enough to fully facilitate or attenuate TSDN responses to target motion. We argue that the TSDN response facilitation could be beneficial in modulating corrective turns during target pursuit.

Significance statementTarget detection in visual clutter is a difficult computational task that insects, with their poor resolution compound eyes and small brains, do successfully and with extremely short behavioral delays. We here show that the responses of target selective descending neurons are attenuated by background motion in the same direction as target motion, but facilitated by opposite direction background motion. This finding is important for understanding conspecific pursuit behavior, since these descending neurons likely control behavioral output. The facilitation that we describe would come into effect if the hoverfly is subjected to background motion in one direction, but the target it is pursuing moves in the opposite direction, and could therefore be used to modulate gaze stabilizing corrective turns.


title: Shotgun scanning glycomutagenesis: a simple and efficient strategy for constructing and characterizing neoglycoproteins

abstract: As a common protein modification, asparagine-linked (N-linked) glycosylation has the capacity to greatly influence the biological and biophysical properties of proteins. However, the routine use of glycosylation at naive sites as a strategy for engineering proteins with advantageous properties is currently limited by our inability to construct large collections of glycoproteins for interrogating the structural and functional consequences of glycan installation. To address this challenge, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis (SSGM) in which DNA libraries encoding all possible glycosylation site variants of a given protein are constructed and subsequently expressed in glycosylation-competent bacteria, thereby enabling rapid determination of glycosylatable sites in the protein. Moreover, the resulting neoglycoproteins can be readily subjected to available medium- to high-throughput assays, making it possible to systematically investigate the structural and functional consequences of glycan conjugation along a protein backbone. The utility of this approach was demonstrated with three different acceptor proteins, namely bacterial immunity protein Im7, bovine pancreatic ribonuclease A, and a human anti-HER2 single-chain Fv antibody, all of which were found to tolerate N-glycan attachment at a large number of positions and with relatively high efficiency. The stability and activity of many glycovariants was measurably altered by the N-linked glycan in a manner that critically depended on the precise location of the modification. Importantly, we anticipate that our workflow for creating and characterizing large ensembles of neoglycoproteins should provide access to unexplored regions of glycoprotein structural space and to custom-made glycoproteins with desirable properties.


title: Nested Stochastic Block Models Applied to the Analysis of Single Cell Data

abstract: Single cell profiling has been proven to be a powerful tool in molecular biology to understand the complex behaviours of heterogeneous system. The definition of the properties of single cells is the primary endpoint of such analysis, cells are typically clustered to underpin the common determinants that can be used to describe functional properties of the cell mixture under investigation. Several approaches have been proposed to identify cell clusters; while this is matter of active research, one popular approach is based on community detection in neighbourhood graphs by optimisation of modularity. In this paper we propose an alternative and principled solution to this problem, based on Stochastic Block Models. We show that such approach not only is suitable for identification of cell groups, it also provides a solid framework to perform other relevant tasks in single cell analysis, such as label transfer. To encourage the use of Stochastic Block Models, we developed a python library, schist, that is compatible with the popular scanpy framework.


title: Size-dependent protein segregation creates a spatial switch for Notch signaling and function

abstract:
Aberrant cleavage of Notch by {gamma}-secretase is implicated in numerous diseases, but how cleavage is regulated in space and time is unclear. Here, we report that cadherin-based adherens junctions (cadAJs) are sites of high cell-surface {gamma}-secretase activity, as well as sites of constrained physical space that excludes {gamma}-secretase substrates having large extracellular domains (ECDs) like Notch. ECD shedding initiates drastic spatial relocalization of Notch to cadAJs, allowing enzyme-substrate interactions and downstream signaling. Spatial mutations by adjusting the ECD size or the physical constraint alter signaling. Dysregulation of this spatial switch promotes precocious differentiation of ventricular zone neural progenitor cells in vivo. We show the generality of this spatial switch for amyloid precursor protein proteolysis. Thus, cadAJs create spatially distinct biochemical compartments regulating cleavage events involving {gamma}-secretase and preventing aberrant activation of receptors.

One Sentence SummaryNotch cleavage by {gamma}-secretase is regulated through dynamic spatial control of receptors, adhesion molecules, and activating proteases


title: Involuntary shifts of spatial attention contribute to behavioral crossmodal distraction - Evidence from oscillatory alpha power and reaction time data

abstract: Imagine you are focusing on the traffic on a busy street to ride your bike safely when suddenly you hear an ambulance siren. This unexpected sound involuntarily captures your attention and interferes with ongoing performance. We tested whether this type of behavioral distraction involves a spatial shift of attention. We measured behavioral distraction and magnetoencephalographic alpha power during a crossmodal paradigm that combined an exogenous cueing task and a distraction task. In each trial, a task-irrelevant sound preceded a visual target (left or right). The sound was usually the same animal sound (i.e., standard sound). Rarely, it was replaced by an unexpected environmental sound (i.e., deviant sound). Fifty percent of the deviants occurred on the same side as the target, and 50% occurred on the opposite side. Participants responded to the location of the target. As expected, responses were slower to targets that followed a deviant compared to a standard, reflecting behavioral distraction. Crucially, this distraction was mitigated by the spatial relationship between the targets and the deviants: responses were faster when targets followed deviants on the same versus different side, indexing a spatial shift of attention. This was further corroborated by a posterior alpha power modulation that was higher in the hemisphere ipsilateral (vs. contralateral) to the location of the attention-capturing deviant. We suggest that this alpha power lateralization reflects a spatial attention bias. Overall, our data support the contention that spatial shifts of attention contribute to behavioral deviant distraction.


title: Three-dimensional Multi-site Random Access Photostimulation (3D-MAP)

abstract: Optical control of neural ensemble activity is crucial for understanding brain function and disease, yet no technology can achieve optogenetic control of very large numbers of neurons at extremely fast rate over a large volume. State-of-the-art multiphoton holographic optogenetics requires high power illumination that only addresses relatively small populations of neurons in parallel. Conversely, one-photon holographic techniques can stimulate more neurons with 2-3 orders lower power, but with a trade-off between resolution and addressable volume. Perhaps most problematically, two-photon holographic optogenetic systems are extremely expensive and sophisticated which has precluded their broader adoption in the neuroscience community. To address this technical gap, we introduce a new one-photon light sculpting technique, Three- Dimensional Multi-site random Access Photostimulation (3D-MAP), that overcomes these limitations by modulating light dynamically, both in the spatial and in the angular domain at multi-kHz rates. We use 3D-MAP to interrogate neural circuits in 3D and demonstrate simultaneous photostimulation and imaging of dozens of user-selected neurons in the intact mouse brain in vivo with high spatiotemporal resolution. 3D-MAP could be broadly adopted for high-throughput all-optical interrogation of brain circuits owing to its powerful combination of scale, speed, simplicity, and cost.


title: Dissociation of tic expression from its neuronal encoding in the striatum during sleep

abstract:
Motor tics, the hallmark of Tourette syndrome, are modulated by different behavioral and environmental factors. A major modulating factor is the sleep-wake cycle in which tics are attenuated to a large extent during sleep. This study demonstrates a similar reduction in tic expression during sleep in an animal model of chronic tic disorders and investigates the underlying neural mechanism. We recorded the neuronal activity during spontaneous sleep-wake cycles throughout continuous GABAA antagonist infusion into the striatum. Analysis of video streams and concurrent kinematic assessments indicated tic reduction during sleep in both frequency and intensity. Extracellular recordings in the striatum revealed a state dependent dissociation between motor tic expression and their macro-level neural correlates ("LFP spikes") during the sleep-wake cycle. LFP spikes, which are highly correlated with tic expression during wakefulness, persisted during tic-free sleep and did not change their properties despite the reduced behavioral expression. Local, micro-level, activity near the infusion site was time-locked to the LFP spikes during wakefulness but this locking decreased significantly during sleep. These results suggest that while LFP spikes encode motor tic generation and feasibility, the behavioral expression of tics requires local striatal neural activity entrained to the LFP spikes, leading to the propagation of the activity to downstream targets and consequently their motor expression. These findings point to a possible mechanism for the modulation of tic expression in TS patients during sleep and potentially during other behavioral states.

SummaryThe expression of motor tics, the defining symptom of Tourette syndrome, is modulated by environmental and behavioral factors. In this study, we explored tic modulation during the sleep-wake cycle and its underlying neurophysiological mechanism, using the rat model of chronic motor tic expression. Behaviorally, during sleep, tic frequency and intensity declined considerably. Physiologically, however, the macro-level neural correlates (termed "LFP spikes") of tics persisted throughout the sleep-wake cycle, whereas the micro-level correlates were reduced during sleep. This dissociation between neuronal activity and its behavioral expression leads not only to a better understanding of tic modulation during sleep but may also suggest potential ways of affecting tic expression during natural behavior and when exposed to external modulating factors.

HighlightsO_LIThe wake-sleep cycle modulates tic expression in chronically tic-expressing rats
C_LIO_LISleep attenuates tics in the rats reminiscent of the phenomenon in TS Patients
C_LIO_LIDuring tic-less sleep tic generation persists via LFP spikes
C_LIO_LIDuring sleep tic expression is blocked via striatal activity desynchronization
C_LI


title: The Filter Detection Task for measurement of breathing-related interoception and metacognition

abstract: The study of the brains processing of sensory inputs from within the body ( interoception) has been gaining rapid popularity in neuroscience, where interoceptive disturbances are thought to exist across a wide range of chronic physiological and psychological conditions. Here we present a task and analysis procedure to quantify specific dimensions of breathing-related interoception, including interoceptive sensitivity (accuracy), decision bias, metacognitive bias, and metacognitive performance. Two major developments address some of the challenges presented by low trial numbers in interoceptive experiments: (i) a novel adaptive algorithm to maintain task performance at 70-75% accuracy; (ii) an extended hierarchical metacognitive model to estimate regression parameters linking metacognitive performance to relevant (e.g. clinical) variables. We demonstrate the utility of the task and analysis developments, using both simulated data and three empirical datasets. This methodology represents an important step towards accurately quantifying interoceptive dimensions from a simple experimental procedure that is compatible with clinical settings.


title: Local thermal environment and warming influence supercooling and drive widespread shifts in the metabolome of diapausing Pieris rapae butterflies

abstract: Global climate change has the potential to negatively impact biological systems as organisms are exposed to novel temperature regimes. Increases in annual mean temperature have been accompanied by disproportionate rates of change in temperature across seasons, and winter is the season warming most rapidly. Yet, we know relatively little about how warming will alter the physiology of overwintering organisms. Here, we simulated future warming conditions by comparing diapausing Pieris rapae butterfly pupae collected from disparate thermal environments and by exposing P. rapae pupae to acute and chronic increases in temperature. First, we compared internal freezing temperatures (supercooling points) of diapausing pupae that were developed in common-garden conditions but whose parents were collected from northern Vermont, USA, or North Carolina, USA. Matching the warmer winter climate of North Carolina, North Carolina pupae had significantly higher supercooling points than Vermont pupae. Next, we measured the effects of acute and chronic warming exposure in Vermont pupae and found that warming induced higher supercooling points. We further characterized the effects of chronic warming by profiling the metabolomes of Vermont pupae via untargeted LC-MS metabolomics. Warming caused significant changes in abundance of hundreds of metabolites across the metabolome. Notably, there were warming-induced shifts in key biochemical pathways, such as pyruvate metabolism, fructose and mannose metabolism, and beta-alanine metabolism, suggesting shifts in energy metabolism and cryoprotection. These results suggest that warming affects various aspects of overwintering physiology in P. rapae and may be detrimental depending on the frequency and variation of winter warming events. Future research is needed to ascertain the extent to which the effects of warming are felt among a broader set of populations of P. rapae, and among other species, in order to better predict how insects may respond to changes in winter thermal environments.


title: TNFa-induced LDL cholesterol accumulation involve elevated LDLR cell surface levels and SR-B1 downregulation in human arterial endothelial cells

abstract: Excess lipid droplets are frequently observed in arterial endothelial cells at sites of advanced atherosclerotic plaques. Here, the role of tumor necrosis factor alpha (TNF) in modulating low density lipoprotein (LDL) content in confluent primary human aortic endothelial cells (pHAECs) was investigated. TNF promoted up to 2 folds increase in cellular cholesterol, which was resistant to ACAT inhibition. The cholesterol increase was associated with increased 125I-LDL surface binding. Using the non-hydrolysable label, Dil, TNF could induce a massive increase in Dil-LDL by over 200 folds. The elevated intracellular Dil-LDL was blocked with excess unlabeled LDL and PCSK9, but not oxidized LDL (oxLDL), receptor associated protein (RAP), or apolipoprotein (apoE) depletion. Moreover, TNF-induced increase of LDL-derived lipids was elevated through lysosome inhibition. Using specific LDLR antibody, the Dil-LDL accumulation was reduced by over 99%. Effects of TNF included LDLR cell surface increase by 138%, and very large increases in ICAM-1 total and surface proteins, respectively. In contrast, that of scavenger receptor B1 (SR-B1) was reduced. Additionally, LDLR antibody bound rapidly in TNF-treated cells by about 30 folds, inducing a migrating shift in the LDLR protein. The effect of TNF on Dil-LDL accumulation was inhibited by the antioxidant tetramethythiourea (TMTU) dose-dependently, but not by inhibitors against NF-{kappa}B, stress kinases, ASK1, JNK, p38, or apoptosis caspases. Grown on transwell inserts, TNF did not enhance apical to basolateral LDL cholesterol or Dil release. It is concluded that TNF promotes LDLR functions through combined increase at the cell surface and SR-B1 downregulation.


title: TNFa-induced LDL cholesterol accumulation involve elevated LDLR cell surface levels and SR-B1 downregulation in human arterial endothelial cellsan arterial endothelial cells


title: Sex-biased islet β cell dysfunction is caused by the MODY MAFA S64F variant by inducing premature aging and senescence in males

abstract: A heterozygous missense mutation producing a variant of the islet {beta}-cell-enriched MAFA transcription factor (Ser(S)64Phe(F) MAFA) was identified in humans who developed adult-onset, {beta}-cell dysfunction (diabetes or insulinomatosis), with men more prone to diabetes. This mutation engenders increased stability to the normally unstable MAFA protein. To obtain insight into how this variant impacts {beta} cell function, we developed a mouse model expressing S64F MafA and found sex-dependent phenotypes, with heterozygous mutant males displaying impaired glucose tolerance while females were slightly hypoglycemic with improved blood glucose clearance. Only heterozygous males showed transiently higher MafA protein levels preceding the onset of glucose intolerance and sex-dependent, differential expression of genes involved in calcium signaling, DNA damage, aging, and senescence. Functional changes in islet calcium handling and signs of islet aging and senescence processes were uniquely observed in male animals. In addition, S64F MAFA expression in human, male EndoC-{beta}H2 {beta} cells accelerated cellular senescence and increased production of senescence-associated secretory proteins compared to cells expressing wild-type MAFA. Together, these results implicate a conserved mechanism of accelerated islet aging and senescence in promoting diabetes in S64F MAFA carriers in a sex-dependent manner.


title: HLF Expression Defines the Human Hematopoietic Stem Cell State

abstract:
Hematopoietic stem cells (HSCs) sustain blood cell homeostasis throughout life and are able to regenerate all blood lineages following transplantation.

Despite this clear functional definition, highly enriched isolation of human HSCs can currently only be achieved through combinatorial assessment of multiple surface antigens. While a number of transgenic HSC reporter mouse strains have been described, no analogous approach to prospectively isolate human HSCs has been reported.

To identify genes with the most selective expression in human HSCs, we profiled population- and single-cell transcriptomes of fresh and ex vivo cultured cord blood derived HSPCs as well as peripheral blood, adult bone marrow and fetal liver. Based on these analyses, we propose the master transcription factor HLF (Hepatic Leukemia Factor) as one of the most specific HSC marker genes.

To directly track its expression in human hematopoietic cells, we developed a genomic HLF reporter strategy, capable of selectively labeling the most immature blood cells on the basis of a single engineered parameter.

Most importantly, HLF-expressing cells comprise all of the stem cell activity in culture and in vivo during serial transplantation.

Taken together, these results experimentally establish HLF as a defining gene of the human hematopoietic stem cell state and outline a new approach to continuously mark these cells with high fidelity.

Key PointsO_LIIn the human blood system, HLF expression is specific to stem cell populations in primary anatomical sites and during ex vivo expansion.
C_LIO_LICRISPR/rAAV6-mediated integration of a genomic HLF-reporter allows selective and stable genetic labeling of human HSCs ex vivo and in vivo.
C_LI

category: developmental biology


title: Valproic Acid-Induced Changes of 4D Nuclear Morphology in Astrocyte Cells

abstract: Histone deacetylase inhibitors, such as valproic acid (VPA), have important clinical therapeutic and cellular reprogramming applications. They induce chromatin re-organization that is associated with altered cellular morphology. However, there is a lack of comprehensive characterization of VPA-induced changes of nuclear size and shape. Here, we quantify 3D nuclear morphology of primary human astrocyte cells treated with VPA over time (hence, 4D). We compared volumetric and surface-based representations and identified seven features that jointly discriminate between normal and treated cells with 85% accuracy on day 7. From day 3, treated nuclei were more elongated and flattened and then continued to morphologically diverge from controls over time, becoming larger and more irregular. On day 7, most of the size and shape descriptors demonstrated significant differences between treated and untreated cells, including a 24% increase in volume and 6% reduction in extent (shape regularity) for treated nuclei. Overall, we show that 4D morphometry can capture how chromatin re-organization modulates the size and shape of the nucleus over time. These nuclear structural alterations may serve as a biomarker for histone (de-)acetylation events and provide insights into mechanisms of astrocytes-to-neurons reprogramming.


title: Limited evidence for parallel evolution among desert adapted Peromyscus deer mice

abstract: Phenotypic plasticity enables an immediate response to changing conditions, but for most species, evolutionary change through adaptation will be more important for long-term survival. Warming climate and increasing desertification urges the identification of genes involved in heat-and dehydration-tolerance to better inform and target biodiversity conservation efforts. Comparisons among extant desert adapted species can highlight parallel or convergent patterns of genome evolution through the identification of shared signatures of selection. We generate chromosome-level genome assembly for the canyon mouse (Peromyscus crinitus) and test for signature of parallel evolution by comparing signatures of selective sweeps across population-level genomic resequencing data from another desert specialist deer mouse (P. eremicus) and a widely-distributed habitat generalist (P. maniculatus), that may locally adapted to arid conditions. We identify few shared candidate loci involved in desert adaptation and do not find support for a shared pattern of parallel evolution. Instead, we hypothesize divergent molecular mechanisms of desert adaptation among deer mice, potentially tied to species-specific historical demography, which may limit or enhance adaptation. We identify a number of candidate loci experiencing selective sweeps in the P. crinitus genome that are implicated in osmoregulation (Trypsin, Prostasin) and metabolic regulation (Kallikrein, eIF2-alpha kinase GCN2, APPL1/2), which may be important to accommodating hot and dry environmental conditions.


title: Exploring rare cellular activity in more than one million cells by a trans-scale-scope

abstract: In many phenomena of biological systems, not a majority, but a minority of cells act on the entire multicellular system causing drastic changes in the system properties. To understand the mechanisms underlying such phenomena, it is essential to observe the spatiotemporal dynamics of a huge population of cells at sub-cellular resolution, which is difficult with conventional tools such as microscopy and flow cytometry. Here, we describe an imaging system named AMATERAS that enables optical imaging with an over-one-centimeter field-of-view and a-few-micrometer spatial resolution. This trans-scale-scope has a simple configuration, composed of a low-power lens for machine vision and a hundred-megapixel image sensor. We demonstrated its high cell-throughput, capable of simultaneously observing more than one million cells. We applied it to dynamic imaging of calcium ions in HeLa cells and cyclic-adenosine-monophosphate in Dictyostelium discoideum, and successfully detected less than 0.01% of rare cells and observed multicellular events induced by these cells.

category: cell biology


title: Single-cell in vivo brain optogenetic stimulation by two-photon excitation fluorescence transfer

abstract: Optogenetic manipulation with single-cell resolution can be achieved by two-photon excitation; however, this frequently requires relatively high laser powers or holographic illumination. Here we developed a practical strategy to improve the efficiency of two-photon stimulation by positioning fluorescent proteins or small fluorescent molecules with high two-photon cross-sections in the vicinity of opsins. This generates a highly localized source of endogenous single-photon illumination that can be tailored to match the optimal opsin absorbance. Through neuronal and vascular stimulation in the live mouse brain, we demonstrate the utility of this technique to achieve more efficient opsin stimulation, without loss of cellular resolution. We also provide a theoretical framework for understanding the potential advantages and constrains of this methodology, with suggestions for future improvements. Altogether, this fluorescence transfer illumination method allows experiments difficult to implement in the live brain such as all-optical neural interrogation and control of regional cerebral blood flow.


title: Optimizing Sequencing Resources in Genotyped Livestock Populations Using Linear Programming

abstract:
BackgroundLow-cost genome-wide single-nucleotide polymorphisms (SNPs) are routinely used in animal breeding programs. Compared to SNP arrays, the use of whole-genome sequence data generated by the next-generation sequencing technologies (NGS) has great potential in livestock populations. However, a large number of animals are required to be sequenced to exploit the full potential of whole-genome sequence data. Thus, novel strategies are desired to allocate sequencing resources in genotyped livestock populations such that the entire population can be sequenced or imputed efficiently.

MethodsWe present two applications of linear programming models called LPChoose for sequencing resources allocation. The first application is to identify the minimum number of animals for sequencing while meeting the criteria that each haplotype in the population is contained in at least one of the animals selected for sequencing. The second is to sequence a fixed number of animals whose haplotypes include as large a proportion as possible of the haplotypes present in the population given a limited sequencing budget.

ResultsIn both applications LPChoose has similar or better performance than some other methods. The linear programming models we proposed are based on rigorous and well defined optimization techniques and easy and straightforward to implement. LPChoose is available as an open-source package.


title: Insights into the secondary structural ensembles of the full SARS-CoV-2 RNA genome in infected cells

abstract: SARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Currently, there are no antiviral drugs with proven efficacy, and development of these treatments are hampered by our limited understanding of the molecular and structural biology of the virus. Like many other RNA viruses, RNA structures in coronaviruses regulate gene expression and are crucial for viral replication. Although genome and transcriptome data were recently reported, there is to date little experimental data on native RNA structures in SARS-CoV-2 and most putative regulatory sequences are functionally uncharacterized. Here we report secondary structure ensembles of the entire SARS-CoV-2 genome in infected cells at single nucleotide resolution using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and the algorithm  detection of RNA folding ensembles using expectation-maximization clustering (DREEM). Our results reveal previously undescribed alternative RNA conformations across the genome, including structures of the frameshift stimulating element (FSE), a major drug target, that are drastically different from prevailing in vitro population average models. Importantly, we find that this structural ensemble promotes frameshifting rates (~40%) similar to in vivo ribosome profiling studies and much higher than the canonical minimal FSE (~20%). Overall, our result highlight the value of studying RNA folding in its native, dynamic and cellular context. The genomic structures detailed here lays the groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics.