title: Cortical ripples provide the conditions for consolidation during NREM sleep in humans

abstract: Hippocampal ripples index the reconstruction of spatiotemporal neuronal firing patterns essential for the consolidation of memories in the cortex during non-rapid eye movement sleep (NREM). Recently, cortical ripples in humans have been shown to enfold the replay of neuron firing patterns during cued recall. Here, we show that cortical ripples also occur during NREM in humans, with similar density, oscillation frequency (~90Hz), and amplitude to waking. Ripples occurred in all cortical regions with similar characteristics, unrelated to putative hippocampal connectivity, and were less dense and robust in higher association areas. Putative pyramidal and interneuron spiking phase-locked to cortical ripples during NREM, with phase delays consistent with ripple generation through pyramidal-interneuron feedback. Cortical ripples were smaller in amplitude than hippocampal ripples, but were similar in density, frequency, and duration. Cortical ripples during NREM typically occurred just prior to the upstate peak, often during spindles. Upstates and spindles have previously been associated with memory consolidation, and we found that cortical ripples grouped co-firing within the window of spike-timing-dependent plasticity. Thus, human NREM cortical ripples are: ubiquitous and stereotyped with a tightly focused oscillation frequency; similar to hippocampal ripples; associated with upstates and spindles; and associated with unit co-firing. These properties are consistent with cortical ripples possibly contributing to memory consolidation and other functions during NREM in humans.


title: Cortical ripples in humans

category: neuroscience


title: On the Contribution of Reproductive and Offspring Investment on Fertility: Human and Animal Societies

abstract: Differences in investment into reproduction or offspring rearing are plentiful throughout the world, from the cells inside our bodies to complex sociological interactions among humans. Such differences can lead to profound impacts on species fitness, fertility, and reproductive rates, sometimes in startling ways. In this paper, we create a simple game-theoretical model to qualitatively investigate the effects of such differential investment. We focus on fertility in human societies and show that more wealthy individuals produce more offspring within a a mating group. However, when assortative mating mechanisms are introduced, this effectively leads to a speciation event, and a higher reproduction rate for poorer individuals is noticed, capturing what we call the "wealthy-to-poor switch". We discuss extensions and implications of this work to nupital gifts in ecology and to clonal competition in cancer cell lines under the influence of treatment.

category: ecology


title: Novel Interpretable Tissue-Specific and Multi-Tissue Transcriptomic Clocks to Infer Aging Mechanisms

abstract:
1.Aging is characterized as a progressive decline in fitness that ultimately results in death. We set out to build both tissue-specific and multi-tissue transcriptomic clocks to make global tissue age predictions in individuals from GTEx. Existing work in the field primarily uses epigenetic clocks as predictors of age, but these models have known issues and are significantly less interpretable than their transcriptomic counterparts. Due to their transcriptomic nature, we can use these models to directly infer mechanisms of aging from their features. Linear regression remains the current standard analysis technique, but we improved upon its baseline performance with modern techniques, exploring both XGBoost and MLPs. We also experimented with using deconvolved cell data for predictions, which account for cellular composition and reduce signal distortion from rare cell types. Since it is known that the heterogeneity of cell types in particular tissues can lead to noise in these models, we proposed using deconvolution as a potential remedy for this problem.

Our results found that MLPs are not well suited for the task due to a lack of training data, but the use of XGBoost is effective at improving the baseline performance of predictions of existing tissue-specific clocks. These models allowed us to directly compute genes most important to age prediction in our models, and we showed that multiple genes found have been independently identified elsewhere to show evidence of correlation with age. Given the small size of our datasets, we were unable to make conclusive determinations about multi-tissue predictors, but preliminary results suggest that the technique shows promise and is worthy of future investigation. Likewise, given our limited deconvolved cell data, we did not currently observe strong results, but we again note that this is an area in need of further investigation.

By improving upon the performance of existing models, we demonstrated that a novel machine learning technique, XGBoost, can be an effective technique to further our understanding of aging mechanisms by extraction of the most relevant genes found in those models. This is significant because the genetic causes of aging are still not fully understood, and research in the field of aging is lacking in comparison to other domains. As the problem of identifying tissues that age at different rates is of specific interest, our tissue-specific models potentially have other applications in this domain, including informing pathologies in tissues that are found to be aging faster, or analyzing how people with similar ages can have vastly different tissue ages. An extended technical presentation of this work can be found here, and a highly simplified non-technical overview presentation can be found here.

category: genomics


title: Responsive robotic prey reveal how predators adapt to predictability in escape tactics

abstract: To increase their chances of survival, prey often respond to predators by being unpredictable when escaping, but the response of predators to such tactics is unknown. We programmed interactive robot-controlled prey to flee from an approaching blue acara predator (Andinoacara pulcher), allowing us to manipulate the predictability of the preys initial escape direction. When repeatedly exposed to predictable prey, the predators adjusted their behaviour before the prey even began to escape: prey programmed to escape directly away were approached more rapidly than prey escaping at an acute angle. These faster approach speeds compensated for a longer time needed to capture such prey during the subsequent pursuit phase, and predators showed greater acceleration when pursuing unpredictable prey. Collectively, these behaviours resulted in the preys predictability having no net effect on the time to capture prey. Rather than minimising capture times, predators adjust their behaviour to maintain an adequate level of performance.

category: animal behavior and cognition


title: Injectable Progranulin-derivative Atsttrin loaded Protein Engineered Gels for Post Traumatic Osteoarthritis

abstract: Protein-based biomaterials offer several advantages over synthetic materials, owing to their unique stimuli-responsive properties, biocompatibility and modular nature. We have successfully developed protein block polymers that consist of elastin like polypeptide (E) and the coiled-coil domain of cartilage oligomeric matrix protein (C). Here, we demonstrate that E5C, a construct consisting of five repeats of E and a single domain of C, is capable of forming a porous networked gel at physiological temperature, making it an excellent candidate for injectable biomaterials. Combination of E5C with Atsttrin, a chondroprotective engineered derivative of anti-inflammatory growth factor progranulin (PGRN), provides a unique biochemical and biomechanical environment to protect against post-traumatic osteoarthritis (PTOA) onset and progression. E5C gel was demonstrated to provide prolonged release of Atsttrin and inhibit chondrocyte catabolism while facilitating anabolic signaling in vitro. We also provide in vivo evidence that prophylactic and therapeutic application of Atsttrin-loaded E5C hydrogels protected against PTOA onset and progression in a rabbit anterior cruciate ligament transection model. Collectively, we have developed a unique protein-based gel capable of minimally invasive, sustained delivery of prospective therapeutics, particularly the PGRN-derivative Atsttrin, for prevention of OA onset and progression.

category: bioengineering


title: OVarFlow: a resource optimized GATK 4 based Open source Variant calling workFlow

abstract:
BackgroundNext generation sequencing technologies are opening new doors to researchers. One application is the direct discovery of sequence variants that are causative for a phenotypic trait or a disease. The detection of an organisms alterations from a reference genome is know as variant calling, a computational task involving a complex chain of software applications. One key player in the field is the Genome Analysis Toolkit (GATK). The GATK Best Practices are commonly referred recipe for variant calling on human sequencing data. Still the fact the Best Practices are highly specialized on human sequencing data and are permanently evolving is often ignored. Reproducibility is thereby aggravated, leading to continuous reinvention of pretended GATK Best Practice workflows.

ResultsHere we present an automatized variant calling workflow, for the detection of SNPs and indels, that is broadly applicable for model as well as non-model diploid organisms. It is derived from the GATK Best Practice workflow for "Germline short variant discovery", without being focused on human sequencing data. The workflow has been highly optimized to achieve parallelized data evaluation and also maximize performance of individual applications to shorten overall analysis time. Optimized Java garbage collection and heap size settings for the GATK applications SortSam, MarkDuplicates, HaplotypeCaller and GatherVcfs were determined by thorough benchmarking. In doing so, runtimes of an example data evaluation could be reduced from 67 h to less than 35 h.

ConclusionsThe demand for standardized variant calling workflows is proportionally growing with the dropping costs of next generation sequencing methods. Our workflow perfectly fits into this niche, offering automatization, reproducibility and documentation of the variant calling process. Moreover resource usage is lowered to a minimum. Thereby variant calling projects should become more standardized, reducing the barrier further for smaller institutions or groups.

category: bioinformatics


title: A clusterability measure for single-cell transcriptomics reveals phenotypic subpopulations

abstract: The ability to discover new cell populations by unsupervised clustering of single-cell transcriptomics data has revolutionized biology. Currently, there is no principled way to decide, whether a cluster of cells contains meaningful subpopulations that should be further resolved. Here we present SIGMA, a clusterability measure derived from random matrix theory, that can be used to identify cell clusters with non-random sub-structure, testably leading to the discovery of previously overlooked phenotypes.


title: NMNAT promotes glioma growth through regulating post-translational modifications of p53 to inhibit apoptosis

abstract: Gliomas are highly malignant brain tumors with poor prognosis and short survival. NAD+ has been shown to impact multiple processes that are dysregulated in cancer; however, anti-cancer therapies targeting NAD+ synthesis have been unsuccessful due to insufficient mechanistic understanding. Here we adapted a Drosophila glial neoplasia model and discovered the genetic requirement for NAD+ synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) in glioma progression in vivo and in human glioma cells. Overexpressing enzymatically active NMNAT significantly promotes glial neoplasia growth and reduces animal viability. Mechanistic analysis suggests that NMNAT interferes with DNA damage-p53-caspase-3 apoptosis signaling pathway by enhancing NAD+-dependent posttranslational modifications (PTMs) poly(ADP-ribosyl)ation (PARylation) and deacetylation of p53. Interestingly, NMNAT forms a complex with p53 and PTM enzyme PARP1 to facilitate PARylation. As PARylation and deacetylation reduce p53 pro-apoptotic activity, our results demonstrate that NMNAT promotes glioma progression through regulating p53 post-translational modifications. Our findings reveal a novel tumorigenic mechanism involving protein complex formation of p53 with NAD+ synthetic enzyme NMNAT and NAD+-dependent PTM enzymes that regulates glioma growth.

category: cancer biology


title: Gaps in global wildlife trade monitoring leave amphibians vulnerable

abstract: As the biodiversity crisis continues, we must redouble efforts to understand and curb pressures pushing species closer to extinction. One major driver is the unsustainable trade of wildlife. Trade in internationally regulated species gains the most research attention, but this only accounts for a minority of traded species and we risk failing to appreciate the scale and impacts of unregulated legal trade. Despite being legal, trade puts pressure on wild species via: direct collection, introduced pathogens, and invasive species. Smaller species-rich vertebrates, such reptiles, fish, and amphibians, may be particularly vulnerable to trading because of gaps in regulations, small distributions, and demand of novel species. Here we combine data from five sources: online web searches in six languages, CITES trade database, LEMIS trade database, IUCN assessments, and a recent literature review, to characterise the global trade in amphibians, and also map use by purpose including meat, pets, medicinal and for research. We show that 1,215 species are being traded (17% of amphibian species), almost three times previous recorded numbers, 345 are threatened, and 100 data deficient or unassessed. Traded species origin hotspots include South American, China, and Central Africa; sources indicate 42% of amphibians are taken from the wild. Newly described species can be rapidly traded (mean time lag of 6.5 years), including threatened and unassessed species. The scale and limited regulation of the amphibian trade, paired with the triptych of connected pressures (collection, pathogens, invasive species), warrants a re-examination of the wildlife trade status-quo, application of the precautionary principle in regards to wildlife trade, and a renewed push to achieve global biodiversity goals.


title: Climate risks to carbon sequestration in US forests

abstract: Forests are currently a substantial carbon sink globally. Many climate change mitigation strategies rely on forest preservation and expansion, but the effectiveness of these approaches hinges on forests sequestering carbon for centuries despite anthropogenic climate change. Yet climate-driven disturbances pose critical risks to the long-term stability of forest carbon. We quantify the key climate drivers that fuel wildfire, drought, and insects, for the United States over 1984-2018 and project future disturbance risks over the 21st century. We find that current risks are widespread and projected to increase across different emission scenarios by a factor of 4-14 for fire and 1.3-1.8 for drought and insects. Our results provide insights for carbon cycle modeling, conservation, and climate policy, underscoring the escalating climate risks facing forests and the need for emissions reductions to mitigate climate change.


title: Remote spatially variant debiased profiling of cell and tissue mechanical properties

abstract: The role of the mechanical environment in defining tissue function, development and growth has been shown to be fundamental. Assessment of the changes in stiffness of tissue matrices at multiple scales has relied mostly on invasive and often specialist equipment such as AFM or mechanical testing devices poorly suited to the cell culture workflow.In this paper, we have developed a novel unbiased passive optical coherence elastography method, exploiting ambient vibrations in the sample that enables real-time noninvasive quantitative profiling of cells and tissues. We demonstrate a robust method that decouples optical scattering and mechanical properties by actively compensating for scattering associated noise bias and reducing variance. The efficiency for the method to retrieve ground truth is validated in silico and in vitro, and exemplified for key applications such as time course mechanical profiling of bone and cartilage spheroids, tissue engineering cancer models, tissue repair models and single cell. Our method is readily implementable with any commercial optical coherence tomography system without any hardware modifications, and thus offers a breakthrough in tissue mechanical assessment for novel on line assessment of spatial mechanical properties for organoids, soft tissues and tissue engineering.


title: Protein contact map prediction using multiple sequence alignment dropout and consistency learning for sequences with less homologs

abstract: The prediction of protein contact map needs enough normalized number of effective sequence (Nf) in multiple sequence alignment (MSA). When Nf is small, the predicted contact maps are often not satisfactory. To solve this problem, we randomly selected a small part of sequence homologs for proteins with large Nf to generate MSAs with small Nf. From these MSAs, input features were generated and were passed through a consistency learning network, aiming to get the same results when using the features generated from the MSA with large Nf. The results showed that this method effectively improves the prediction accuracy of protein contact maps with small Nf.


title: Protein contact map prediction using multiple sequence alignment dropout and consistency learning for sequences with fewer homologs


title: A graphical, interactive and GPU-enabled workflow to process long-read sequencing data

abstract: We present a graphical cloud-enabled workflow for fast, interactive analysis of nanopore sequencing data using GPUs. Users customize parameters, monitor execution and visualize results through an accessible graphical interface. To facilitate reproducible deployment, we use Docker containers and provide an Amazon Machine Image (AMI) with all software and drivers pre-installed for GPU computing on the cloud. We observe a 34x speedup and a 109x reduction in costs for the rate-limiting basecalling step in the analysis of blood cancer cell line data. The graphical interface and greatly simplified deployment facilitate the adoption of GPUs for rapid, cost-effective analysis of long-read sequencing.


title: Functional plasticity coupled with structural predispositions in auditory cortex shape successful music category learning

abstract: Categorizing sounds into meaningful groups helps listeners more efficiently process the auditory scene and is a foundational skill for speech perception and language development. Yet, how auditory categories develop in the brain through learning, particularly for nonspeech sounds, is not well understood. Here, we asked musically naive listeners to complete a brief ([~]20 min) training session where they learned to identify sounds from a nonspeech continuum (minor-major 3rd musical intervals). We used multichannel EEG to track behaviorally relevant neuroplastic changes in the auditory event-related potentials (ERPs) pre- to post-training. To rule out mere exposure-induced changes, neural effects were evaluated against a control group of 14 nonmusicians who did not undergo training. We also compared individual categorization performance with structural volumetrics of bilateral primary auditory cortex (PAC) from MRI to evaluate neuroanatomical substrates of learning. Behavioral performance revealed steeper (i.e., more categorical) identification functions in the posttest that correlated with better training accuracy. At the neural level, improvement in learners behavioral identification was characterized by smaller P2 amplitudes at posttest, particularly over right hemisphere. Critically, learning-related changes in the ERPs were not observed in control listeners, ruling out mere exposure effects. Learners also showed smaller and thinner PAC bilaterally, indicating superior categorization was associated with structural differences in primary auditory brain regions. Collectively, our data suggest successful auditory categorical learning of nonspeech sounds is characterized by short-term functional changes (i.e., greater post-training efficiency) in sensory coding processes superimposed on preexisting structural differences in bilateral auditory cortex.


title: Scale-free behavioral dynamics directly linked with scale-free cortical dynamics

abstract:
Naturally occurring body movements and collective neural activity both exhibit complex dynamics, often with scale-free, fractal spatiotemporal structure. Scale-free dynamics of both brain and behavior are each associated with functional benefits to the organism. Despite their similarities, scale-free brain activity and scale-free behavior have been studied separately, without a unified explanation. Here we show that scale-free dynamics of behavior and certain subsets of cortical neurons are strongly related. Surprisingly, the scale-free neural subsets exhibit stochastic winner-take-all competition with other neural subsets. This observation is inconsistent with current theory of critical phenomena in neural systems. We develop a computational model which incorporates known cell-type-specific circuit structure, explaining our findings with a new type of criticality. Our results establish neural underpinnings of scale-free behavior and clear behavioral relevance of scale-free neural activity.

Popular SummaryOver the past 2 decades many measurements of brain activity have revealed a peculiar property; the activity is statistically similar when examined at different scales. This self-similar, fractal nature of brain activity has attracted the interest of neuroscientists and physicists alike. In physics, fractal structure shows up in certain non-living systems when they are operating near the tipping point of a phase transition (e.g. liquid water turning into gaseous vapor). This led to the criticality hypothesis, which posits that the brain may also operate near the tipping point of a phase transition, i.e. near criticality.

Initially the criticality hypothesis was very exciting at a basic level - physical principles of phase transitions promised to help us understand how the brain works. However, a important question naturally arose and remains unresolved. What do the scale-free, fractal fluctuations of brain activity have to do with the behavior of the organism? Is it just background noise in the brain or is there something fractal-like about how we behave and interact with the world?

Here we show for the first time that scale-free fluctuations of brain activity are directly related to moment-to-moment fluctuations in behavior. In fact, we show that both the brain activity and mouse behavior have very similar fractal structure. These results take the criticality hypothesis to a new level of biological and, perhaps, psychological relevance. Scale-free brain dynamics are not just background noise; they correspond to the dynamics of behavior.


title: Predicting the N400 ERP component using the Sentence Gestalt model trained on a large scale corpus

abstract: The N400 component of the event related brain potential is widely used to investigate language and meaning processing. However, despite much research the components functional basis remains actively debated. Recent work showed that the update of the predictive representation of sentence meaning (semantic update, or SU) generated by the Sentence Gestalt model (McClelland, St. John, & Taraban, 1989) consistently displayed a similar pattern to the N400 amplitude in a series of conditions known to modulate this event-related potential. These results led Rabovsky, Hansen, and McClelland (2018) to suggest that the N400 might reflect change in a probabilistic representation of meaning corresponding to an implicit semantic prediction error. However, a limitation of this work is that the model was trained on a small artificial training corpus and thus could not be presented with the same naturalistic stimuli presented in empirical experiments. In the present study, we overcome this limitation and directly model the amplitude of the N400 elicited during naturalistic sentence processing by using as predictor the SU generated by a Sentence Gestalt model trained on a large corpus of texts. The results reported in this paper corroborate the hypothesis that the N400 component reflects the change in a probabilistic representation of meaning after every word presentation. Further analyses demonstrate that the SU of the Sentence Gestalt model and the amplitude of the N400 are influenced similarly by the stochastic and positional properties of the linguistic input.


title: Fluid intelligence and naturalistic task impairments after focal brain lesions

abstract: Classical executive tasks, such as Wisconsin card-sorting and verbal fluency, are widely used as tests of frontal lobe control functions. Since the pioneering work of Shallice and Burgess (1991), it has been known that complex, naturalistic tasks can capture deficits that are missed in these classical tests. Matching this finding, deficits in several classical tasks are predicted by loss of fluid intelligence, linked to damage in a specific cortical "multiple-demand" (MD) network, while deficits in a more naturalistic task are not. To expand on these previous results, we examined the effect of focal brain lesions on three new tests - a modification of the previously-used Hotel task, a new test of task switching after extended delays, and a test of decision-making in imagined real-life scenarios. As potential predictors of impairment we measured volume of damage to a priori MD and default mode (DMN) networks, as well as cortical damage outside these networks. Deficits in the three new tasks were substantial, but were not explained by loss of fluid intelligence, or by volume of damage to either MD or DMN networks. Instead, deficits were associated with diverse lesions, and not strongly correlated with one another. The results confirm that naturalistic tasks capture cognitive deficits beyond those measured by fluid intelligence. We suggest, however, that these deficits may not arise from specific control operations required by complex behaviour. Instead, like everyday activities, complex tasks combine a rich variety of interacting cognitive components, bringing many opportunities for processing to be disturbed.


title: microRNA-138 controls hippocampal interneuron function and short-term memory

abstract: The proper development and function of neuronal circuits relies on a tightly regulated balance between excitatory and inhibitory (E/I) synaptic transmission, and disrupting this balance can cause neurodevelopmental disorders, e.g. schizophrenia. microRNA-dependent gene regulation in pyramidal neurons is important for excitatory synaptic function and cognition, but its role in inhibitory interneurons is poorly understood. Here, we identify miR-138-5p as a regulator of short-term memory and inhibitory synaptic transmission in the hippocampus. Sponge-mediated miR-138-5p inactivation specifically in parvalbumin (PV)-expressing interneurons impairs spatial recognition memory and enhances GABAergic synaptic input onto pyramidal neurons. Cellular and behavioural phenotypes associated with miR-138-5p inactivation are paralleled by an upregulation of the schizophrenia-associated Erbb4, which we validated as a direct miR-138-5p target gene. Our findings suggest that miR-138-5p is a critical regulator of PV interneuron function, with implications for cognition and schizophrenia. More generally, they provide evidence that microRNAs orchestrate neural circuit development by fine-tuning both excitatory and inhibitory synaptic transmission.


title: Visuo-proprioceptive integration and recalibration with multiple visual stimuli

abstract: To organize the plethora of sensory signals from our environment into a coherent percept, our brain relies on the processes of multisensory integration and sensory recalibration. We here asked how visuo-proprioceptive integration and recalibration are shaped by the presence of more than one potentially relevant visual stimulus, hence paving the way to studying multisensory perception under more naturalistic settings with multiple signals per sensory modality. By manipulating the spatio-temporal correspondence between the hand position and two visual stimuli during a cursor-control task, we contrasted two alternative accounts: that only the temporally more proximal signal enters integration and recalibration similar to a winner-takes-all process, or that the influences of both visual signals superpose. Our results show that integration - and likely also recalibration - are shaped by the superposed contributions of multiple stimuli rather than by only individual ones.


title: LMO7-dependent apical constriction requires the binding of the Myosin heavy chain

abstract: Apical constriction, or reduction of the apical domain, underlies many morphogenetic events during development, such as furrow or tube formation. Actomyosin complexes play an essential role in apical constriction, however the detailed analysis of molecular mechanisms is still pending. Here we show that Lim domain only protein 7 (Lmo7), a multidomain adaptor at apical junctions, promotes apical constriction in the Xenopus superficial ectoderm, whereas apical domain size increases in Lmo7-depleted cells. Lmo7 is localized to and promotes the formation of the circumferential actomyosin belt adjacent to apical junctions. We find that Lmo7-dependent apical constriction requires the RhoA-ROCK-Non-muscle myosin II (NMII) pathway. Strikingly, Lmo7 binds and recruits NMII heavy chains to apical junctions. Lmo7 overexpression altered the subcellular distribution of Wtip, a sensor of mechanical tension. Our findings suggest that Lmo7 serves as a scaffold organizing the actomyosin network to regulate contractility at apical junctions.

category: cell biology


title: Lmo7 recruits myosin II heavy chain to induce apical constriction in Xenopus ectoderm


title: The effect of magnesium on calcium binding to cardiac troponin C related hypertrophic cardiomyopathy mutants

abstract:
Cardiac troponin C (cTnC) is the calcium (Ca2+) sensing component of the troponin complex. Binding of Ca2+ to cTnC triggers a cascade of myofilament conformational changes that culminate in force production. Mutations in cTnC linked to hypertrophic myocardial myopathy (HCM) induce a a greater degree and duration of Ca2+ binding, which may underly the hypertrophic phenotype. Recent evidence from our laboratories demonstrated novel modifications of cTnC Ca2+ binding by cellular magnesium (Mg2+) that we hypothesize may be of significance in promoting HCM.

Regulation of contraction has long been thought to occur exclusively through Ca2+ binding to site II of cTnC. However, abundant cellular Mg2+ is a potential competitor for binding to the same sites; work by several groups also suggests this is possible. We have used isothermal titration calorimetry (ITC) to explore the thermodynamic properties associated with the interaction between Ca2+/Mg2+ and site II of cTnC; these experiments demonstrated that physiological concentrations of Mg2+ may compete with Ca2+ to bind site II of cTnC.

In experiments reported here, we studied a series of mutations in cTnC thought to be causal in HCM. Three mutants (A8V, L29Q, and A31S) slightly elevated the affinity for both Ca2+ and Mg2+, whereas other mutants (L48Q, Q50R, and C84Y), that are closer to the C-terminal domain and surrounding the EF hand binding motif of site II had a more significant effect on affinity and the thermodynamics of the binding interaction.

To the best of our knowledge, this work is the first to explore the role of Mg2+ in modifying the Ca2+ affinity ofcTnC mutations linked to HCM. Our results indicate a physiologically significant role for cellular Mg2+ at baseline conditions and when elevated on the control of the dynamics of contraction by modifications in the Ca2+ binding properties of cTnC.


title: Transcriptomic profiling of tissue environments critical for post-embryonic patterning and morphogenesis of zebrafish skin

abstract: Regulation of neural crest derived pigment cells and dermal cells that form skin appendages is broadly similar across vertebrate taxa. In zebrafish, organized pigment stripes and an array of calcified scales form simultaneously in the skin during post-embryonic development. Understanding mechanisms that regulate stripe patterning and dermal morphogenesis may lead to discovery of fundamental mechanisms that govern development of animal form. To learn about cell types and potential signaling interactions that govern skin patterning and morphogenesis we generated and analyzed single cell transcriptomes of skin with genetic or induced defects in pigmentation and squamation. These data reveal a previously undescribed population of ameloblast-like epidermal cells, suggest hormonal control of epithelial-mesenchymal signaling, clarify the signaling network that governs scale papillae development, and identify the hypodermis as a crucial pigment cell support environment. These analyses provide new insights into the development of skin and pigmentation and highlight the utility of zebrafish for uncovering essential features of post-embryonic development in vertebrates.

category: developmental biology


title: A gene expression panel for estimating age in males and females of the sleeping sickness vector Glossina morsitans

abstract: Many vector-borne diseases are controlled by methods that kill the insect vectors responsible for disease transmission. Recording the age structure of vector populations provides information on mortality rates and vectorial capacity, and should form part of the detailed monitoring that occurs in the wake of control programmes, yet tools for obtaining estimates of individual age remain limited. We investigate the potential of using markers of gene expression to predict age in tsetse flies, which are the vectors of deadly and economically damaging African trypanosomiases. We use RNAseq to identify candidate expression markers, and test these markers using qPCR in laboratory-reared Glossina morstians morsitans of known age. Measuring the expression of six genes was sufficient to obtain a prediction of age with root mean squared error of less than 8 days, while just two genes were sufficient to classify flies into age categories of [≤] 15 and >15 days old. Further testing of these markers in field-caught samples and in other species will determine the accuracy of these markers in the field.


title: One Hundred and Fifty Years of Warming on Caribbean Coral Reefs

abstract: Anthropogenic climate change is rapidly altering the characteristics and dynamics of biological communities. This is especially apparent in marine systems as the worlds oceans are warming at an unprecedented rate, causing dramatic changes to coastal marine systems, especially on coral reefs of the Caribbean. We used three complementary ocean temperature databases (HadISST, Pathfinder, and OISST) to quantify change in thermal characteristics of Caribbean coral reefs over the last 150 years (1871-2020). These sea surface temperature (SST) databases included combined in situ and satellite-derived SST (HadISST, OISST), as well as satellite-only observations (Pathfinder) at multiple spatial resolutions. We also compiled a Caribbean coral reef database identifying 5,326 unique reefs across the region. We found that Caribbean reefs have warmed on average by 0.20 {degrees}C per decade since 1987, the calculated year that rapid warming began on Caribbean reefs. Further, geographic variation in warming rates ranged from 0.17 {degrees}C per decade on Bahamian reefs to 0.26 {degrees}C per decade on reefs within the Southern and Eastern Caribbean ecoregions. If this linear rate of warming continues, these already threatened ecosystems would warm by an additional 1.6 {degrees}C on average by 2100. We also found that marine heatwave (MHW) events are increasing in both frequency and duration across the Caribbean. Caribbean coral reefs now experience on average 5 MHW events annually, compared to 1 per year in the early 1980s. Combined, these changes have caused a dramatic shift in the composition and function of Caribbean coral reef ecosystems. If reefs continue to warm at this rate, we are likely to lose even the remnant Caribbean coral reef communities of today in the coming decades.


title: A spatially aware likelihood test to detect sweeps from haplotype distributions

abstract: The inference of positive selection in genomes is a problem of great interest in evolutionary genomics. By identifying putative regions of the genome that contain adaptive mutations, we are able to learn about the biology of organisms and their evolutionary history. Here we introduce a composite likelihood method that identifies recently completed or ongoing positive selection by searching for extreme distortions in the spatial distribution of the haplotype frequency spectrum along the genome relative to the genome-wide expectation taken as neutrality. Furthermore, the method simultaneously infers two parameters of the sweep: the number of sweeping haplotypes and the "width" of the sweep, which is related to the strength and timing of selection. We demonstrate that this method outperforms the leading haplotype-based selection statistics. As a positive control, we apply it to two well-studied human populations from the 1000 Genomes Project and examine haplotype frequency spectrum patterns at the LCT and MHC loci. We also apply it to a data set of brown rats sampled in NYC and identify genes related to olfactory perception. To facilitate use of this method, we have implemented it in user-friendly open source software.

category: evolutionary biology


title: Social selection within aggregative multicellular development drives morphological evolution

abstract: The evolution of developmental systems might be shaped by both historical differences in developmental features and social selection, among other factors. In aggregative multicellularity, development is itself a social process in which unicellular organisms cooperate in carrying out complex developmental programs. In some aggregative systems, development culminates in the construction of spore-packed fruiting bodies. Fruiting body development in myxobacteria often unfolds within genetically and behaviorally diverse conspecific cellular environments that can include social defection and warfare. Here we use the bacterium Myxococcus xanthus to test whether the character of the cellular environment during aggregative development shapes morphological evolution. We manipulated the cellular composition of Myxococcus development in an experiment in which evolving populations initiated from a single ancestor repeatedly co-developed with one of several non-evolving partners - a benign cooperator, one of three cheaters or one of three antagonists. Fruiting body morphology was found to diversify as a function of developmental partners, revealing adaptation specific to distinct cellular environments. Collectively, antagonistic partners selected for higher levels of robust fruiting body formation than did cheaters or the benign cooperator. Moreover, even small degrees of genetic divergence between the distinct cheater partners were sufficient to drive treatment-level morphological divergence. Co-developmental partners not only shaped mean trait evolution but also determined the magnitude and dynamics of stochastic morphological diversification and subsequent convergence. In sum, we find that even few genetic differences affecting developmental and social features can greatly impact the morphological evolution of multicellular bodies and experimentally demonstrate that microbial warfare can promote cooperation.


title: One Cell At a Time: A Unified Framework to Integrate and Analyze Single-cell RNA-seq Data

abstract: 1The surge of single-cell RNA sequencing technologies gives rise to the abundance of large single-cell RNA-seq datasets at the scale of hundreds of thousands of single cells. Integrative analysis of large-scale scRNA-seq datasets has the potential of revealing de novo cell types as well as aggregating biological information. However, most existing methods fail to integrate multiple large-scale scRNA-seq datasets in a computational and memory efficient way. We hereby propose OCAT, One Cell At a Time, a graph-based method that sparsely encodes single-cell gene expressions to integrate data from multiple sources without most variable gene selection or explicit batch effect correction. We demonstrate that OCAT efficiently integrates multiple scRNA-seq datasets and achieves the state-of-the-art performance in cell type clustering, especially in challenging scenarios of non-overlapping cell types. In addition, OCAT efficaciously facilitates a variety of downstream analyses, such as differential gene analysis, trajectory inference, pseudotime inference and cell inference. OCAT is a unifying tool to simplify and expedite the analysis of large-scale scRNA-seq data from heterogeneous sources.


title: Clone expansion of mutation-driven clonal hematopoiesis is associated with aging and metabolic dysfunction in individuals with obesity

abstract:
AimsHaematopoietic clones caused by somatic mutations with [≥]2% variant allele frequency (VAF), known as clonal haematopoiesis of indeterminate potential (CHIP), increase with age and have been linked to risk of haematological malignancies and cardiovascular disease. Recent observations suggest that smaller clones are also associated with adverse clinical outcomes. Our aims were to determine the prevalence of clonal haematopoiesis driven by clones of variable sizes, and to examine the development of clones over time in relation to age and metabolic dysregulation over up to 20 years in individuals with obesity.

Methods and ResultsWe used an ultrasensitive single-molecule molecular inversion probe sequencing assay to identify clonal haematopoiesis driver mutations (CHDMs) in blood samples from individuals with obesity from the Swedish Obese Subjects study. In a single-timepoint dataset with samples from 1050 individuals, we identified 273 candidate CHDMs in 216 individuals, with VAF ranging from 0.01% to 31.15% and CHDM prevalence and clone sizes increasing with age. Longitudinal analysis over 20 years in CHDM-positive samples from 40 individuals showed that small clones can grow over time and become CHIP. VAF increased on average by 7% (range -4% to 27%) per year. Rate of clone growth was positively associated with insulin resistance (R=0.40, P=0.025) and low circulating levels of high-density lipoprotein-cholesterol (HDL-C) (R=-0.68, P=1.74E-05).

ConclusionOur results show that haematopoietic clones can be detected and monitored before they become CHIP and indicate that insulin resistance and low HDL-C, well-established cardiovascular risk factors, are associated with clonal expansion in individuals with obesity.

Translational perspectivesClonal haematopoiesis-driver mutations are somatic mutations in haematopoietic stem cells that lead to clones detectable in peripheral blood. Haematopoietic clones with a variant allele frequency (VAF) [≥]2%, known as clonal haematopoiesis of indeterminate potential (CHIP), are recognized as an independent cardiovascular risk factor. Here, we show that smaller clones are prevalent, and also correlate with age. Our longitudinal observations in individuals with obesity over 20 years showed that more than half of all clone-positive individuals show growing clones and clones with VAF <2% can grow and become CHIP. Importantly, clone growth was accelerated in individuals with insulin resistance and low high-density lipoprotein-cholesterol (HDL-C).

Translational outlook 1: Haematopoietic clones can be detected and monitored before they become CHIP.

Translational outlook 2: The association between insulin resistance and low HDL-C with growth of haematopoietic clones opens the possibility that treatments improving metabolism, such as weight loss, may reduce growth of clones and thereby cardiovascular risk.

One Sentence SummaryIn obesity, the growth rate of mutation-driven haematopoietic clones increased with insulin resistance and low HDL-C, both known risk factors for cardiovascular disease.


title: Expansion of mutation-driven haematopoietic clones is associated with insulin resistance and low HDL-cholesterol in individuals with obesity


title: Single-cell epigenomic tracing of lifelong endothelial cell plasticity across mouse organs

abstract:
Endothelial cells (ECs) across ages and tissues are highly heterogeneous in developmental origins, structures, functions, and cellular plasticity. Here, we applied CoBATCH for single-cell epigenomic tracing of dynamic EC lineage histories in five mouse organs from development to ageing. Our analyses showed that epigenomic memory reflects both developmental origins and tissue-restricted specialization of EC sublineages but with varying time lengths across organs. To gain insights into cellular plasticity of ECs, we identified bivalent chromatin occupancy of otherwise mutually exclusive EC- (ERG) and mesenchymal-specific (TWIST1/SNAI1) transcription factors promoting endothelial-to-mesenchymal transition. We further revealed that pseudotime trajectories by histone modifications H3K36me3 and H3K27ac faithfully recapitulate short- and long-range EC fate change over senescence, respectively. Together, our data provide a unique exploration of chromatin-level cell fate regulation of organotypic EC lineages across the lifespan.

One-Sentence SummarySingle-cell chromatin binding is examined for tracing endothelial cell lineages in mouse organs across the lifespan.


title: Mycolactone enhances the Ca2+ leakage from endoplasmic reticulum by trapping Sec61 translocons in a Ca2+ permeable state

abstract: The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of, the major translocon subunit Sec61, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61 because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61 in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon "breathing" during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactones stabilisation of the latter underpins the phenotype observed.

category: biochemistry


title: Programming large target genomic deletion and concurrent insertion via a prime editing-based method: PEDAR

abstract: Genomic insertions, duplications, and insertion/deletions (indels) account for ~14% of human pathogenic mutations. Current gene editing methods cannot accurately or efficiently correct these abnormal genomic rearrangements, especially larger alterations (>100 bp). Thus, developing a method to accurately delete insertions/duplications and repair the deletion junction could improve the scope of gene therapies. Here, we engineer a novel gene editor, PE-Cas9, by conjugating Cas9 nuclease to reverse transcriptase. Combined with two prime editing guide RNAs (pegRNAs) targeting complementary DNA strands, PE-Cas9 can direct the replacement of a genomic fragment, ranging from to ~1-kb to >10-kb, with a desired sequence at the target site without requiring an exogenous DNA template. In a reporter cell line, this PE-Cas9-based deletion and repair (PEDAR) method restored mCherry expression through in-frame deletion of a disrupted GFP sequence. We further show that PEDAR efficiency could be enhanced by using pegRNAs with high cleavage activity or increasing transfection efficiency. In tyrosinemia mice, PEDAR removed a 1.38-kb pathogenic insertion within the Fah gene and precisely repaired the deletion junction to restore FAH expression in liver. This study highlights PEDAR as a tool for correcting pathogenic mutations.


title: Unsupervised weights selection for optimal transport based dataset integration

abstract: A formulation of the dataset integration problem describes the task of aligning two or more empirical distributions sampled from sources of the same kind, so that records of similar object end up close to one another. We propose a variant of the optimal transport (OT)- and Gromov-Wasserstein (GW)-based dataset integration algorithm introduced in SCOT [Demetci et al., 2020]. We formulate a constrained quadratic program to adjust sample weights before OT or GW so that weighted point density is close to be uniform over the point cloud, for a given kernel. We test this method with one synthetic and two real-life datasets from single-cell biology. Weights adjustment allows distributions with similar effective supports but different local densities to be reliably integrated, which is not always the case with the original method. This approach is entirely unsupervised, scales well to thousands of samples and does not depend on dimensionality of the ambient space, which makes it efficient for the analysis of single-cell datasets in biology. We provide an open-source implementation of this method in a Python package, woti.


title: A de novo protein structure prediction by iterative partition sampling, topology adjustment, and residue-level distance deviation optimization

abstract:
MotivationWith the great progress of deep learning-based inter-residue contact/distance prediction, the discrete space formed by fragment assembly cannot satisfy the distance constraint well. Thus, the optimal solution of the continuous space may not be achieved. Designing an effective closed-loop continuous dihedral angle optimization strategy that complements the discrete fragment assembly is crucial to improve the performance of the distance-assisted fragment assembly method.

ResultsIn this article, we proposed a de novo protein structure prediction method called IPTDFold based on closed-loop iterative partition sampling, topology adjustment and residue-level distance deviation optimization. First, local dihedral angle crossover and mutation operators are designed to explore the conformational space extensively and achieve information exchange between the conformations in the population. Then, the dihedral angle rotation model of loop region with partial inter-residue distance constraints is constructed, and the rotation angle satisfying the constraints is obtained by differential evolution algorithm, so as to adjust the spatial position relationship between the secondary structures. Lastly, the residue distance deviation is evaluated according to the difference between the conformation and the predicted distance, and the dihedral angle of the residue is optimized with biased probability. The final model is generated by iterating the above three steps. IPTDFold is tested on 462 benchmark proteins, 24 FM targets of CASP13, and 20 FM targets of CASP14. Results show that IPTDFold is significantly superior to the distance-assisted fragment assembly method Rosetta_D (Rosetta with distance). In particular, the prediction accuracy of IPTDFold does not decrease as the length of the protein increases. When using the same FastRelax protocol, the prediction accuracy of IPTDFold is significantly superior to that of trRosetta without orientation constraints, and is equivalent to that of the full version of trRosetta.

AvailabilityThe source code and executable are freely available at https://github.com/iobio-zjut/IPTDFold.

Contactzgj@zjut.edu.cn


title: Probing DNA - transcription factor interactions using single-molecule fluorescence detection in nanofluidic devices

abstract: Single-molecule fluorescence detection offers powerful ways to study biomolecules and their complex interactions. Here, we combine nanofluidic devices and camera-based, single-molecule Forster resonance energy transfer (smFRET) detection to study the interactions between plant transcription factors of the auxin response family (ARF) and DNA oligonucleotides that contain target DNA response elements. In particular, we show that the binding of the unlabelled ARF DNA binding domain (ARF-DBD) to donor and acceptor labelled DNA oligonucleotides can be detected by changes in the FRET efficiency and changes in the diffusion coefficient of the DNA. In addition, our data on fluorescently labelled ARF-DBDs suggest that, at nanomolar concentrations, ARF-DBDs are exclusively present as monomers. In general, the fluidic framework of freely diffusing molecules minimizes potential surface-induced artifacts, enables high-throughput measurements and proved to be instrumental in shedding more light on the interactions between ARF-DBDs monomers and between ARF-DBDs and their DNA response element.

category: biophysics


title: Chlorophyll fluorescence: How the quality of information about PAM instrument parameters may affect our research

abstract:
Chlorophyll a fluorescence is a powerful indicator of photosynthetic energy conversion in plants and photosynthetic microorganisms. One of the most widely used measurement techniques is Pulse Amplitude Modulation (PAM) fluorometry. Unfortunately, parameter settings of PAM instruments are often not completely described in scientific articles although their variations, however small these may seem, can influence measurements. We show the effects of parameter settings on PAM measurements. We first simulated fluorescence signals using a previously published computational model of photosynthesis. Then, we validated our findings experimentally. Our analysis demonstrates how the kinetics of non-photochemical quenching (NPQ) induction and relaxation are affected by different settings of PAM instrument parameters. Neglecting these parameters may mislead data interpretation and derived hypotheses, hamper independent validation of the results, and cause problems for mathematical formulation of underlying processes. Given the uncertainties inflicted by this neglect, we urge PAM users to provide detailed documentation of measurement protocols. Moreover, to ensure accessibility to the required information, we advocate minimum information standards that can serve both experimental and computational biologists in our efforts to advance system-wide understanding of biological processes. Such specification will enable launching a standardized database for plant and data science communities.

HighlightPAM fluorometry measurement is sensitive to instrument settings and protocols. Yet, protocols are published incompletely. We urge to reach an agreement on minimal protocol information of PAM experiments to be shared publicly.

category: plant biology


title: Perception of soft materials relies on physics-based object representations: Behavioral and computational evidence

abstract: When encountering objects, we readily perceive not only low-level properties (e.g., color and orientation), but also seemingly higher-level ones - including aspects of physics (e.g., mass). Perhaps nowhere is this contrast more salient than in the perception of soft materials such as cloths: the dynamics of these objects (including how their three-dimensional forms vary) are determined by their physical properties such as stiffness, elasticity, and mass. Here we hypothesize that the perception of cloths and their physical properties must involve not only image statistics, but also abstract object representations that incorporate "intuitive physics". We provide behavioral and computational evidence for this hypothesis. We find that humans can visually match the stiffness of cloths with unfamiliar textures from the way they undergo natural transformations (e.g. flapping in the wind) across different scenarios. A computational model that casts cloth perception as mental physics simulation explains important aspects of this behavior.


title: A novel cholinergic neural pathway and its role in the drug relapse

abstract: The lateral parabrachial nucleus (LPB) is critical hub implicated in the control of food intake, reward and aversion. Here, we identified a novel cholinergic projection from choline acetyltransferase (ChAT)-positive neurons in external portion of the lateral parabrachial nucleus (eLPBChAT) to {gamma}-aminobutyric acid (GABA) neurons in central nucleus of amygdala (CeAGABA), activation of which could block methamphetamine (METH)-primed conditioned place preference (CPP) in mice.


title: RTK-dependent inducible degradation of mutant PI3K alpha drives GDC-0077 (Inavolisib) efficacy

abstract: PIK3CA is one of the most frequently mutated oncogenes; the p110 protein it encodes plays a central role in tumor cell proliferation and survival. Small molecule inhibitors targeting the PI3K p110 catalytic subunit have entered clinical trials, with early-phase GDC-0077 (Inavolisib) studies showing anti-tumor activity and a manageable safety profile in patients with PIK3CA-mutant, hormone receptor-positive breast cancer as a single agent or in combination therapy. Despite this, preclinical studies have shown that PI3K pathway inhibition releases negative feedback and activates receptor tyrosine kinase signaling, reengaging the pathway and attenuating drug activity. Here we discover that GDC-0077 and taselisib more potently inhibit mutant PI3K pathway signaling and cell viability through unique HER2-dependent degradation. Both are more effective than other PI3K inhibitors at maintaining prolonged pathway suppression, resulting in enhanced apoptosis and greater efficacy. This unique mechanism against mutant p110 reveals a new strategy for creating inhibitors that specifically target mutant tumors with selective degradation of the mutant oncoprotein and also provide a strong rationale for pursuing PI3K degraders in patients with HER2-positive breast cancer.


title: ABA homeostasis and long-distance translocation is redundantly regulated by ABCG ABA importers

abstract: The effects of abscisic acid (ABA) on plant growth, development and response to the environment depend on local ABA concentrations. Here, we exploited a genome-scale amiRNA screen, targeting the Arabidopsis transportome, to show that ABA homeostasis is regulated by two previously unknown ABA transporters. ABCG17 and ABCG18 are localized to the plasma membranes of leaf mesophyll and stem cortex cells to redundantly promote ABA import, leading to conjugated inactive ABA sinks, thus restricting stomatal closure. ABCG17 and ABCG18 double knockdown revealed that the transporters encoded by these genes not only limit stomatal aperture size, conductance and transpiration while increasing water-use efficiency but also control ABA translocation from the shoot to the root to regulate lateral root emergence. The proposed ABCG17- and ABCG18-dependent ABA glucosyl ester shoot sink mechanism is restrained under abiotic stress conditions to further activate the ABA responses.


title: Mechanistic Gene Networks Inferred from Single-Cell Data are Better Predictors than Neural Networks.

abstract:
AO_SCPLOWBSTRACTC_SCPLOWWith advances in single-cell techniques, measuring gene dynamics at cellular resolution has become practicable. In contrast, the increased complexity of data has made it more challenging computationally to unravel underlying biological mechanisms. Thus, it is critical to develop novel computational methods capable of dealing with such complexity and of providing predictive deductions from such data. Many methods have been developed to address such challenges, each with its own advantages and limitations. We present an iterative regression algorithm for inferring a mechanistic gene network from single-cell data. Using this regression, we infer a developmental model for the gene dynamics in Drosophila melanogaster blastoderm embryo. Our results show that the predictive power of the inferred model is higher than that of other models inferred with least squares and ridge regressions.

Furthermore, model predictions of the gene dynamics are more accurate than predictions made with neural networks. This holds true even in the limit of small sample sizes. We compare predictions for various gene knockouts with published experimental results, finding substantial agreement. We also make predictions for gene dynamics under various gene network perturbations, impossible in non-mechanistic models.

category: systems biology


title: cAMP controls a trafficking mechanism that directs the neuron specificity and subcellular placement of electrical synapses

abstract: Electrical synapses are established between specific neurons and within distinct subcellular compartments, but the mechanisms that direct gap junction assembly in the nervous system are largely unknown. Here we show that a transcriptional program tunes cAMP signaling to direct the neuron-specific assembly and placement of electrical synapses in the C. elegans motor circuit. For these studies, we use live cell imaging to visualize electrical synapses in vivo and a novel optogenetic assay to confirm that they are functional. In VA motor neurons, the UNC-4 transcription factor blocks expression of cAMP antagonists that promote gap junction miswiring. In unc-4 mutants, VA electrical synapses are established with an alternative synaptic partner and are repositioned from the VA axon to soma. We show that cAMP counters these effects by driving gap junction trafficking into the VA axon for electrical synapse assembly. Thus, our experiments in an intact nervous system establish that cAMP regulates gap junction trafficking for the biogenesis of electrical synapses.


title: A behavioral screen for mediators of age-dependent TDP-43 neurodegeneration identifies SF2/SRSF1 among a group of potent suppressors in both neurons and glia.

abstract:
Cytoplasmic aggregation of Tar-DNA/RNA binding protein 43 (TDP-43) occurs in 97 percent of amyotrophic lateral sclerosis (ALS), about 40 percent of frontotemporal dementia (FTD) and in many cases of Alzheimers disease (AD). Cytoplasmic TDP-43 inclusions are seen in both sporadic and familial forms of these disorders, including those cases that are caused by repeat expansion mutations in the C9orf72 gene. To identify downstream mediators of TDP-43 toxicity, we expressed human TDP-43 in a subset of Drosophila motor neurons. Such expression causes age-dependent deficits in negative geotaxis behavior. Using this behavioral readout of locomotion, we conducted an shRNA suppressor screen and identified 32 transcripts whose knockdown was sufficient to ameliorate the neurological phenotype. The majority of these suppressors also substantially suppressed the negative effects on lifespan seen with glial TDP-43 expression. In addition to identification of a number of genes whose roles in neurodegeneration were not previously known, our screen also yielded genes involved in chromatin regulation and nuclear/import export-pathways that were previously identified in the context of cell based or neurodevelopmental suppressor screens. A notable example is SF2, a conserved orthologue of mammalian SRSF1, an RNA binding protein with roles in splicing and nuclear export. Our identification SF2/SRSF1 as a potent suppressor of both neuronal and glial TDP-43 toxicity also provides a convergence with C9orf72 expansion repeat mediated neurodegeneration, where this gene also acts as a downstream mediator.

Author SummaryLoss of nuclear function of TDP-43 and its mislocalization into cytoplasmic inclusions are central features to a suite of neurodegenerative disorders. We screened 2700 Drosophila genes to identify downstream mediators that suppress an age-dependent motor dysfunction phenotype when they are knocked down by RNA interference. We identified both previously implicated pathways and several novel genes whose knock down is sufficient to dramatically and robustly rescue TDP-43 toxicity both in neuronal and glial contexts. Notably, we report that SF2/SRSF1, which was previously reported as a suppressor of C9orf72 hexanucleotide expansion repeat toxicity, also is a potent suppressor of TDP-43 mediated neurodegeneration.


title: Transcriptional, epigenetic, and functional reprogramming of blood monocytes in non-human primates following chronic alcohol drinking

abstract: Chronic heavy drinking (CHD) of alcohol is a known risk factor for increased susceptibility to bacterial and viral infection as well as impaired wound healing. Evidence suggests that these defects are mediated by a dysregulated inflammatory response originating from myeloid cells, notably monocytes and macrophages, but the mechanisms remain poorly understood. Our ability to study CHD is impacted by the complexities of human drinking patterns and behavior as well as comorbidities and confounding risk factors for patients with alcohol use disorders. To overcome these challenges, we utilize a translational rhesus macaque model of voluntary ethanol self-administration that closely recapitulates human drinking patterns and chronicity. In this study, we examined the effects of CHD on blood monocytes and alveolar macrophages in control and CHD female macaques after 12 months of daily ethanol consumption. While monocytes from CHD female macaques generated a hyper-inflammatory response to ex vivo LPS stimulation, their response to E.Coli was dampened. In depth scRNA-Seq analysis of purified monocytes revealed significant shifts in classical monocyte subsets with accumulation of cells expressing markers of hypoxia (HIF1A) and inflammation (NFkB signaling pathway) in CHD macaques. The increased presence of monocyte subsets poised to generate a hyperinflammatory response was confirmed by the epigenetic analysis which revealed higher accessibility of promoter regions that regulate genes involved in cytokine signaling pathways. Finally, alveolar macrophages (AM) from the same animals produced higher levels of inflammatory mediators in response to LPS stimulation, but reduced ability to phagocytose bacteria. Collectively, data presented in this manuscript demonstrate that CHD primes monocytes and tissue-resident macrophages towards a more hyper-inflammatory immune response with compromised functional abilities, which could be used in diagnostic purposes or preventative measures for patients with alcohol use disorders.

category: immunology


title: A Trio of Ubiquitin Ligases Sequentially Drive Ubiquitylation and Autophagic Degradation of Dysfunctional Yeast Proteasomes

abstract:
As central effectors of ubiquitin (Ub)-mediated proteolysis, proteasomes are regulated at multiple levels, including degradation of unwanted or dysfunctional particles via autophagy (termed proteaphagy). In yeast, inactive proteasomes are exported from the nucleus, sequestered into cytoplasmic aggresomes via the Hsp42 chaperone, extensively ubiquitylated, and then tethered to the expanding phagophore by the autophagy receptor Cue5. Here, we demonstrate the need for ubiquitylation driven by the trio of Ub ligases (E3s) San1, Rsp5 and Hul5, which, together with their corresponding E2s, work sequentially to promote nuclear export and Cue5 recognition. Whereas San1 functions prior to nuclear export, Rsp5 and Hul5 likely decorate aggresome-localized proteasomes in concert. Ultimately, topologically complex Ub chain(s) containing both K48 and K63 Ub-Ub linkages are assembled, mainly on the regulatory particle, to generate autophagy-competent substrates. As San1, Rsp5, Hul5, Hsp42, and Cue5 also participate in general proteostasis, proteaphagy likely engages an essential mechanism for eliminating inactive/misfolded proteins.

HIGHLIGHTSO_LIUbiquitylation is essential for the autophagic turnover of dysfunctional proteasomes.
C_LIO_LIThe San1, Rsp5 and Hul5 E3s act sequentially to drive proteaphagy.
C_LIO_LIThe E2s Ubc1, Ubc4 and Ubc5 are collectively required.
C_LIO_LIBoth K48- and K63-mediated Ub-Ub linkages are assembled for efficient proteaphagy.
C_LI


title: Dual functions of ZmGI1 in the photoperiodic flowering pathway and salt stress responses in maize

abstract: The circadian clock perceives photoperiodic changes and initiates processes leading to floral transition. GIGANTEA (GI) primarily functions as a principal clock component that integrates environmental cues into regulation of growth and development in Arabidopsis. However, it is unclear whether ZmGIs regulate photoperiodic flowering and abiotic stress response. Here, we demonstrated that the expression of ZmGI1 depicted a typical circadian pattern and was differentially expressed under LDs and SDs in photoperiodic sensitive and insensitive maize lines. The transcription level was significantly and positively correlated with days to silking and photoperiodic sensitivity in maize. Moreover, natural variation in ZmGI1 was associated with maize photoperiod response and the fine-tuning of plant development traits. Overexpression of ZmGI1Huangzao4 induced early flowering and enhanced salt tolerance in Arabidopsis relative to the wild-type and gi mutants. ZmGI1 formed a protein complex with ZmFKF1 and acted as a positive regulator of flowering time by regulating CONSTANS transcription in the photoperiod pathway. The ZmGI1/ZmThox complex regulates oxidative stress induced by salt stress via a redox balance pathway. Over all, we have provided compelling evidence to suggest that ZmGI1 is a pleotropic gene whose expression depicts a typical circadian rhythmic pattern and regulates flowering time and confers salt stress tolerance.


title: A rapid method to quantify small-scale vegetation patch structure to complement conventional quadrat surveys

abstract:
AimsVegetation sampling typically involves the use of quadrats, often 1m2, to estimate species cover-abundance. Such surveys do not generally record small-scale vegetation patch structure at sub-quadrat scales, for example 10 cm2. Here we test a simple method to quantify patch structure that complements conventional techniques. We compare the two methods, and analyse metrics derived from small-scale patch surveys with environment / management data.

LocationNorthumberland, United Kingdom

MethodsWe recorded cover-abundance of all species in an upland moor with 1m2 quadrats. These were divided into 100  sub-quadrats, 10 x 10 cm, and the dominant and subdominant species identified. Patch metrics (number, area and shape) for individual species recorded as dominant or subdominant in the sub-quadrat survey were analysed using multivariate generalised linear models with environmental and management data. Sub-quadrat data were also aggregated for each quadrat, to create species composition data. The two sets of compositional data, from whole-quadrat and sub-quadrat aggregations, were compared via Procrustes rotation of ordination scores.

ResultsPatch number, area and shape for dominant and subdominant species were all significantly affected by soil pH, soil water content, slope and elevation. Effects of proximity to sheep tracks and drainage ditches were less consistent amongst species. Ordinations of vegetation data from conventional and sub-quadrats were similar, with significant Procrustes R-squared of 67% and 70% for dominant and subdominant species respectively.

ConclusionsSub-quadrat surveys can easily be used to complement existing whole-quadrat surveys at little cost in time or resources. Their patch metrics can provide additional insights into the environmental and management drivers that may affect the growth of individual plants or clumps, potentially in relation to plant traits, and thus alter the overall community composition. The methods we describe can readily be adapted to other sizes of quadrats and sub-quadrats in a wide range of vegetation communities.


title: Robust detection of natural selection using a probabilistic model of tree imbalance

abstract:
Neutrality tests such as Tajimas D (Tajima, 1989) and Fay and Wus H (Fay and Wu, 2000) are standard implements in the population genetics toolbox. One of their most common uses is to scan the genome for signals of natural selection. However, it is well understood that deviance measures like D and H are confounded by other evolutionary forces--in particular, population expansion--that may be unrelated to selection. Because they are not model-based, it is not clear how to deconfound these statistics in a principled way.

In this paper we derive new likelihood-based methods for detecting natural selection which are robust to confounding by fluctuations in effective population size. At the core of our method is a novel proba-bilistic model of tree imbalance, which generalizes Kingmans coales-cent to allow certain aberrant tree topologies to arise more frequently than is expected under neutrality. We derive a frequency spectrum-based estimator which can be used in place of D, and also extend to the case where genealogies are first estimated. We benchmark our meth-ods on real and simulated data, and provide an open source software implementation.

category: genetics


title: ThruTracker: Open-Source Software for 2-D and 3-D Animal Video Tracking

abstract:
O_LITracking animal movement patterns using videography is an important tool in many biological disciplines ranging from biomechanics to conservation. Reduced costs of technology such as thermal videography and unmanned aerial vehicles has made video-based animal tracking more accessible, however existing software for processing acquired video limits the application of these methods.
C_LIO_LIHere, we present a novel software program for high-throughput 2-D and 3-D animal tracking. ThruTracker provides tools to allow video tracking under a variety of conditions with minimal technical expertise or coding background and without the need for paid licenses. Notable capabilities include calibrating the intrinsic properties of thermal cameras; tracking and counting hundreds of animals at a time; and the ability to make 3-D calibrations without dedicated calibration objects. Automated 2-D and 3-D workflows are integrated to allow for analysis of largescale datasets.
C_LIO_LIWe tested ThruTracker with two case studies. The 2-D workflow is demonstrated by counting bats emerging from bridges and caves using thermal Videography. Tests show that ThruTracker has a similar accuracy compared to humans under a variety of conditions. The 3-D workflow is shown for making accurate calibrations for tracking bats and birds at wind turbines using only the wind turbine itself as a calibration object.
C_LIO_LIThruTracker is a robust software program for tracking moving animals in 2-D and 3-D. Major applications include counting animals such as bats, birds, and fish that form large aggregations, and documenting movement trajectories over medium spatial scales ([~]100,000 m3). When combined with emerging technologies, we expect videographic techniques to continue to see widespread adoption for an increasing range of biological applications.
C_LI


title: Total Biosynthesis of Triacsin Featuring an N-hydroxytriazene Pharmacophore

abstract: Triacsins are an intriguing class of specialized metabolites possessing a conserved N-hydroxytriazene moiety not found in any other known natural products. Triacsins are notable as potent acyl-CoA synthetase inhibitors in lipid metabolism, yet their biosynthesis has remained elusive. Through extensive mutagenesis and biochemical studies, we here report all enzymes required to construct and install the N-hydroxytriazene pharmacophore of triacsins. Two distinct ATP-dependent enzymes were revealed to catalyze the two consecutive N-N bond formation reactions, including a glycine-utilizing hydrazine-forming enzyme, Tri28, and a nitrous acid-utilizing N-nitrosating enzyme, Tri17. This study paves the way for future mechanistic interrogation and biocatalytic application of enzymes for N-N bond formation.


title: Nuclear lamin isoforms differentially contribute to LINC complex-dependent nucleocytoskeletal coupling and whole cell mechanics

abstract: The ability of a cell to regulate its mechanical properties is central to its function. Emerging evidence suggests that interactions between the cell nucleus and cytoskeleton influence cell mechanics through poorly understood mechanisms. Here we show that A- and B-type nuclear lamin isoforms distinctively modulate both nuclear and cellular volume and selectively stabilize the linker of nucleoskeleton and cytoskeleton (LINC) complexes that couple the nucleus to cytoskeletal actin and vimentin. We reveal, further, that loss of each of the four-known lamin isoforms in the mouse embryonic fibroblasts differentially affects cortical and cytoplasmic stiffness as well as cellular contractility, and then propose a LINC complex mediated model that explains these impaired mechanical phenotypes. Finally, we demonstrate that loss of each lamin isoform softens the nucleus in a manner that correlates with loss of heterochromatin. Together, these findings uncover distinctive roles for each lamin isoform in maintaining cellular and nuclear mechanics.


title: Motor patterning, ion regulation and Spreading Depolarization during CNS shutdown induced by experimental anoxia in Locusta migratoria.

abstract: Anoxia induces a reversible coma in insects. Coma onset is triggered by the arrest of mechanisms responsible for maintaining membrane ion homeostasis in the CNS, resulting in a wave of neuronal and glial depolarization known as spreading depolarization (SD). Different methods of anoxia influence the behavioural response but their effects on SD are unknown. We investigated the effects of CO2, N2, and H2O on the characteristics of coma induction and recovery in Locusta migratoria. Water immersion delayed coma onset and recovery, likely due to involvement of the tracheal system and the nature of asphyxiation but otherwise resembled N2 delivery. The main difference between N2 and CO2 was that CO2 hastened onset of neural failure and SD and delayed recovery. In the CNS, this was associated with CO2 inducing an abrupt and immediate decrease of interstitial pH and increase of extracellular [K+]. Recording of the transperineurial potential showed that SD propagation and a postanoxic negativity (PAN) were similar with both gases. The PAN increased with ouabain treatment, likely due to removal of the counteracting electrogenic effect of Na+/K+-ATPase, and was inhibited by bafilomycin, a proton pump inhibitor, suggesting that it was generated by the electrogenic effect of a Vacuolar-type ATPase (VA). Muscle fibres depolarized by [~]20 mV, which happened more rapidly with CO2 compared with N2. Wing muscle motoneurons depolarized nearly completely in two stages, with CO2 causing more rapid onset and slower recovery than N2. Other parameters of SD onset and recovery were similar with the two gases. Electrical resistance across the ganglion sheath increased during anoxia and at SD onset. We provisionally attribute this to cell swelling reducing the dimensions of the interstitial pathway from neuropil to the bathing saline. Neuronal membrane resistance decreased abruptly at SD onset indicating opening of an unidentified membrane conductance. Consideration of the intracellular recording relative to the saline suggests that the apical membrane of perineurial glia depolarizes prior to neuron depolarization. We propose that SD is triggered by events at the perineurial sheath and then propagates laterally and more deeply into the neuropil. We conclude that the fundamental nature of SD is not dependent on the method of anoxia however the timing of onset and recovery are influenced; water immersion is complicated by the tracheal system and CO2 delivery has more rapid and longer lasting effects, associated with severe interstitial acidosis.

category: physiology


title: Spatiotemporal 3D image registration for mesoscale studies of brain development.

abstract: Comparison of brain samples representing different developmental stages often necessitates registering the samples to common coordinates. Although the available software tools are successful in registering 3D images of adult brains, registration of perinatal brains remains challenging due to rapid growth-dependent morphological changes and variations in developmental pace between animals. To address these challenges, we propose a multi-step algorithm for the registration of perinatal brains. First, we optimized image preprocessing to increase the algorithms sensitivity to mismatches in registered images. Second, we developed an attention-gated simulated annealing (Monte Carlo) procedure capable of focusing on the differences between perinatal brains. Third, we applied classical multidimensional scaling (CMDS) to align ("synchronize") brain samples in time, accounting for individual development paces. We tested this multi-step algorithm on 28 samples of whole-mounted perinatal mouse brains (P0 - P9) and observed accurate registration results. Our computational pipeline offers a runtime of several minutes per brain on a personal computer and automates brain registration tasks including mapping brain data to atlases, comparison of averaged experimental groups, and monitoring brain development dynamics.

category: neuroscience


title: Structural and cellular transcriptome foundations of human brain disease

abstract: Genes associated with risk for brain disease exhibit characteristic expression patterns that reflect both anatomical and cell type relationships. Brain-wide transcriptomic patterns of disease risk genes provide a molecular based signature for identifying disease association, often differing from common phenotypic classification. Analysis of adult brain-wide transcriptomic patterns associated with 40 human brain diseases identified five major transcriptional patterns, represented by tumor-related, neurodegenerative, psychiatric and substance abuse, and two mixed groups of diseases. Brain disease risk genes exhibit unique anatomic transcriptomic signatures, based on differential co-expression, that often uniquely identify the disease. For cortical expressing diseases, single nucleus data in the middle temporal gyrus reveals cell type expression gradients separating neurodegenerative, psychiatric, and substance abuse diseases. By homology mapping of cell types across mouse and human, transcriptomic disease signatures are found largely conserved, but with psychiatric and substance abuse related diseases showing important specific species differences. These results describe the structural and cellular transcriptomic landscape of disease in the adult brain, highlighting significant homology with the mouse yet indicating where human data is needed to further refine our understanding of disease-associated genes.


title: Brace root phenotypes predict root lodging susceptibility and the contribution to anchorage in maize

abstract: Plant mechanical failure (lodging) causes global yield losses of 7-66% in cereal crops. We have previously shown that the above-ground nodal roots (brace roots) in maize are critical for anchorage. However, it is unknown how brace root phenotypes vary across genotypes and the functional consequence of this variation. This study quantifies the contribution of brace roots to anchorage, brace root traits, plant height, and root lodging susceptibility in 52 maize inbred lines. We show that the contribution of brace roots to anchorage and root lodging susceptibility varies among genotypes and this contribution can be explained by plant architectural variation. Additionally, supervised machine learning models were developed and show that multiple plant architectural phenotypes can predict the contribution of brace roots to anchorage and root lodging susceptibility. Together these data define the plant architectures that are important in lodging resistance and show that the contribution of brace roots to anchorage is a good proxy for root lodging susceptibility.


title: Multiple brace root phenotypes promote anchorage and limit root lodging in maize


title: CV2CoV, an enhanced mRNA-based SARS-CoV-2 vaccine candidate, supports higher protein expression and improved immunogenicity in rats

abstract: More than a year after emergence of the SARS-CoV-2 pandemic, multiple first-generation vaccines are approved and available for vaccination. Still, many challenges remain. The ongoing vaccination programs across the globe suffer from insufficient vaccine supply. The virus is adapting to the human host and novel variants are circulating that are neutralised less efficiently by antibodies raised against ancestral SARS-CoV-2 variants. Here, we describe CV2CoV, a second-generation mRNA vaccine developed for enhanced protein expression and immunogenicity. CV2CoV supports increased levels of protein expression in cell culture compared to our clinical candidate CVnCoV. Vaccination with CV2CoV induces high levels of virus neutralising antibodies with accelerated kinetics in rats. Robust antibody responses are reflected in significant cross-neutralisation of circulating SARS-CoV-2 variants of concern, i.e. B.1.1.7 and B.1.351. Together, these results underline the value of CV2CoV as next-generation SARS-CoV-2 mRNA vaccine.


title: Plasmacytoid dendritic cells produce type I interferon and reduce viral replication in airway epithelial cells after SARS-CoV-2 infection

abstract:
Infection with SARS-CoV-2 has caused a pandemic of unprecedented dimensions. SARS-CoV-2 infects airway and lung cells causing viral pneumonia. The importance of type I interferon (IFN) production for the control of SARS-CoV-2 infection is highlighted by the increased severity of COVID-19 in patients with inborn errors of type I IFN response or auto-antibodies against IFN-. Plasmacytoid dendritic cells (pDCs) are a unique immune cell population specialized in recognizing and controlling viral infections through the production of high concentrations of type I IFN. In this study, we isolated pDCs from healthy donors and showed that pDCs are able to recognize SARS-CoV-2 and rapidly produce large amounts of type I IFN. Sensing of SARS-CoV-2 by pDCs was independent of viral replication since pDCs were also able to recognize UV-inactivated SARS-CoV-2 and produce type I IFN. Transcriptional profiling of SARS-CoV-2 and UV-SARS-CoV-2 stimulated pDCs also showed a rapid type I and III IFN response as well as induction of several chemokines, and the induction of apoptosis in pDCs. Moreover, we modeled SARS-CoV-2 infection in the lung using primary human airway epithelial cells (pHAEs) and showed that co-culture of pDCs with SARS-CoV-2 infected pHAEs induces an antiviral response and upregulation of antigen presentation in pHAE cells. Importantly, the presence of pDCs in the co-culture results in control of SARS-CoV-2 replication in pHAEs. Our study identifies pDCs as one of the key cells that can recognize SARS-CoV-2 infection, produce type I and III IFN and control viral replication in infected cells.

ImportanceType I interferons (IFNs) are a major part of the innate immune defense against viral infections. The importance of type I interferon (IFN) production for the control of SARS-CoV-2 infection is highlighted by the increased severity of COVID-19 in patients with defects in the type I IFN response. Interestingly, many cells are not able to produce type I IFN after being infected with SARS-CoV-2 and cannot control viral infection. In this study we show that plasmacytoid dendritic cells are able to recognize SARS-CoV-2 and produce type I IFN, and that pDCs are able to help control viral infection in SARS-CoV-2 infected airway epithelial cells.


title: CsoDIAq Software for Direct Infusion Shotgun Proteome Analysis

abstract: New mass spectrometry data collection methods require new computational tools. Direct Infusion Shotgun Proteome Analysis (DISPA) is a new paradigm for expedited mass spectrometry-based proteomics, but the original data analysis workflow was onerous. Here we introduce CsoDIAq, a user-friendly software package for the identification and quantification of peptides and proteins from DISPA data. In addition to establishing a complete and automated analysis workflow with a graphical user interface, CsoDIAq introduces algorithmic concepts to improve peptide identification speed and sensitivity. These include spectra pooling to reduce search time complexity, and a new spectrum-spectrum match score called match count and cosine (MaCC), which improves target discrimination in a target-decoy analysis. We further show that reanalysis after fragment mass tolerance correction increased the number of peptide identifications. Finally, we adapt CsoDIAq to standard LC-MS DIA, and show that it outperforms other spectrum-spectrum matching software.


title: Structural basis for template switching by a group II intron-encoded non-LTR-retroelement reverse transcriptase

abstract: Reverse transcriptases (RTs) can template switch during cDNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-seq, and may contribute to retroelement fitness by enabling continuous cDNA synthesis on damaged templates. Here, we determined an X-ray crystal structure of a template-switching complex of a group II intron RT bound simultaneously to an acceptor RNA and donor RNA template/DNA heteroduplex with a 1-nt 3-DNA overhang. The latter mimics a completed cDNA after non-templated addition (NTA) of a nucleotide complementary to the 3 nucleotide of the acceptor as required for efficient template switching. The structure showed that the 3 end of the acceptor RNA binds in a pocket formed by an N-terminal extension (NTE) present in non-long-terminal-repeat (LTR)-retroelement RTs and the RT fingertips loop, with the 3 nucleotide of the acceptor base paired to the 1-nt 3-DNA overhang and its penultimate nucleotide base paired to the incoming dNTP at the RT active site. Analysis of structure-guided mutations identified amino acids that contribute to acceptor RNA binding and a phenylalanine near the RT active site that mediates NTA. Mutation of the latter residue decreased multiple sequential template switches in RNA-seq. Our results provide new insights into the mechanisms of template switching and NTA by RTs, suggest how these reactions could be improved for RNA-seq, and reveal common structural features for template switching by non-LTR-retroelement RTs and viral RNA-dependent RNA polymerases.


title: A protein microarray-based in vitro transglutaminase assay platform for epitope mapping and peptide discovery

abstract: Transglutaminases (TGs) are a family of crosslinking enzymes catalyzing the formation of intra- and inter-molecular glutamine-lysine isopeptide bonds in a calcium dependent manner. Activation of transglutaminases is pathogenically associated with severe human diseases including neurodegenerations, cardiovascular diseases, and autoimmune diseases. Although continuous efforts determining the enzymes substrate preference have been witnessed, a high-throughput assay platform with the "omic" efficiency is still missing for the global identification of substrate-specific TG modification sites. In this study we report a protein microarray-based in vitro TG assay platform for rapid and large-scale (up to 30000 reactions per chip) determination of the glutamine (Q)-bearing TG modification motifs. With this platform we identified the Q16 in superoxide dismutase 1 and Q109 in alpha-synuclein as the primary modification sites for tissue transglutaminase (TG2), the most ubiquitous member of the enzyme family. Of particular interest, we found a close match between the modification motifs and published vaccine epitope sequences in alpha-synuclein, implying an essential and intrinsic role transglutaminase might play in the determination of immunodominant epitopes. Our data collectively suggest the glutamine and its follow-up five residues on the C terminal of a protein compose a minimal determinant motif for TG2 modification and the TG2 modification motifs determined by our platform could finally correspond to the substrates epitope sequences in antigen processing. To screen for site-specific interfering peptides and assist gene editing-based protein engineering, we optimized through onchip amino-acid scanning the TG2 modification motif QQIV in the extracellular matrix protein fibronectin and obtained 14 variants with significantly higher TG2 reactivity that might serve as the competitive inhibitor peptides and 1 with lower reactivity. We further confirmed this optimization approach with 12-mer peptides, the longest ones that could be synthesized on the chip. Taken together, our synthetic transglutaminase assay platform might be able to deliver a precise epitope blueprint for immunotherapeutic targeting and provide proof-of-concept and directional studies for TG-based peptide discovery and protein design.

category: synthetic biology


title: A protein microarray-based in vitro transglutaminase assay platform for epitope mapping and peptide discovery: implication for transglutaminase-mediated immunodominant determination


title: Dual origin and multiple neuropeptidergic trajectories of hypothalamic POMC progenitors revealed by developmental single-cell transcriptomics

abstract: Proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus are essential to regulate food intake and energy balance. However, the ontogenetic transcriptional programs that specify the identity and functioning of these neurons are poorly understood. Here, we use scRNAseq to define the transcriptomes characterizing POMC progenitors in the developing hypothalamus and TRAP-seq to analyze the subsequent translatomes of mature POMC neurons. Our data show that Pomc-expressing neurons originate from multiple developmental pathways expressing distinct combinations of transcription factors. The predominant cluster, featured by high levels of Pomc and Prdm12 transcripts represents the precursor of canonical arcuate POMC neurons. Additional clusters of progenitors expressing lower levels of Pomc mature into different neuronal phenotypes characterized by distinct combinations of transcription factors, neuropeptides, processing enzymes, cell surface and nuclear receptors. We conclude that the genetic programs specifying the identity and differentiation of arcuate POMC neurons are diverse and generate a heterogeneous repertoire of neuronal phenotypes early in development that continue to evolve postnatally.


title: Developmental single-cell transcriptomics of hypothalamic POMC progenitors reveal the genetic trajectories of multiple neuropeptidergic phenotypes


title: Composition and Biophysical Properties of the Sorting Platform Pods in the Shigella Type III Secretion System

abstract: Shigella flexneri, causative agent of bacillary dysentery (shigellosis), uses a type III secretion system (T3SS) as its primary virulence factor. The T3SS injectisome delivers effector proteins into host cells to promote entry and create an important intracellular niche. The injectisomes cytoplasmic sorting platform (SP) is a critical assembly that contributes to substrate selection and energizing secretion. The SP consists of oligomeric Spa33 "pods" that associate with the basal body via MxiK and connect to the Spa47 ATPase via MxiN. The pods contain heterotrimers of Spa33 with one full-length copy associated with two copies of a C-terminal domain (Spa33C). The structure of Spa33C is known, but the precise makeup and structure of the pods in situ remains elusive. We show here that recombinant wild-type Spa33 can be prepared as a heterotrimer that forms distinct stable complexes with MxiK and MxiN. In two-hybrid analyses, association of the Spa33 complex with these proteins occurs via the full-length Spa33 component. Furthermore, these complexes each have distinct biophysical properties. Based on these properties, new high-resolution cryo-electron tomography data and architectural similarities between the Spa33 and flagellar FliM-FliN complexes, we provide a preliminary model of the Spa33 heterotrimers within the SP pods. From these findings and evolving models of SP interfaces and dynamics in the Yersinia and Salmonella T3SS, we suggest a model for SP function in which two distinct complexes come together within the context of the SP to contribute to form the complete pod structures during the recruitment of T3SS secretion substrates.

category: microbiology


title: Vector-borne Trypanosoma brucei parasites develop in artificial human skin and persist as skin tissue forms

abstract: Transmission of Trypanosoma brucei by tsetse flies involves the deposition of the cell cycle-arrested metacyclic life cycle stage into mammalian skin at the site of the flys bite. In the skin, the metacyclic parasites reactivate and differentiate into proliferative trypanosomes before colonizing the hosts blood and tissues. We generated an advanced human skin equivalent and used tsetse flies to naturally infect the artificial skin with trypanosomes. We detailed the chronological order of the parasites development in the skin, and found a rapid activation of metacyclic trypanosomes and differentiation to proliferative parasites. Single-cell parasite transcriptomics were used to document the biological events during differentiation and host invasion at five different timepoints. After the establishment of a proliferative trypanosome population in the skin, the parasites entered a reversible quiescent state characterized by slow replication and a strongly reduced metabolism. We termed these quiescent trypanosomes skin tissue forms (STF), a parasite population that may play an important role in maintaining the infection over long time periods and in asymptomatic infected individuals.