title: Invasion genetics of the longhorn crazy ant: the global expansion of a double-clonal reproduction system

abstract: Reproduction mode represents a key determinant for success of biological invasion as it influences the genetic variation and evolutionary potential of introduced populations. The worlds most widespread invasive ant, Paratrechina longicornis, was found to display an unusual double-clonal reproduction system, whereby both males and queens were produced clonally, while workers are produced sexually. Despite its worldwide distribution, the origin of this ant species and the prevalence of the double-clonal reproductive system across the ants geographic range remain unknown. To retrace the evolutionary history of this global invasive species and its reproductive system, we examined genetic variation and characterized the mode of reproduction of P. longicornis sampled worldwide using both microsatellite genotyping and mitochondrial DNA sequencing approaches. Analyses of global genetic variations indicate that the Indian subcontinent is a genetic diversity hotspot of this species, suggesting that this geographic area is at least part of its native range. Our analyses revealed that inferred native and introduced populations both exhibit double-clonal reproduction. Remarkably, queens and males worldwide belong to two separate, non-recombining clonal lineages. Workers are highly heterozygous and first-generation inter- lineage hybrids, a pattern strongly supportive of a strict worldwide prevalence of double clonality. By maintaining heterozygosity in the worker force, this unusual genetic system allows P. longicornis to avoid inbreeding during colonization bottlenecks and may have acted as an adaptive trait linked to the species invasion success.


title: Selection on the fly: short term adaptation to an altered sexual selection regime in Drosophila pseudoobscura

abstract:
Experimental evolution studies are powerful approaches to unveil the evolutionary history of lab populations. Such studies have shed light on how selection changes phenotypes and genotypes. Most of these studies have not examined the time course of adaptation under sexual selection manipulation, by resequencing the populations genomes at multiple time points. Here, we analyse allele frequency trajectories in Drosophila pseudoobscura where we altered their sexual selection regime for 200 generations and sequenced pooled populations at 5 time points. The intensity of sexual selection was either relaxed in monogamous populations (M) or elevated in polyandrous lines (E). We present a comprehensive study of how selection alters population genetics parameters at the chromosome and gene level. We investigate differences in the effective population size - Ne - between the treatments, and perform a genome-wide scan to identify signatures of selection from the time-series data.

We found genomic signatures of adaptation to both regimes in D. pseudoobscura. There are more significant variants on E lines as expected from stronger sexual selection. However, we found that the response on the X chromosome was substantial in both treatments, only more marked in E and restricted to chromosome arm XR in M. Ne is lower on the X at the start of the experiment, which might indicate a swift adaptive response at the onset of selection. Additionally, we show that the third chromosome was also affected by elevated polyandry. Its distal end harbours a region showing a strong signal of adaptive divergence in E lines.

category: evolutionary biology


title: Coevolution of Patch Selection in Stochastic Environments

abstract: Species live and interact in patchy landscapes where environmental conditions vary both in time and space. In the face of this spatial-temporal heterogeneity, species may co-evolve how they select habitat patches. Under equilibrium conditions, coevolution of patch-selection is predicted to give rise to ideal-free distributions of all species: their per-capita growth rates are zero in occupied patches and negative in unoccupied patches. While ideal-free distributions explain observed empirical patterns including enemy-free space and the ghost of competition past, they do not explain why some species occupy sink patches, why competitors have overlapping spatial ranges, or why predators avoid highly productive patches. To understand these patterns, we analyze stochastic Lotka-Volterra models accounting for spatial heterogeneity, environmental stochasticity, and any number of interacting species. We derive an analytically tractable characterization of coevolutionarily stable strategies (coESS) for patch-selection and introduce a numerical algorithm for solving for a coESS. The analytic characterization shows whenever there is selection for a species to occupy multiple patches, their local stochastic growth rates will be negative in the occupied patches i.e. all populations are sink populations. Applying our methods to models of antagonistic interactions reveals that environmental stochasticity can partially exorcize the ghost of competition past, select for new forms of enemy-free and victimless space, and generate hydra effects over evolutionary time scales. To provide additional perspective on our results, we discuss how they relate to the Modern Portfolio Theory of economics. Importantly, our results highlight how environmental stochasticity can reverse or amplify selective forces due to species interactions and spatial heterogeneity.


title: Inbreeding depression in an outbred nine-spined stickleback (Pungitius pungitius) population

abstract: Inbreeding depression refers to the reduced fitness of offspring produced by related individuals and is expected to be rare in large outbred populations. When it occurs, marked fitness loss is possible as large populations can carry large loads of recessive harmful mutations which are normally sheltered at the heterozygous state. Using experimental cross data and genome-wide identity-by-descent (IBD) relationships from an outbred marine nine-spined stickleback (Pungitius pungitius) population, we documented a significant decrease in offspring survival probability with increasing parental IBD sharing associated with an average inbreeding load (B) of 15.896. Interestingly, we found that this relationship was also underlined by a positive effect of paternal inbreeding coefficient on offspring survival, suggesting that certain combinations of parental inbreeding and genetic relatedness among mates may promote offspring survival. Apart from demonstrating substantial inbreeding load in an outbred population, the results also highlight the potential caveat associated with artificial establishment of families in experimental studies: wild founder individuals are often - and perhaps mistakenly - assumed to be unrelated.


title: Hybrid-derived weedy rice maintains adaptive combinations of alleles associated with seed dormancy

abstract: Hybridization is a widespread phenomenon in plants and is a pathway for the evolution of adaptive traits. However, this process may also affect the persistence of combinations of adaptive alleles evolved through natural selection when hybridization occurs between adapted and non-adapted populations. Hybridization between weedy and cultivated rice has been confirmed with an adaptive introgression of deep seed dormancy alleles from cultivated rice. In this study, we explored the influence of hybridization on the conservation of combinations of adaptive alleles by evaluating the natural variation in and the genetic structure of genomic regions associated with seed dormancy. Based on sequence variation in the genomic regions associated with seed dormancy, we revealed that hybrid-derived weedy rice strains maintained most of the adaptive combinations for this trait that were observed in the parental weedy rice, despite equal representation of the parental weedy and cultivated rice in the whole genome sequence. Moreover, the hybrid-derived weedy rice strains had deeper seed dormancy than their parental weedy rice strains. This study suggests that hybridization between weedy rice (having adaptive allelic combinations for seed dormancy) and cultivated rice (having non-adaptive combinations) generates weedy rice strains that express deep seed dormancy caused by genome stabilization through the removal of alleles derived from cultivated rice, in addition to the adaptive introgression of deep seed dormancy alleles derived from cultivated rice. Thus, hybridization between adapted and non-adapted populations seems to be reinforcing the trajectory towards the evolution of adaptive traits.


title: Evolution of transposable element-derived enhancer activity

abstract: Many transposable elements (TEs) contain transcription factor binding sites and are implicated as potential regulatory elements. However, TEs are rarely functionally tested for regulatory activity, which in turn limits our understanding of how TE regulatory activity has evolved. We systematically tested the human LTR18A subfamily for regulatory activity using massively parallel reporter assay (MPRA) and found AP-1 and C/EBP-related binding motifs as drivers of enhancer activity. Functional analysis of evolutionarily reconstructed ancestral sequences revealed that LTR18A elements have generally lost regulatory activity over time through sequence changes, with the largest effects occurring due to mutations in the AP-1 and C/EBP motifs. We observed that the two motifs are conserved at higher rates than expected based on neutral evolution. Finally, we identified LTR18A elements as potential enhancers in the human genome, primarily in epithelial cells. Together, our results provide a model for the origin, evolution, and co-option of TE-derived regulatory elements.

category: genetics


title: minute: A MINUTE-ChIP data analysis workflow

abstract:
Quantitative ChIP-seq methods are essential for accurately characterizing and comparing genome-wide DNA-protein interactions across samples. Procedures that enable such quantitative comparisons involve addition of spike-in chromatin or recombinant nucleosome material, or a multiplexed process using barcoding of chromatin fragments. ChIP-seq analysis workflows typically require a number of computational steps involving multiple tools in order to reach interpretable results, and quantitative analyses require additional steps that ensure scaling of the processed output according to the quantitative measurements. Crucially, the different quantitative approaches have unique analysis requirements reflecting the disparate experimental workflows, hence no universal analysis pipeline exists for quantitative ChIP-seq. Here, we developed minute, a user-friendly computational workflow to easily process multiplexed ChIP data that handles the specific needs of quantitative ChIP. minute enables transformation of raw, multiplexed FASTQ files into a set of normalized, scaled bigWig files that can serve as a basis for a quantitative, comparative downstream analysis. minute is implemented in Python and Snakemake and paired with a Conda environment, to facilitate usability and reproducibility in different platforms.

Source code of minute is available on GitHub: https://github.com/NBISweden/minute

category: bioinformatics


title: MHC-I binding affinity derived metrics fail to predict tumor specific neoantigen immunogenicity

abstract: Tumor-specific antigens emerging through somatic genomic rearrangements, known as neoantigens, play a critical role in current anticancer immunotherapy. They may or may not elicit an immune response when presented on the tumor cell surface bound to the MHC-I molecule, whose strength has been assumed as an indicator of immunogenicity. Several in silico peptide-MHC-I binding affinity predictors are used to prioritize putative immunogenic neoantigens to be experimentally and clinically explored either as biomarkers or targets for anticancer vaccines. This claims for a fair evaluation of such predictors, making essential the development of appropriate databases with experimentally validated, immunogenic/non-immunogenic neoantigens. Thus far, such a database is lacking. We herein present ITSNdb, a new and curated immunogenic neoantigen database and use it to benchmark current neoantigen immunogenicity predictors. Benchmark results failed to support the application of the predicted peptide- MHC-I binding affinity or its derived metrics as a tool to estimate neoantigen immunogenicity and the tumor neoantigen burden as an immunotherapy response biomarker. Moreover, binding affinity based immunogenicity definition leads to identifying wild-type peptide counterparts as predictors of immunotherapy response. We demonstrate that MHC-I binding affinity is insufficient to define neoantigen immunogenicity, despite being necessary for neoantigen tumor cell presentation suggesting that a paradigm shift for the emergence of new rules to identify immunogenic neoantigens is required.


title: Effect of ambient fluid rheology on oscillatory instabilities in filament-motor systems

abstract: Filaments and filament bundles such as microtubules or actin interacting with molecular motors such as dynein or myosin constitute a common motif in biology. Synthetic mimics, examples being artificial muscles and reconstituted active networks, also feature active filaments. A common feature of these filament-motor systems is the emergence of stable oscillations as a collective dynamic response. Here, using a combination of classical linear stability analysis and non-linear numerical solutions, we study the dynamics of a minimal filament-motor system immersed in model viscoelastic fluids. We identify steady states, test the linear stability of these states, derive analytical stability boundaries, and investigate emergent oscillatory solutions and their properties. We show that the interplay between motor activity, aggregate elasticity and fluid viscoelasticity allows for stable oscillations or limit cycles to bifurcate from steady states. For highly viscous Newtonian media, frequencies at onset decay with viscosity  as [Formula]. In viscoelastic fluids that have the same viscosity as the Newtonian fluid but additionally allow for temporary energy storage, emergent limit cycles are associated with higher frequencies. The magnitude of the increase in the frequency depends on motor mechanochemistry and the interplay between fluid relaxation time-scales and time-scales associated with motor binding and unbinding. Our results suggest that stability and dynamical response in filamentous active systems may be controlled by tailoring the rheology of the ambient environment.

category: biophysics


title: Pharmacokinetics of Sustained-Release Buprenorphine and Extended-Release Buprenorphine in Mice with Surgical Catheterization

abstract: The Guide for the Care and Use of Laboratory Animals strongly encourages the use of pharmaceutical grade chemicals and analgesics. The extra-label use of sustained-release buprenorphine (SRB) is commonly administered to rodents to mitigate moderate-to-severe pain. An FDA-indexed buprenorphine formulation, known as extended-release buprenorphine (XRB), has recently become available and is currently the only pharmaceutical grade slow-release buprenorphine approved for use in mice and rats. However, no studies have directly compared the pharmacokinetic (PK) parameters and therapeutic efficacy of SRB and XRB in surgically catheterized mice. Thus, we compared the plasma buprenorphine concentrations and PK parameters of SRB and XRB in mice after surgical catheterization. We hypothesized that mice treated with SRB or XRB would have circulating buprenorphine concentrations exceeding the therapeutic threshold for up to 72-hours post-operatively. Male and female C57Bl/6J mice were anesthetized, treated with either SRB (1 mg/kg, SC, once) or XRB (3.25 mg/kg, SC, once) and underwent surgical catheterization. At 6, 24, 48, and 72 h after SRB or XRB administration, arterial blood samples were collected. Post-operative weight loss was similar between groups with a decline of 11.7 {+/-} 1.6 and 12.3 {+/-} 0.7% in males and 7.6 {+/-} 2.2 and 8.1 {+/-} 1.1% (mean {+/-} SEM) in females treated with SRB and XRB, respectively. Both SRB and XRB maintained circulating buprenorphine concentrations above the therapeutic level of 1.0 ng/mL for 72 h after administration. XRB buprenorphine concentrations were significantly greater (3-4-fold) than SRB concentrations at 6, 24, and 48 h, commensurate with the increase dose concentration of XRB to SRB. These results support the use of either SRB or XRB for the alleviation of postoperative pain in mice. The new availability of FDA-indexed XRB increases the options for safe and effective pharmaceutical grade analgesia in rodents.

category: pharmacology and toxicology


title: Does ankle push-off correct for errors in anterior-posterior foot placement relative to center-of-mass state?

abstract: I.Understanding the mechanisms humans use to stabilize walking is vital for predicting falls in elderly. Modelling studies identified two of them: foot placement control and push-off control. Foot placement control relies on center-of-mass (CoM) state, by stepping in the direction of CoM movement, which has been found in human gait. Push-off control has been suggested to modulate the deviations of CoM trajectories from the desired trajectories in perturbed human walking, but it is unknown whether this mechanism is also used in steady-state (unperturbed) walking. To test this, we considered the covariance between CoM state and anterior-posterior foot placement as a measure of foot placement accuracy and investigated whether the difference between the actual and the ideal foot placement based on the CoM state at heel strike may serve as a feedback signal for push-off control. We found the (ankle) push-off torque to be correlated to the foot placement error in 30 participants when walking at normal and slow speed, with mean correlation values of up to 0.45. Our study hence suggests that humans use a push-off strategy for correcting foot placement errors in steady-state walking.

category: physiology


title: Adenosine A1R/A3R Agonist AST-004 Reduces Brain Infarction in Mouse and Rat Models of Acute Ischemic Stroke

abstract: Acute ischemic stroke (AIS) is the second leading cause of death globally. No Food and Drug Administration (FDA) approved therapies exist targeting cerebroprotection following stroke. Our group recently reported significant cerebroprotection with the adenosine A1/A3 receptor agonist, AST-004, in a transient stroke model in non-human primates (NHP) and in a preclinical mouse model of traumatic brain injury (TBI). However, the specific receptor pathway activated was only inferred based on in vitro binding studies. The current study investigated the underlying mechanism of AST-004 cerebroprotection in two independent models of AIS: permanent photothrombotic stroke in mice and transient middle cerebral artery occlusion (MCAO) in rats. AST-004 treatments across a range of doses were cerebroprotective and efficacy could be blocked by A3R antagonism, indicating a mechanism of action that does not require A1R agonism. The high affinity A3R agonist MRS5698 was also cerebroprotective following stroke, but not the A3R agonist Cl-IB-MECA under our experimental conditions. AST-004 efficacy was blocked by the astrocyte specific mitochondrial toxin fluoroacetate, confirming an underlying mechanism of cerebroprotection dependent on astrocyte mitochondrial metabolism. An increase in A3R mRNA levels following stroke suggested an intrinsic cerebroprotective response that was mediated by A3R signaling. Together, these studies confirm certain A3R agonists, such as AST-004, are promising new therapeutics for the treatment of AIS.

category: neuroscience


title: Mixture of Learning Strategies Underlies Rodent Behavior in Dynamic Foraging

abstract: In volatile foraging environments, animals need to adapt their learning in accordance with the uncertainty of the environment and knowledge of the hidden structure of the world. In these contexts, previous studies have distinguished between two types of strategies, model-free learning, where reward values are updated locally based on external feedback signals, and inference-based learning, where an internal model of the world is used to make optimal inferences about the current state of the environment. Distinguishing between these strategies during the dynamic foraging behavioral paradigm has been a challenging problem for studies of reward-guided decisions, due to the diversity in behavior of model-free and inference-based agents, as well as the complexities that arise when animals mix between these types of strategies. Here, we developed two solutions that jointly tackle these problems. First, we identified four key behavioral features that together benchmark the switching dynamics of agents in response to a change in reward contingency. We performed computational simulations to systematically measure these features for a large ensemble of model-free and inference-based agents, uncovering an organized structure of behavioral choices where observed behavior can be reliably classified into one of six distinct regimes in the two respective parameter spaces. Second, to address the challenge that arises when animals use multiple strategies within single sessions, we developed a novel state-space method, block Hidden Markov Model (blockHMM), to infer switches in discrete latent states that govern the choice sequences across blocks of trials. Our results revealed a remarkable degree of mixing between different strategies even in expert animals, such that model-free and inference-based learning modes often co-existed within single sessions. Together, these results invite a re-evaluation of the stationarity of behavior during dynamic foraging, provide a comprehensive set of tools to characterize the evolution of learning strategies, and form the basis of understanding neural circuits involved in different modes of behavior within this domain.


title: The Influence of Neural activity and Neural Cytoarchitecture on Cerebrovascular Arborization: A Computational Model

abstract:
Normal functioning of the brain relies on a continual and efficient delivery of energy by a vast network of cerebral blood vessels. The bidirectional coupling between neurons and blood vessels consists of vasodilatory energy demand signals from neurons to blood vessels, and the retrograde flow of energy substrates from the vessels to neurons, which fuel neural firing, growth and other housekeeping activities in the neurons. Recent works indicate that, in addition to the functional coupling observed in the adult brain, the interdependence between the neural and vascular networks begins at the embryonic stage, and continues into subsequent developmental stages. The proposed Vascular Arborization Model (VAM) captures the effect of neural cytoarchitecture and neural activity on vascular arborization.

The VAM describes three important stages of vascular tree growth: (i) The prenatal growth phase, where the vascular arborization depends on the cytoarchitecture of neurons and non-neural cells, (ii) the post-natal growth phase during which the further arborization of the vasculature depends on neural activity in addition to neural cytoarchitecture, and (iii) the settling phase, where the fully grown vascular tree repositions its vascular branch points or nodes to ensure minimum path length and wire length. The vasculature growth depicted by VAM captures structural characteristics like vascular volume density, radii, mean distance to proximal neurons in the cortex. VAM-grown vasculature agrees with the experimental observation that the neural densities do not covary with the vascular density along the depth of the cortex but predicts a high correlation between neural areal density and microvascular density when compared over a global scale (across animals and regions).

To explore the influence of neural activity on vascular arborization, the VAM was used to grow the vasculature in neonatal rat whisker barrel cortex under two conditions: (i) Control, where the whiskers were intact and (ii) Lesioned, where one row of whiskers was cauterized. The model captures a significant reduction in vascular branch density in lesioned animals compared to control animals, concurring with experimental observation.


title: Selective V1-to-V4 communication of attended stimuli mediated by attentional effects in V1

abstract: Selective attention implements preferential routing of attended stimuli, likely through increasing the influence of the respective synaptic inputs on higher-area neurons. As the inputs of competing stimuli converge onto postsynaptic neurons, presynaptic circuits might offer the best target for attentional top-down influences. If those influences enabled presynaptic circuits to selectively entrain postsynaptic neurons, this might lead to selective routing. Indeed, when two visual stimuli induce two gamma rhythms in V1, only the gamma induced by the attended stimulus entrains gamma in V4. Here, we modeled this selective entrainment with a Dynamic Causal Model for Cross-Spectral Densities and found that it can be explained by attentional modulation of intrinsic V1 connections. Specifically, local inhibition was decreased in the granular input layer and increased in the supragranular output layer of the V1 circuit that processed the attended stimulus. Thus, presynaptic attentional influences and ensuing entrainment were sufficient to mediate selective routing.


title: Brain Capillary Pericytes are Metabolic Sentinels that Control Blood Flow through KATP Channel Activity

abstract: Capillary pericytes and their processes cover [~]90% of the total length of the brains capillary bed. Despite their abundance, little is known of pericyte function, and their contributions to the control of brain hemodynamics remain unclear. Here, we report that deep capillary pericytes possess a mechanistic  energy switch that, when activated by a decrease in glucose, elicits robust KATP channel activation to increase blood flow and protect energy substrate availability. We demonstrate that pharmacological activation of KATP channels profoundly hyperpolarizes capillary pericytes and leads to dilation of upstream penetrating arterioles and arteriole-proximate capillaries covered with contractile pericytes, leading to an increase in local capillary blood flow. Stimulation of a single capillary pericyte with a KATP channel agonist is sufficient to evoke this response, which is mediated via KIR channel-dependent retrograde propagation of hyperpolarizing electrical signals. Genetic inactivation of pericyte KATP channels via expression of a dominant-negative version of KIR6.1 eliminates these effects. Critically, we show that lowering extracellular glucose below 1 mM evokes dramatic KATP channel-mediated pericyte hyperpolarization. Inhibiting glucose uptake by blocking GLUT1 transporters in vivo also activates this energy switch to increase pericyte KATP channel activity, dilate arterioles and increase blood flow. Together, our findings recast capillary pericytes as metabolic sentinels that respond to local energy deficits by robustly increasing blood flow to protect metabolic substrate delivery to neurons and prevent energetic shortfalls.


title: Spontaneous myocarditis in mice predisposed to autoimmune disease: Including Vaccination-induced onset

abstract: Non-obese diabetic (NOD)/ShiLtJ mice, like biobreeding rats, are used as an animal model for type 1 diabetes. Diabetes develops in NOD mice as a result of insulitis, a leukocytic infiltrate of the pancreatic islets. The onset of diabetes is associated with a moderate glycosuria and a non-fasting hyperglycemia. Previously, in non-obese diabetic (NOD)/ShiLtJ mice spontaneously developing type 1 diabetes, the possible involvement of decreased expression of LMP2/{beta}1i, an immunoproteasome {beta} subunit, and associated decreased expression of NF-{kappa}B1 (also called as p50) in the development of type 1 diabetes was argued between our research team and other research groups. In response to these arguments, we created NOD mice in which NF-{kappa}B1 expression is not consistently observed. Unexpectedly, most NOD Nf{kappa}b1 homozygote mice were found to die by the 8th week of age due to the development of severe myocarditis. Furthermore, in all NOD Nf{kappa}b1 heterozygote mice, the onset of Insulitis was observed from 4 months of age. In addition, in NOD Nf{kappa}b1 heterozygote mice, an increase in cTnT due to vaccination with influenza or HBV vaccine was observed without gender difference. Now, we found a direct involvement of decreased expression of NF-{kappa}B1 in the development of autoimmune diseases in NOD/ShiLtJ mice. Therefore, we would like to introduce new research results on autoimmune diseases, including findings on important risk factors for the development of myocarditis observed after vaccination with mRNA-based COVID-19 vaccine.

category: immunology


title: A novel method accounting for predictor uncertainty and model transferability of invasive species distribution models

abstract:
Predicting novel ranges of non-native species is a critical component to understanding the biosecurity threat posed by pests and diseases on economic, environmental and social assets. Species distribution models (SDMs) are often employed to predict the potential ranges of exotic pests and diseases in novel environments and geographic space. To date, researchers have focused on model complexity, data available for model fitting, the size of the geographic area to be considered and how the choice of model impacts results. These investigations are coupled with considerable examination of how model evaluation methods and test scores are influenced by these choices. An area that remains under-discussed is how to account for uncertainty in predictor selection while also selecting variables that increase a models ability to predict to novel environments (model transferability).

Here we propose a novel method to finesse this problem by using multiple simple (bivariate) models to search for the candidate sets of predictor variables that are likely to produce transferable models. Once identified, each set is then used to construct 2-dimensional niche envelopes of pest presence/absence. This process ultimately results in a number of possible models that can be used to predict pest potential distributions, however, rather than relying on a single model, we ensemble these models in an attempt to account for predictor uncertainty. We apply this method to both virtual species and real species data, and find that it generally performs well against conventional approaches for statistically fitting numerous variables in a single model. While our methods only consider simple ecological relationships of species to environmental predictors, they allow for increased model transferability because they reduce the likelihood of over-fitting and collinearity issues. Simple models are also likely to be more conservative (over-predict potential distributions) relative to complex models containing many covariates - making them more appropriate for risk-averse applications such as biosecurity. The approach we have explored transforms a model selection problem, for which there is no true correct answer amongst the typically distal covariates on offer, to one of model uncertainty. We argue that increased model transferability at the expense of model interpretation is perhaps more important for effective rapid predictions and management of non-native species and biological invasions.

category: ecology


title: Functional Assays Reclassify Suspected Splice-Altering Variants of Uncertain Significance in Mendelian Channelopathies

abstract:
BackgroundRare protein-altering variants in SCN5A, KCNQ1, and KCNH2 are major causes of Brugada Syndrome (BrS) and the congenital Long QT Syndrome (LQTS). While splice-altering variants lying outside 2-bp canonical splice sites can cause these diseases, their role remains poorly described.

ObjectiveWe implemented two functional assays to assess 12 recently reported putative splice-altering variants of uncertain significance (VUS) and 1 likely pathogenic (LP) variant without functional data observed in BrS and LQTS probands.

MethodsWe deployed minigene assays to assess the splicing consequences of 10 variants. Three variants incompatible with the minigene approach were introduced into control induced pluripotent stem cells (iPSCs) by CRISPR genome editing. We differentiated cells into iPSC-derived cardiomyocytes (iPSC-CMs) and studied splicing outcomes by reverse transcription-polymerase chain reaction (RT-PCR). We used the American College of Medical Genetics and Genomics functional assay criteria (PS3/BS3) to reclassify variants.

ResultsWe identified aberrant splicing, with presumed disruption of protein sequence, in 8/10 variants studied using the minigene assay and 1/3 studied in iPSC-CMs. We reclassified 9 VUS to LP, 1 VUS to Likely Benign, and 1 LP variant to pathogenic.

ConclusionsFunctional assays reclassified splice-altering variants outside canonical splice sites in BrS- and LQTS-associated genes.


title: A comprehensive dimensional reduction framework to learn single-cell phenotypic topology uncovers T cell diversity

abstract: The core task when analyzing single-cell data (SCD) is dimensional reduction (DR), which aims to find latent signals encoding biological heterogeneity. Here, we dissected DR steps to build TopOMetry, a machine learning framework that learns latent data topology to perform DR in a modular fashion and show that current analysis practices are biased due to the non-uniformity and non-linearity of the geometry underlying SCD. We used TopOMetry to analyze SCD from peripheral blood mononuclear cells (PBMC), and consistently found a plethora of unreported T CD4 subpopulations that are missed by current workflows across datasets and diseases. We also found comparable T CD4 diversity in the human cerebrospinal fluid. The proposed framework scalates to millions of cells, excels at discovering fine-grained local structure, and holds natural connections to clustering and trajectory inference. We hope this powerful tool will accelerate biomedical discovery and inspire new methods to learn and explore phenotypic topology.


title: SIMMAM 3.0 - Updating the Toolbox for the Conservation of Marine Mammals

abstract: SIMMAM (Sistema de Apoio ao Monitoramento de Mamiferos Marinhos) has been available for marine mammal researchers and environmental agencies in Brazil since 2005. However, it had to be updated, to adapt to new standards and data sharing policies. SIMMAM 3.0 uses more modern technologies (PHP 7.4, Symfony 5.x, PostgreSQL 11.x, PostGIS 3.x, Leaflet 1.7) and is compliant to DarwinCore (DwC) standard and able to exchange data with other data portals that use it. Its database now holds more than 44000 marine mammal records and is an important tool in the decision-making process of Brazilian environmental agencies.


title: Photons guided by axons may enable backpropagation-based learning in the brain

abstract: Despite great advances in explaining synaptic plasticity and neuron function, a complete understanding of the brains learning algorithms is still missing. Artificial neural networks provide a powerful learning paradigm through the backpropagation algorithm which modifies synaptic weights by using feedback connections. Backpropagation requires extensive communication of information back through the layers of a network. This has been argued to be biologically implausible and it is not clear whether backpropagation can be realized in the brain. Here we suggest that biophotons guided by axons provide a potential channel for backward transmission of information in the brain. Biophotons have been experimentally shown to be produced in the brain, yet their purpose is not understood. We propose that biophotons can propagate from each post-synaptic neuron to its pre-synaptic one to carry the required information backward. To reflect the stochastic character of biophoton emissions, our model includes the stochastic backward transmission of teaching signals. We demonstrate that a three-layered network of neurons can learn the MNIST handwritten digit classification task using our proposed backpropagation-like algorithm with stochastic photonic feedback. We model realistic restrictions and show that our system still learns the task for low rates of biophoton emission, information-limited (one bit per photon) backward transmission, and in the presence of noise photons. Our results suggest a new functionality for biophotons and provide an alternate mechanism for backward transmission in the brain.


title: Angiotensin blockade enhances motivational reward learning via enhancing ventral striatal prediction error and frontostriatal communication

abstract: Adaptive human learning utilizes reward prediction errors (RPEs) that scale the differences between expected and actual outcomes to optimize future choices. Depression has been linked with biased RPE signaling and an exaggerated impact of negative outcomes on learning which may promote amotivation and anhedonia. The present proof-of-concept study combined computational modelling and multivariate decoding with neuroimaging to determine the influence of the angiotensin II type 1 receptor antagonist losartan on learning from positive or negative outcomes and the underlying neural mechanisms in healthy humans. In a double-blind, between-subjects, placebo-controlled pharmaco-fMRI experiment, 61 healthy male participants (losartan, n=30; placebo, n=31) underwent a probabilistic selection reinforcement learning task incorporating a learning and transfer phase. Losartan improved choice accuracy for the hardest stimulus pair relative to the placebo group during learning. Computational modelling revealed that losartan reduced the learning rate for negative outcomes and increased exploitatory choice behaviors while preserving learning for positive outcomes. These behavioral patterns were paralleled on the neural level by increased RPE signaling in orbitofrontal-striatal regions and enhanced positive outcome representations in the ventral striatum (VS) following losartan. In the transfer phase, losartan accelerated response times and enhanced VS functional connectivity with left dorsolateral prefrontal cortex when approaching maximum rewards. These findings elucidate the potential of losartan to reduce the impact of negative outcomes during learning and subsequently facilitate motivational approach towards maximum rewards in the transfer of learning. The mechanism could help to normalize biased reward learning characteristic of depression.


title: Glassy phase in dynamically-balanced neural networks

abstract: We present a novel mean-field theory for balanced neuronal networks with arbitrary levels of symmetry in the synaptic connectivity. The theory determines the fixed point of the network dynamics and the conditions for its stability. The fixed point becomes unstable by increasing the synaptic gain beyond a critical value that depends on the level of symmetry. Beyond this critical gain, for positive levels of symmetry, we find a previously unreported phase. In this phase, the dynamical landscape is dominated by a large number of marginally-stable fixed points. As a result, the network dynamics exhibit non-exponential relaxation and ergodicity is broken. We discuss the relevance of such a glassy phase for understanding dynamical and computational aspects of cortical operation.


title: Generation and proof-of-concept for allogeneic CD123 CAR-Delta One T (DOT) cells in Acute Myeloid Leukemia

abstract:
Chimeric Antigen Receptor (CAR)-T cells have emerged as a breakthrough treatment for relapse/refractory (r/r) hematological tumors, showing impressive complete remission rates in B-cell malignancies. However, around 50% of the patients relapse before 1-year post-treatment. T-cell "fitness" is critical to prolong the persistence and activity of the adoptively transferred product. Allogeneic T cells from healthy donors are less dysfunctional or exhausted than autologous patient-derived T cells, enabling a very attractive and cost-effective "off-the-shelf" therapy option. In this context, Delta One T cells (DOTs), a recently described cellular product based on MHC/HLA-independent V{delta}1+ {gamma}{delta} T cells generated from the peripheral blood of healthy donors, represent a robust platform of allogeneic effector T cells. Here we generated and pre-clinically validated 4-1BB-based CAR-DOTs directed against the IL-3 chain receptor (CD123), a target antigen widely expressed on acute myeloid leukemia (AML) blasts. CD123CAR-DOTs showed vigorous, superior to control DOTs, cytotoxicity against AML cell lines and primary samples both in vitro and in vivo. Continuous administration of IL-15 supported the long-term persistence of a single-dose CD123CAR-DOTs in patient-derived xenograft models, sustaining their anti-leukemic efficacy as demonstrated in a re-challenge assay in vivo. Our results provide proof-of-concept for an allogeneic next-generation therapy based on CD123CAR-DOTs for r/r AML patients.

KEY POINTS- CD123CAR-DOTs exert specific and robust cytotoxicity in vitro and anti-leukemic activity in vivo against AML cell lines and primary cells.
- CD123CAR-DOTs show IL-15-dependent long-term persistence in vivo and vigorous anti-leukemic activity upon re-challenge.


title: SLC15A4 favors inflammasome function via mTORC1 signaling and autophagy restraint in dendritic cells

abstract: Phagocytosis is the first step in the assessment of foreign microbes or particles and enables activation of innate immune pathways such as the inflammasome. However, missing links between phagosomes and inflammasomes remain to be discovered. We show that in murine dendritic cells (DCs) the lysosomal histidine/peptide solute carrier transporter SLC15A4, associated with human inflammatory disorders, is recruited to phagosomes and is required for optimal inflammasome activity after infectious or sterile stimuli. Dextran sodium sulphate-treated SLC15A4-deficient mice exhibit decreased colon inflammation, reduced IL-1{beta} production by intestinal DCs and increased autophagy. Similarly, SLC15A4-deficient DCs infected with Salmonella typhimurium show reduced caspase-1 cleavage and IL-1{beta} production. This correlates with peripheral NLRC4 inflammasome assembly and increased autophagy. Overexpression of constitutively active mTORC1 rescues decreased IL-1{beta} levels and caspase-1 cleavage, and restores perinuclear inflammasome positioning. Our findings suggest that SLC15A4 is a novel link that couples phagocytosis with inflammasome perinuclear assembly and inhibition of autophagy through phagosomal content sensing. Our data also reveal the previously unappreciated importance of mTORC1 signaling pathways to promote and sustain inflammasome activity.


title: Mutation-Attention (MuAt): deep representation learning of somatic mutations for tumour typing and subtyping

abstract: Cancer genome sequencing enables accurate classification of tumours and tumour sub-types. However, prediction performance is still limited using exome-only sequencing and for tumor types with low somatic mutation burden such as many pediatric tumours. Moreover, the ability to leverage deep representation learning in discovery of tumour entities remains unknown. We introduce here Mutation-Attention (MuAt), a deep neural network to learn representations of simple and complex somatic alterations for prediction of tumour types and subtypes. MuAt achieved prediction accuracy of 89% for whole genomes (24 tumour types) and 64% for whole exomes (20 types), and a top-5 accuracy of 97% and 90%, respectively. Tumour representations learnt by MuAt included tumour entities such as acral melanoma, SHH-activated medulloblastoma, SPOP-associated prostate cancer, microsatellite instability, and MUTYH-associated pancreatic endocrine tumours although these tumour subtypes and subgroups were not used as training labels. Integrated representations of somatic alterations hold significant potential to drive discovery of novel tumour entities and clinical application.

category: genomics


title: Mapping Multiple Factors Mediated Chromatin Interactions to Assess Regulatory Network and Dysregulation of Lung Cancer-Related Genes

abstract: Studies on the lung cancer genome are indispensable for developing a cure for lung cancer. Whole-genome resequencing, genome-wide association studies, and transcriptome sequencing have greatly improved our understanding of the cancer genome. However, dysregulation of long-range chromatin interactions in lung cancer remains poorly described. To better understand the three-dimensional (3D) genomic interaction features of the lung cancer genome, we used the A549 cell line as a model system. The generated high-resolution data revealed chromatin interactions associated with RNA polymerase II (RNAPII), CCCTC-binding factor (CTCF), enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), and histone 3 lysine 27 trimethylation (H3K27me3) using specific antibodies and long-read chromatin interaction analysis by paired-end tag sequencing (ChIA-PET). The EZH2/H3K27me3-mediated interactions further silenced target genes, either through loops or domains, and showed distributions along the genome distinct from and complementary to those associated with RNAPII. We found that cancer-related genes were highly enriched in chromatin interactions. We identified abnormal interactions associated with oncogenes and tumor suppressors, such as additional repressive interactions on FOXO4 and promoter - promoter interactions between NF1 and RNF135. Knockout of abnormal interactions reversed the dysregulation of cancer-related genes, suggesting that chromatin interactions are essential for proper expression of lung cancer-related genes. These findings demonstrate the 3D landscape and gene regulatory relationships of the lung cancer genome.


title: Limited effects of age on the use of the ankle and counter-rotation mechanism in the sagittal plane

abstract: Two mechanisms can be used to accelerate the center of mass (CoM) to control the CoM in relation to the base of support during standing. The first is applying ankle moments to shift the center of pressure (CoP), which has been coined the "CoP mechanism". The second is changing the angular momentum around the CoM to change the direction of the ground reaction force, i.e., the "counter-rotation mechanism". At both the beginning and the end of the lifespan, problems with postural control are common. In this study, we asessed anteroposterior balance performance and the related use of these postural control mechanisms in children, younger adults, and older adults. Sixteen pre-pubertal children (6-9y), 17 younger adults (18-24y) and eight older adults (65-80y) performed bipedal upright standing trials of 16 seconds on a rigid surface and on three balance boards that could freely move in the sagittal plane, varying in height (15-19 cm) of the board above the point of contact with the floor. Full body kinematics were measured. Performance related outcome measures, i.e., the number of trials with balance loss and the Root Mean Square (RMS) of the time series of the CoM acceleration were calculated. Additionally, the RMS of the time series of the CoM acceleration due to the CoP and counter-rotation mechanism and the contributions of the CoP and the counter-rotation mechanism to the CoM acceleration (in %) in the sagittal plane were calculated. Furthermore, selected kinematic measures, i.e., the orientation of the board and the head and the Mean Power Frequency of balance board orientation and of CoM acceleration were calculated. Compared to younger adults, children and older adults showed a poorer balance performance, reflected by a greater RMS of CoM accelerations and more balance loss in older adults. Across age groups and conditions, the contribution of the CoP mechanism to the total CoM acceleration was dominant, i.e., 95%-108%. The contribution of the counter-rotation mechanism ranged between 19%-31% (with totals higher than 100% indicating opposite effects of both mechanisms). We suggest that the contribution of the counter-rotation mechanism is limited, since the counter-rotation mechanism would conflict with stabilizing the orientation of the head in space. Furthermore, children did use the counter-rotation mechanism relatively more to accelerate the CoM compared to younger adults. Possibly this reflects that they are still learning to limit the contribution of the counter-rotation mechanism to the same extent as adults.

category: bioengineering


title: Probabilistic Brain Extraction in MR Images via Conditional Generative Adversarial Networks

abstract: Brain extraction, which refers to the task of segmenting brain tissue in an MR image of a subject, forms an essential first step for many quantitative neuroimaging applications. These include quantifying grey and white matter volumes, monitoring neurological diseases like multiple sclerosis (MS) and Alzheimers disease, and estimating brain atrophy. Over the years several algorithms that automate brain extraction have been proposed. More recently, novel image-to-image deep learning methods have been implemented for this task, and have demonstrated significant gains in accuracy and robustness. However, to our knowledge, none of these algorithms account for the uncertainty that is inherent in brain extraction. Motivated by this, we propose a novel, probabilistic deep learning algorithm for brain extraction that recasts this task as a Bayesian inference problem, and then utilizes a conditional generative adversarial network (cGAN) to solve it. The input to the generator network is an MR image of the head and the output is a collection of images of the brain that are drawn from a probability density conditioned on the input image. These images are used to generate a pixel-wise mean image, which serves as the best guess for an image of the brain, and a pixel-wise standard deviation image, which quantifies the uncertainty in the prediction. We test this algorithm on head MR images of fifty subjects and demonstrate that it is more accurate than a commonly used brain extraction tool, and that its performance compares well with the current state of the art in deep learning algorithms. We also demonstrate the utility of the estimates of uncertainty generated by the algorithm.


title: Fluximplied: A novel approach integrates rate limiting steps and differential expression for pathway analysis

abstract:
BackgroundMany tools exist to perform a rigorous pathway analysis, though traditional gene set enrichment analysis remains among the most common. While useful for many applications, one common situation where it is less so is the metabolic profiling of bulk omics datasets. Rate limiting steps in more linear pathways are the main determinant of flux through these pathways, but differential expression of the enzymes that catalyze these steps is usually not differentially weighted in pathway analysis. Fluximplied was built to perform pathway analysis with rate limiting steps in mind to assess the implied flux through a number of well validated metabolic pathways.

ResultsA database of rate limiting steps and their associated pathway was constructed. Using publicly available human RNA sequencing data from liver, putamen, and adipose tissue, fluximplied generally corroborated the pathway analysis. When comparing two CD8 T cell subsets from mouse lung, our algorithm confirmed previous findings that were not found previously.

ConclusionFluximplied is an accessible tool for pathway analysis which is intended to assist the user with hypothesis generation. Unlike traditional approaches to pathway analysis, it specifically queries a database of rate limiting steps in order to infer flux through canonical metabolic pathways.


title: UniFuncNet: a flexible network annotation framework

abstract:
SummaryFunctional annotation is an integral part in the analysis of organisms, as well as of multi-species communities. A common way to integrate such information is using biological networks. However, current data integration network tools are heavily dependent on a single source of information, which might strongly limit the amount of relevant data contained within the network. Here we present UniFuncNet, a network annotation framework that dynamically integrates data from multiple biological databases, thereby enabling data collection from various sources based on user preference. This results in a flexible and comprehensive data retrieval framework for network based analyses of omics data. Importantly, UniFuncNets data integration methodology allows for the output of a non-redundant composite network and associated metadata. In addition, a workflow exporting UniFuncNets output to the graph database management system Neo4j was implemented, which allows for efficient querying and analysis.

AvailabilitySource code is available at https://github.com/PedroMTQ/UniFuncNet.


title: Non-ribosomal peptide synthetase domain boundary identification and new motifs discovery based on motif-intermotifs standardized architecture

abstract: Non-ribosomal peptide synthetase (NRPS) is a diverse family of biosynthetic enzymes for the assembly of bioactive peptides. Its repetitive modular structure inspired many reengineering efforts. Despite the continued expansion of microbial sequencing data, varied standards for annotating domains and modules make data-driven discoveries in NRPSs challenging. In this work, we presented a motif-and-intermotif architecture of NRPSs by partitioning the condensation (C), adenylation (A), thiolation (T) and thioesterase (TE) domains. This "standardization" enables systematic evaluation of functional couplings from a large number of NRPS sequences, resulting in the discovery of novel motifs such as glycine (G)-motif with functional implications for structural flexibility. As a proof of concept, the function of G-motif in controlling the activity of NRPS was verified by mutation experiments in an antitumor fumiquinazoline pathway. Additionally, coevolution studies clarify significant barriers associated with reengineering NRPSs, and comparison of sequences between species reveals interactions between phylogeny and substrate specificity. Our findings give an in-depth analysis and statistical insights into the sequencing characteristics of NRPSs, paving the path for future data-driven discoveries.


title: The identification of two newly discovered fluorescent proteins in human glioblastoma

abstract: Glioblastoma (GBM) is a common malignancy featured by an extremely strong proliferation with enriched genetic information. Due to the rapid proliferation and the unstable genome-induced evolutionary potential, unique proteins may be expressed in GBM cells under the certain influence of the microenvironment. We therefore speculated that fluorescent proteins exist in GBM cells. During the immunofluorescence staining assay, we accidently discovered autofluorescence in primary GBM cells without fluorescent labeling, which were further validated as 2 newly discovered fluorescent proteins excited by 467 nm and 378 nm wavelength, respectively, namely human fluorescent protein and (HFP1, HFP2). Fluorescence colocalization and fluorescence resonance energy transfer (FRET) results showed the tight interaction of HFP1 and HFP2, and their synergistic effect. Our results for the first time identified 2 newly discovered fluorescent proteins in GBM cells, and clarified their chemical properties.

category: cancer biology


title: Heterogeneous multimeric metabolite ion species observed in LC-MS based metabolomics data sets

abstract: Covalent or non-covalent heterogeneous multimerization of molecules associated with extracts from biological samples analyzed via LC-MS is quite difficult to recognize/annotate and therefore the prevalence of multimerization remains largely unknown. In this study, we utilized 13C labeled and unlabeled Pichia pastoris extracts to recognize heterogeneous multimers. More specifically, between 0.8% and 1.5% of the biologically-derived features detected in our experiments were confirmed to be heteromers, about half of which we could successfully annotate with monomeric partners. Interestingly, we found specific chemical classes such as nucleotides to disproportionately contribute to heteroadducts. Furthermore, we compiled these compounds into the first MS/MS library that included data from heteromultimers to provide a starting point for other labs to improve the annotation of such ions in other metabolomics data sets. Then, the detected heteromers were also searched in publicly accessible LC-MS datasets available in Metabolights, Metabolomics WB, and GNPS/MassIVE to demonstrate that these newly annotated ions are also relevant to other public datasets. Furthermore, in additional datasets (Triticum aestivum, Fusarium graminearum, and Trichoderma reesei) our developed workflow also detected 0.5% to 4.9% of metabolite features to originate from heterodimers, demonstrating heteroadducts to be present in metabolomics studies at a low percentage.

category: systems biology


title: Model-guided design of the diversity of a synthetic human gut community

abstract: Microbial communities have tremendous potential as therapeutics. However, a major bottleneck is manufacturing high-diversity microbial communities with desired species compositions. We develop a two-stage, model-guided framework to produce microbial communities with target species compositions. We apply this method to optimize the diversity of a synthetic human gut community. The first stage exploits media components to enable uniform growth responses of individual species and the second stage uses a design-test-learn cycle with initial species abundance as a control point to manipulate community composition. Our designed culture conditions yield 91% of the maximum possible diversity. Leveraging these data, we construct a dynamic ecological model to guide the design of lower-order communities with desired temporal properties over a longer timescale. In sum, a deeper understanding of how microbial community assembly responds to changes in environmental factors, initial species abundances, and inter-species interactions can enable the predictable design of community dynamics.


title: Glucagon-like peptide-1 receptor differentially controls mossy cell activity across the dentate gyrus longitudinal axis

abstract: Understanding the role of dentate gyrus (DG) mossy cells (MCs) in learning and memory has rapidly evolved due to increasingly precise methods for targeting MCs and for in vivo recording and activity manipulation in rodents. These studies have shown MCs are highly active in vivo, strongly remap to contextual manipulation, and that their inhibition or hyperactivation impairs pattern separation and location or context discrimination. What is not well understood is how MC activity is modulated by neurohormonal mechanisms, which might differentially control the participation of MCs in cognitive functions during discrete states, such as hunger or satiety. In this study, we demonstrate that glucagon-like peptide-1 (GLP-1), a neuropeptide produced in the gut and the brain that regulates food consumption and hippocampal-dependent mnemonic function, might regulate MC function through selective expression of its receptor, GLP-1R. RNA-seq demonstrated that most Glp1r in hippocampal principal neurons is expressed in MCs, and in situ hybridization revealed strong expression of Glp1r in hilar neurons. Glp1r-ires-Cre mice crossed with Ai14D reporter mice followed by co-labeling for the MC marker GluR2/3 revealed that almost all MCs in the ventral DG expressed Glp1r and that almost all Glp1r-expressing hilar neurons were MCs. However, only ~60% of dorsal DG MCs expressed Glp1r, and Glp1r was also expressed in small hilar neurons that were not MCs. Consistent with this expression pattern, peripheral administration of the GLP-1R agonist exendin-4 (5 g/kg) increased cFos expression in ventral but not dorsal DG hilar neurons. Finally, whole-cell patch-clamp recordings from ventral MCs showed that bath application of exendin-4 (200 nM) depolarized MCs and increased action potential firing. Taken together, this study identifies a potential neurohormonal mechanism linking a critically important satiety signal with activity of MCs in the ventral DG that might have functional effects on learning and memory during distinct states.


title: Microarray profiling of hypothalamic gene expression changes in Huntington's disease mouse models

abstract: Structural changes and neuropathology in the hypothalamus have been suggested to contribute to the non-motor manifestations of Huntingtons disease (HD), a neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene. In the present study, we investigated whether transcriptional changes would be part of hypothalamic pathology induced by the disease-causing huntingtin (HTT) protein. We performed microarray analysis using the Affymetrix platform on total hypothalamic RNA isolated from two HD mouse models and their littermate controls; BACHD mice with ubiquitous expression of full-length mutant HTT (mHTT) and wild-type mice with targeted hypothalamic overexpression of either wild-type HTT (wtHTT) or mHTT fragments. To analyze microarray datasets (34760 variables) and obtain functional implications of differential expression patterns, we used Linear Models for Microarray Data (limma) followed by Gene Set Enrichment Analysis (GSEA) using ClusterProfiler. Limma identified 735 and 721 significantly differentially expressed genes (adjusted p < 0.05) in hypothalamus of AAV datasets wtHTT vs control and mHTT vs control. In contrast, for BACHD datasets and the AAV mHTT vs. wtHTT dataset, none of the genes were differentially expressed (adjusted p-value > 0.05 for all probe IDs). In AAV groups, from the combined limma with GSEA using ClusterProfiler, we found both shared and unique gene sets and pathways for mice with wtHTT overexpression compared to mice with mHTT overexpression. mHTT caused widespread suppression of neuroendocrine networks, as evident by GSEA enrichment of GO-terms related to neurons and/or specific neuroendocrine populations. Using qRT-PCR, we confirmed that mHTT overexpression caused significant downregulation of key enzymes involved in neuropeptide synthesis, including histidine and dopa decarboxylases, compared to wtHTT overexpression. Multiple biosynthetic pathways such as sterol synthesis were among the top shared processes, where both unique and shared genes constituted leading-edge subsets. In conclusion, mice with targeted overexpression of HTT (wtHTT or mHTT) in the hypothalamus show dysregulation of pathways, of which there are subsets of shared pathways and pathways unique to either wtHTT or mHTT overexpression.


title: Optogenetic stimulation of medial septal glutamatergic neurons modulates theta-gamma coupling in the hippocampus

abstract:
The oscillatory activity of the hippocampus and parahippocampal structures encodes information during cognition. Multiple researchers emphasize hippocampal cross-frequency theta-gamma coupling (TGC) as a basic mechanism for information processing, retrieval, and consolidation of long-term and working memory. While the role of entorhinal afferents in the modulation of hippocampal TGC is widely accepted, the influence of other main input to the hippocampus, from the medial septal area (MSA, the pacemaker of the hippocampal theta rhythm) is poorly understood. This area innervates multiple targets in the hippocampus, so information about the participation of MSA in cross-frequency interactions might be critical for an understanding of neural coding.

Optogenetics allows us to explore how different neuronal populations of septohippocampal circuits control neuronal oscillations in vivo. It was shown that optogenetic activation of glutamatergic projections to the hippocampus with theta-frequency stimuli paces the hippocampal theta rhythm. Here we investigated the influence of phasic activation of MSA glutamatergic neurons expressing channelrhodopsin II on theta-gamma coupling in the hippocampus. Transgenic mice B6.Cg-Tg (Thy1-COP4/EYFP)18Gfng/J were used in this experiment. Expression of ChRII in MSA was verified immunohistochemically and the optic fibers just above MSA and the recording electrodes in the CA1 hippocampal field were implanted. During the experiment, local field potentials of MSA and hippocampus of freely behaving mice were modulated by 470nm light flashes with theta frequency (2-10) Hz.

It was shown that both the power and the strength of modulation of gamma rhythm nested on hippocampal theta waves depend on the frequency of stimulation. The modulation of the amplitude of slow gamma rhythm (30-50 Hz) prevailed over modulation of fast gamma (55-100 Hz) during flash trains and the observed effects were specific for theta stimulation of MSA. We discuss the possibility that phasic depolarization of septal glutamatergic neurons controls theta-gamma coupling in the hippocampus and plays a role in memory retrieval and consolidation.


title: Developmental phenomics suggests that H3K4 monomethylation catalyzed by Trr functions as a phenotypic capacitor

abstract:
How epigenetic modulators of gene regulation affect the development and evolution of animals has been difficult to ascertain. Despite the widespread presence of histone 3 lysine 4 monomethylation (H3K4me1) on enhancers, hypomethylation appears to have minor effects on animal development and viability. In this study, we performed quantitative, unbiased and multi-dimensional explorations of key phenotypes on Drosophila melanogaster with genetically induced hypomethylation. Hypomethylation reduced transcription factor enrichment in nuclear microenvironments, leading to reduced gene expression, and phenotypes outside of standard laboratory conditions. Our developmental phenomics survey further showed that H3K4me1 hypomethylation led to context-dependent changes in morphology, metabolism, and behavior. Therefore, H3K4me1 may contribute to phenotypic evolution as a phenotypic capacitor by buffering the effects of chance, genotypes and environmental conditions on transcriptional enhancers.

Quote"Developmental biologists are often not so much opposed to a role for ecology as they simply ignore it" -Doug Erwin1

category: developmental biology


title: Targeted deletion of uterine glandular Foxa2 induces embryonic diapause in mice

abstract: Embryonic diapause is a reproductive strategy in which embryo development and growth is temporarily arrested within the uterus to ensure the survival of neonates and mothers during unfavorable conditions. Pregnancy is reinitiated when conditions become favorable for neonatal survival. The mechanism of how the uterus enters diapause remains unclear. Mice with uterine deletion of Foxa2, a transcription factor, are infertile. In this study, we show that dormant blastocysts are recovered from these mice on day 8 of pregnancy with persistent uterine Msx1 expression, a gene critical to maintaining the uterine quiescent state, suggesting that these mice enter embryonic diapause. Leukemia inhibitory factor (LIF) can resume implantation in these mice. Although estrogen is critical for implantation in progesterone (P4)-primed uterus, our current model reveals that FOXA2-independent estrogenic effects are detrimental to sustaining uterine quiescence. Interestingly, P4 and anti-estrogen can prolong uterine quiescence in the absence of FOXA2. While we find that Msx1 expression persists in the uterus deficient in Foxa2, the complex relationship of FOXA2 with Msx genes and estrogen receptors remains to be explored.


title: Noninvasive stimulation of the ventromedial prefrontal cortex modulates rationality of human decision-making

abstract: The framing-effect is a bias that affects decision-making depending on whether the available options are presented with positive or negative connotations. Even when the outcome of two choices is equivalent, people have a strong tendency to avoid the negatively framed option because losses are perceived about twice as salient as gains of the same amount (i.e. loss-aversion). The ventromedial prefrontal cortex (vmPFC) is crucial for rational decision-making, and dysfunctions in this region have been linked to cognitive biases, impulsive behavior and gambling addiction. Using a financial decision-making task in combination with magnetoencephalographic neuroimaging, we show that excitatory compared to inhibitory non-invasive transcranial direct current stimulation (tDCS) of the vmPFC reduces framing-effects while improving the assessment of loss-probabilities, ultimately leading to increased overall gains. Behavioral and neural data consistently suggest that this improvement in rational decision-making is predominately a consequence of reduced loss-aversion. These findings recommend further research towards clinical applications of vmPFC-tDCS in addictive disorders.


title: The pyruvate dehydrogenase complex regulates mitophagic selectivity independently of its catalytic activity

abstract: The mitophagic degradation of mitochondrial matrix proteins is selective, reflecting a pre-engulfment sorting step. This selectivity is regulated through phosphorylation of mitochondrial matrix proteins by the matrix kinases Pkp1 and Pkp2, which in turn appear to be regulated by the phosphatase Aup1/Ptc6. However, these proteins also regulate the phosphorylation and catalytic activity of the yeast pyruvate dehydrogenase complex. To understand the relationship between these two functions, we tested the effect of deleting PDA1 on mitophagic selectivity. We report that pda1{Delta} cells show a mitophagy selectivity phenotype nearly identical to that of the pkp1{Delta} pkp2{Delta} double mutant. However, this is not due to a role for pyruvate dehydrogenase enzymatic activity in regulating mitophagy. Rather, our data suggest a novel mechanism wherein the pyruvate dehydrogenase complex directly regulates its cognate kinases and phosphatases, independent of its catalytic activity, to determine the phosphorylation state of mitochondrial matrix proteins in response to metabolic cues.

category: cell biology


title: Long-term T cell perturbations and waning antibody levels in individuals needing hospitalization for COVID-19

abstract: COVID-19 is being extensively studied, and much remains unknown regarding the long-term consequences of the disease on immune cells. The different arms of the immune system are interlinked, with humoral responses and the production of high-affinity antibodies being largely dependent on T cell immunity. Here, we longitudinally explored the effect COVID-19 has on T cell populations and the virus-specific T cells, as well as neutralizing antibody responses, for 6-7 months following hospitalization. The CD8+ TEMRA and exhausted CD57+CD8+ T cells were markedly affected with elevated levels that lasted long into convalescence. Further, markers associated with T-cell activation were upregulated at the inclusion, and in the case of CD69+CD4+ T cells this lasted all through the study duration. The levels of T cells expressing negative immune checkpoint molecules were increased in COVID-19 patients and sustained for a prolonged duration following recovery. Within 2-3 weeks after symptom onset, all COVID-19 patients developed anti-nucleocapsid IgG and spike-neutralizing IgG as well as SARS-CoV-2-specific T cell responses. In addition, we found alterations in follicular T helper (TFH) cell populations, such as enhanced TFH-TH2 following recovery from COVID-19. Our study revealed significant and long-term alterations in T cell populations and key events associated with COVID-19 pathogenesis.


title: The effect of waning on antibody levels and memory B cell recall following SARS-CoV-2 infection or vaccination

abstract: As of March 2022, there have been over 450 million reported SARS-CoV-2 cases worldwide, and more than 4 billion people have received their primary series of a COVID-19 vaccine. In order to longitudinally track SARS-CoV-2 antibody levels in people after vaccination or infection, a large-scale COVID-19 sero-surveillance progam entitled SPARTA (SeroPrevalence and Respiratory Tract Assessment) was established early in the pandemic. Anti-RBD antibody levels were tracked in more than 1,000 people. There was no significant decrease in antibody levels during the first 14 months after infection in unvaccinated participants, however, significant waning of antibody levels was observed following vaccination, regardless of previous infection status. Moreover, participants who were pre-immune to SARS-CoV-2 prior to vaccination seroconverted to significantly higher antibody levels, and antibodies were maintained at significantly higher levels than in previously infected, unvaccinated participants. This pattern was entirely due to differences in the magnitude of the initial seroconversion event, and the rate of antibody waning was not significantly different based on the pre-immune status. Participants who received a third (booster) dose of an mRNA vaccine not only increased their anti-RBD antibody levels [~]14-fold, but they also had [~]3 times more anti-RBD antibodies compared to the peak of their antibody levels after receiving their primary vaccine series. In order to ascertain whether the presence of serum antibodies is important for long-term seroprotection, PBMCs from 13 participants who lost all detectable circulating antibodies after vaccination or infection were differentiated into memory cells in vitro. There was a significant recall of memory B cells in the absence of serum antibodies in 70% of the vaccinated participants, but not in any of the infected participants. Therefore, there is a strong connection between anti-RBD antibody levels and the effectiveness of memory B cell recall.


title: A novel anti-PD-L1/IL-15 immunocytokine overcomes resistance to PD-L1 blockade and elicits potent antitumor immunity

abstract: Despite the demonstrated immense potential of immune checkpoint inhibitors in various types of cancers, only a minority of patients respond to these therapies. Immunocytokines designed to deliver an immune-activating cytokine directly to the immunosuppressive tumor microenvironment (TME) and block the immune checkpoint simultaneously may provide a strategic advantage over the combination of two single agents. To increase response rate to checkpoint blockade, in this study we developed a novel immunocytokine (LH01) composed of the antibody against programmed death-ligand 1 (PD-L1) fused to IL-15 receptor alpha-sushi domain/IL-15 complex. We demonstrate that LH01 efficiently binds mouse or human PD-L1 and maintains IL-15 stimulatory activity. In syngeneic mouse models, LH01 showed improved antitumor efficacy and safety versus anti-PD-L1 plus LH02 (Fc-Sushi-IL15) combination and overcame resistance to anti-PD-L1 treatment. Mechanistically, the dual anti-immunosuppressive function of LH01 led to activation of both the innate and adaptive immune response and decreased levels of transforming growth factor-{beta}1 (TGF-{beta}1) within the TME. Furthermore, combination therapy with LH01 and bevacizumab exerts synergistic antitumor effects in HT29 colorectal xenograft model. Collectively, our results provide supporting evidence that fusion of anti-PD-L1 and IL-15 might be a potent strategy to treat patients with cold tumors or resistance to checkpoint blockade.


title: Broad neutralization of SARS-CoV-2 variants by circular mRNA producing VFLIP-X spike in mice

abstract: Next-generation COVID-19 vaccines are critical due to the ongoing evolution of SARS-CoV-2 virus and waning duration of the neutralizing antibody response against current vaccines. The mRNA vaccines mRNA-1273 and BNT162b2 were developed using linear transcripts encoding the prefusion-stabilized trimers (S-2P) of the wildtype spike, which have shown a reduced neutralizing activity against the variants of concern B.1.617.2 and B.1.1.529. Recently, a new version of spike trimer, termed VFLIP has been suggested to possess native-like glycosylation, and greater pre-fusion trimeric stability as opposed to S-2P. Here, we report that the spike protein VFLIP-X, containing six rationally substituted amino acids to reflect emerging variants (K417N, L452R, T478K, E484K, N501Y and D614G), offers a promising candidate for a next-generation SARS-CoV-2 vaccine. Mice immunized by a circular mRNA (circRNA) vaccine prototype producing VFLIP-X elicited neutralizing antibodies for up to 7 weeks post-boost against SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs). In addition, a balance in TH1 and TH2 responses was achieved by immunization with VFLIP-X. Our results indicate that the VFLIP-X delivered by circRNA confers humoral and cellular immune responses, as well as neutralizing activity against broad SARS-CoV-2 variants.


title: High-resolution secretory timeline from vesicle formation at the Golgi to fusion at the plasma membrane in S. cerevisiae

abstract: Most of the components in the yeast secretory pathway have been studied, yet a high resolution temporal timeline of their participation is lacking. Here we define the order of acquisition, lifetime, and release of critical components involved in late secretion from the Golgi to the plasma membrane. Of particular interest is the timing of the many reported effectors of the secretory vesicle Rab protein Sec4, including the myosin-V Myo2, the exocyst complex, the lgl homolog Sro7, and the small yeast-specific protein Mso1. At the trans-Golgi network (TGN) Sec4s GEF, Sec2, is recruited to Ypt31-positive compartments, quickly followed by Sec4 and Myo2 and vesicle formation. While transported to the bud tip, the entire exocyst complex, including Sec3, is assembled on to the vesicle. Before fusion, vesicles tether for 5s, during which the vesicle retains the exocyst complex and stimulates lateral recruitment of Rho3 on the plasma membrane. Sec2 and Myo2 are rapidly lost, followed by recruitment of cytosolic Sro7, and finally the SM protein Sec1, which appears for just 2 seconds prior to fusion. Perturbation experiments reveal an ordered and robust series of events during tethering that provide insights into the function of Sec4 and effector exchange.


title: Development of a novel high-throughput screen for the identification of new inhibitors of protein S-acylation

abstract: Protein S-acylation is a reversible post-translational modification that modulates the localisation and function of many cellular proteins. S-acylation is mediated by a family of zinc finger DHHC domain-containing proteins encoded by 23 distinct ZDHHC genes in the human genome. These enzymes catalyse S-acylation in a two-step process involving "auto-acylation" of the cysteine residue in the catalytic DHHC motif followed by transfer of the acyl chain to a substrate cysteine. S-acylation is essential for many fundamental physiological processes, and there is growing interest in zDHHC enzymes as novel drug targets for a range of disorders. However, there is currently a lack of chemical modulators of S-acylation either for use as tool compounds or for potential development for therapeutic purposes. In this study, we developed and implemented a novel FRET-based high throughput assay for the discovery of compounds that interfere with auto-acylation of zDHHC2, an enzyme that is implicated in neuronal S-acylation pathways. A screen of >350,000 compounds identified two related tetrazole containing compounds (TTZ-1 and -2) that inhibited both zDHHC2 auto-acylation and substrate S-acylation in cell-free systems. Furthermore, these compounds were also active in HEK293T cells, where they inhibited substrate S-acylation mediated by different zDHHC enzymes, with some apparent isoform selectivity. Resynthesis of the hit compounds confirmed their activity, providing sufficient quantities of material for further investigations. The assays developed herein provide novel strategies to screen for zDHHC inhibitors, and the identified compounds add to the chemical toolbox for interrogating the cellular activities of S-acylation and zDHHC enzymes.


title: AtEH/Pan1 proteins drive phase separation of the TPLATE complex and clathrin polymerisation during plant endocytosis

abstract: Endocytosis is the process by which cells internalise molecules from their cell surface via plasma membrane-derived vesicles. In plants, clathrin-mediated endocytosis requires the evolutionarily ancient TSET/TPLATE complex (TPC), which was lost in metazoan and fungal lineages. TPC is required for membrane bending, but how TPC functions in the initiation of endocytosis and clathrin assembly is unclear. Here we used live-cell imaging and biochemical approaches to investigate the function of the Arabidopsis thaliana TPC subunit AtEH1/Pan1. Using in vitro and in vivo experiments we found that AtEH/Pan1 proteins can self-assemble into condensates through phase separation, which is influenced by both structured and intrinsically disordered regions. The proteome composition of these condensates revealed many key endocytic components which are selectively recruited via prion-like- and IDR-based interactions, including the ESCRT-0 TOM-Like proteins. Furthermore, AtEH/Pan1 condensates selectively nucleate on the plasma membrane by binding specific phospholipid species that are recognised by their EH domains. Visualization of the ultrastructure of the endocytic condensates via CLEM-ET revealed that the coat protein clathrin can assemble into lattices within condensates. Our results reveal that AtEH/Pan1 proteins act as scaffolds to direct endocytic machinery to specific plasma membrane regions to initiate internalisation. These findings provide new insight into the interplay between membranes and protein condensates.


title: Mother cells control daughter cell proliferation in intestinal organoids to minimize proliferation fluctuations

abstract:
During renewal of the intestine, cells are continuously generated by proliferation of stem and transit amplifying cells. To ensure homeostasis, proliferation and differentiation must be tightly balanced, with any bias towards proliferation resulting in uncontrolled exponential growth. Yet, the inherently stochastic nature of cells raises the question how such fluctuations are limited. We addressed it using a novel experimental approach. Through time-lapse microscopy, we tracked all individual cells in crypts of growing intestinal organoids for multiple generations, allowing full reconstruction of the underlying lineage dynamics in space and time. We found that proliferative behavior was highly symmetric between sister cells, with both sisters either jointly ceasing or continuing to proliferate. Simulations revealed that such symmetric proliferative behavior minimizes cell number fluctuations, and explained the observed constant numbers of proliferating cells even as crypts increased in size considerably. Proliferative symmetry was preserved for sisters with different distances to Paneth cells, which provide short-range proliferative signals. Hence, proliferative symmetry did not reflect positional symmetry in the crypt, but rather control through the lineage by the mother cell. Clone size distributions obtained in mice confirmed symmetry of proliferation also in vivo. Our results indicate a concrete mechanism by which the intestinal epithelium balances proliferation and differentiation with minimal fluctuations, and suggests that it may be more broadly relevant for other tissues.

Significance statementTissue maintenance requires constant birth of proliferating cells to offset their loss through terminal differentiation. While any misbalance between birth and loss of proliferating cells could lead to their overgrowth or depletion, the decision to proliferate or not is stochastic, causing fluctuations. How is stable maintenance ensured despite these fluctuations? We study cell proliferation by a novel approach, tracking all cells in crypts of intestinal organoids to fully reconstruct cell lineages. Combined with simulations, our results imply that fluctuations in cell proliferation are minimized by a mechanism that couples strong symmetry in proliferative behavior between sister cells with the partitioning of crypts in compartments of different proliferative potential. This mechanism likely applies to a broader class of stem cell systems.


title: Substrate trapping approach identifies TRIM25 ubiquitination targets involved in diverse cellular and antiviral processes

abstract: The tripartite motif (TRIM) family of E3 ubiquitin ligases is well known for its roles in antiviral restriction and innate immunity regulation, in addition to many other cellular pathways. In particular, TRIM25-mediated ubiquitination affects both carcinogenesis and antiviral response. While individual substrates have been identified for TRIM25, it remains unclear how it regulates diverse processes. Here we characterized a mutation, R54P, critical for TRIM25 catalytic activity, which we successfully utilized to "trap" substrates. We demonstrated that TRIM25 targets proteins implicated in stress granule formation (G3BP1/2), nonsense-mediated mRNA decay (UPF1), and nucleoside synthesis (NME1). R54P abolishes TRIM25 inhibition of alphaviruses independently of the host interferon response, suggesting that this antiviral effect is a direct consequence of ubiquitination. Consistent with that, we observed diminished antiviral activity upon knockdown of several TRIM25-R54P specific interactors including NME1. Our findings highlight that multiple substrates mediate the cellular and antiviral activities of TRIM25, illustrating the multi-faceted role of this ubiquitination network in diverse biological processes.


title: En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration

abstract: The mouse digit tip is a complex tissue that is capable of regeneration after amputation. How the regenerated digit tip is patterned is unknown, but a long-standing hypothesis in the field of regeneration proposes that developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits strong dorsal-ventral (DV) polarity, so we focus on Engrailed 1 (En1) and LIM homeobox transcription factor 1B (Lmx1b), two well-studied transcription factors necessary for DV patterning during limb development. We investigate if En1 and Lmx1b are re-expressed during regeneration in a developmental-like spatially restricted pattern, and if they direct DV morphology of the regenerated digit tip. We find that both En1 and Lmx1b are expressed in the regenerating mouse digit tip epithelium and mesenchyme, respectively, but without DV polarity. We use conditional genetics and quantitative analysis of digit tip bone morphology to determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning of the regenerate. Collectively, our data suggest that while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.


title: Sea anemone Frizzled receptors play partially redundant roles in the oral-aboral axis patterning

abstract: Canonical Wnt (cWnt) signaling is involved in a plethora of basic developmental processes such as endomesoderm specification, gastrulation and patterning the main body axis. To activate the signal, Wnt ligands form complexes with LRP5/6 and Frizzled receptors, which leads to nuclear translocation of {beta}-catenin and transcriptional response. In Bilateria, the expression of different Frizzled genes is often partially overlapping, and their functions are known to be redundant in several developmental contexts. Here we demonstrate that all four Frizzled receptors take part in the cWnt-mediated oral-aboral axis patterning in the cnidarian Nematostella vectensis but show partially redundant functions. However, we do not see evidence for their involvement in the specification of the endoderm - an earlier event likely relying on maternal, intracellular {beta}-catenin signaling components. Finally, we demonstrate that the main Wnt ligands crucial for the early oral-aboral patterning are Wnt3 and Wnt4. Comparison of our data to the knowledge from other models suggests that distinct but overlapping expression domains and partial functional redundancy of cnidarian and bilaterian Frizzled genes may represent a shared ancestral trait.


title: Differential expression of Nanog 3UTR and coding region mRNA components defines an embryonic stem cell niche

abstract: The known molecular heterogeneity among pluripotent embryonic stem cells (ESCs) in culture may be maladaptive or a beneficial determinant of normal biology. In other systems such as hair and intestine, normal and dynamic heterogeneity among stem cells is necessary for balancing stem cell division and quiescence in order to maintain the stem cell pool. In ESCs, Sox2, Oct4 and Nanog (the SON genes) critically regulate pluripotency but their role in stem cell heterogeneity or pool maintenance is not clear. We previously reported an additional level of gene expression, the unexpected differential expression of mRNA coding region (CDS) and cognate 3'untranslated region (3UTR) components, often revealing novel developmental patterns. Here we show dramatic, patterned, concentric, differential expression of pluripotent gene 3UTR and CDS mRNA components in both undifferentiated ESCs in culture and in developing human and mouse embryos. This differential expression reveals robust pluripotent ESC heterogeneity that provides the underpinnings of a previously unrecognized ESC cell niche structure. A hallmark of the niche is the presence of cells expressing high levels of Nanog 3UTR but only low or no Nanog CDS at structure perimeters, We used a novel bioinformatic approach combining the 3UTR to CDS ratio and gene expression, and identified unique ESC clusters. High Nanog 3UTR cells have lower cell cycle activity and higher quiescence than other ESCs and express a distinct complement of cellular histone marks. These quiescent perimeter cells exchange with interior domains, opening the possibility that they act as cell replenishment reservoirs in the niche or the embryo. Functionally, the Nanog 3UTR, independent of its CDS, is critical, as its selective deletion decreases ESC cell division while increasing trophectoderm markers. Lastly, self-organization of ESCs according to pluripotent gene differential 3UTR and CDS expression is modified by activin signaling, indicating extrinsic modulation of the niche structure. Pluripotent embryonic stem cells, actively display regulated, dynamic differential expression of SON gene mRNA components, allowing the formation of a heterogenous, ordered, stem cell niche bordered by quiescent hub stem cells, thus illuminating the role of SON genes in pluripotent embryonic stem cell pool maintenance. More generally this suggests that differential mRNA component expression may widely define cell types that exist in non-permanent, different states in either development or disease.


title: Inherent dynamics of maltose binding protein (MBP) are immune to the native environment.

abstract: Biophysical characterizations of proteins typically rely on a reductionistic approach, studying proteins in a highly purified from and in absence of their natural cellular environment. Little is known about how the highly crowded conditions prevalent within living cells influence the dynamic structures proteins on the molecular level. To address this outstanding question, we characterize here the dynamic behavior of the periplasmic model protein MBP from Escherichia coli in situ, confined in the native lumen of bacterial outer membrane vesicles. To this end we determine the dynamics of side-chain methyl groups of MBP across several timescales and compare them to purified in vitro MBP. We find that the inherent dynamics of MBP are surprisingly insensitive to the native cellular environment and that the molecular motion of the protein is mainly impacted on a global level.


title: Gill developmental program in the teleost mandibular arch

abstract: Whereas no known living vertebrate possesses gills derived from the jaw-forming mandibular arch, it has been proposed that the jaw arose through modifications of an ancestral mandibular gill. Here we show that the zebrafish pseudobranch, which regulates blood pressure in the eye, develops from mandibular arch mesenchyme and first pouch epithelia and shares gene expression, enhancer utilization, and developmental gata3 dependence with the gills. Combined with work in chondrichthyans, our findings in a teleost fish point to the presence of a mandibular pseudobranch with serial homology to gills in the last common ancestor of jawed vertebrates, consistent with a gill origin of vertebrate jaws.


title: A non-invasive method for time-lapse imaging of microbial interactions and biofilm dynamics

abstract:
Complex interactions between microbial populations can greatly affect the overall properties of a microbial community, sometimes leading to cooperation and mutually beneficial coexistence, or to competition and the death or displacement of particular organisms or sub-populations. Interactions between different biofilm populations are highly relevant in diverse scientific areas, from antimicrobial resistance to microbial ecology. The utilization of modern microscopic techniques has provided new and interesting insight into how bacteria interact at the cellular level to form and maintain microbial biofilms. However, our ability to follow complex intra- and inter-species interactions in vivo at the microscopic level has remained somewhat limited. Here, we detail BacLive, a novel non-invasive method for tracking bacterial growth and biofilm dynamics using high resolution fluorescence microscopy and an associated ImageJ processing macro (https://github.com/BacLive) for easier data handling and image analysis. Finally, we provide examples of how BacLive can be used in the analysis of complex bacterial communities.

ImportanceCommunication and interactions between single cells are continuously defining the structure and composition of microbial communities timely and spatially. Methods routinely used to the study of these communities at cellular level rely on sample manipulation what deprives from microscopic time lapse experiments on a given sample. BacLive is conceived as a method for the non-invasive study of the formation and development of bacterial communities, such as biofilms, and the dynamics of formation of specialized subpopulations in time-lapse experiments. In addition we prove that BacLive largely simplifies the analysis of the data generated.

category: microbiology


title: A bipartite, low-affinity roadblock domain-containing GAP complex regulates bacterial front-rear polarity

abstract: The Ras-like GTPase MglA is a key regulator of front-rear polarity in rod-shaped Myxococcus xanthus cells. MglA-GTP localizes to the leading cell pole and stimulates assembly of the two motility machineries. MglA-GTP localization is spatially constrained by its cognate GEF, the RomR/RomX complex, and GAP, the MglB Roadblock-domain protein. RomR/RomX and MglB localize similarly with low and high concentrations at the leading and lagging poles, respectively. Yet, GEF activity dominates at the leading and GAP activity at the lagging pole by unknown mechanisms. Here, we identify RomY as a co-GAP that stimulates MglB GAP activity. The MglB/RomY interaction is low affinity, restricting complex formation to the lagging pole with the high MglB concentration. Our data support a model wherein RomY, by forming a low-affinity complex with MglB, ensures that MglB GAP activity is spatially precisely confined to the lagging pole, thereby constraining MglA-GTP to the leading pole establishing front-rear polarity.


title: Acute Cytomegalovirus Infection Modulates the Intestinal Microbiota and Targets Intestinal Epithelial Cells

abstract: Primary and recurrent cytomegalovirus (CMV) infections frequently cause CMV colitis in immunocompromised as well as inflammatory bowel disease (IBD) patients. Additionally, colitis occasionally occurs upon primary CMV infection in patients who are apparently immunocompetent. In both cases, the underlying pathophysiologic mechanisms are largely elusive - in part due to the lack of adequate access to specimens. We employed the mouse cytomegalovirus (MCMV) model to probe into the association between CMV and colitis. During acute primary MCMV infection of immunocompetent mice, the gut microbial composition was affected within the first 5 days post-infection as manifested by an altered ratio of the Firmicutes to Bacteroidetes phyla. Interestingly, these microbial changes incited with high-titer MCMV replication in the colon, mild crypt necrosis and increased colonic pro-inflammatory cytokine levels. Further analyses revealed that murine and human intestinal epithelial cell line as well as primary intestinal crypt cells/ organoids represent direct targets of CMV accompanied by increased cell mortality upon infection. Accordingly, in vivo MCMV infection disrupted the intestinal epithelial barrier and increased apoptosis of intestinal epithelial cells. In summary, our data show that CMV induces colitis in immunocompetent hosts by altering the intestinal homeostasis.