title: Rapid and robust directed differentiation of mouse epiblast stem cells into definitive endoderm and forebrain organoids

abstract:
Directed differentiation of pluripotent stem cells (PSCs) is a powerful model system for deconstructing embryonic development. Although mice are the most advanced mammalian model system for genetic studies of embryonic development, state-of-the-art protocols for directed differentiation of mouse PSCs into defined lineages tend to be slower and generate target cell types with lower purity than analogous protocols for human PSCs, limiting their application as models for mechanistic studies of development. Here, we examine the potential of mouse epiblast stem cells (EpiSCs) cultured in media containing Wnt pathway inhibitors (primed ground state conditions) as a starting point for directed differentiation. As a proof-of-concept, we focused our efforts on two specific cell/tissue types that have proven difficult to generate efficiently and reproducibly from mouse embryonic stem cells: definitive endoderm and neural organoids. First, we developed a new protocol that can rapidly generate nearly pure definitive endoderm from EpiSCs. Second, we developed a protocol for generating forebrain organoids that model the development of prethalamic and hippocampal neurons. These significantly improved differentiation models present new possibilities for combining mouse genetic tools and resources with in vitro differentiation to characterize the molecular and cellular mechanisms of embryonic development.

SUMMARY STATEMENTNew optimized protocols for directed differentiation of mouse epiblast stem cells into definitive endoderm and forebrain-patterned organoids.


title: Single cell quantification of ribosome occupancy in early mouse development

abstract: Technological limitations precluded transcriptome-wide analyses of translation at single cell resolution. To solve this challenge, we developed a novel microfluidic isotachophoresis approach, named RIBOsome profiling via IsoTachoPhoresis (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key regulatory mechanism of genes involved in centrosome organization and N6-methyladenosine modification of RNAs. Our high coverage measurements enabled the first analysis of allele-specific ribosome engagement in early development and led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes. Finally, by integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle stage oocytes is the predominant determinant of protein abundance in the zygote. Taken together, these findings resolve the long-standing paradox of low correlation between RNA expression and protein abundance in early embryonic development. The novel Ribo-ITP approach will enable numerous applications by providing high coverage and high resolution ribosome occupancy measurements from ultra-low input samples including single cells.

category: developmental biology


title: Game Of Theories: An Agent-Based Model Of How Variation In Contest Cost-Benefits Determines The Evolution Of Contest Resolution Rules

abstract: Game-theory based models are used to understand rules that animals use to settle contests over indivisible resources. However, the empirical literature of contests indicates controversial support to models, with some species supporting different models and other species showing no support to any model. Since strategies used to resolve contests may have different associated costs, it is possible that different conditions have determined the evolution of distinct assessment strategies used by animals. We used an agent-based model to explore the importance of the following conditions: resource availability, probability of reproduction with resource, and damage costs on evolution of assessment strategies. We used self- and mutual-assessment models as a heurist framework to build agents with different assessment strategies. In our model, agents competed for resources in scenarios with different combinations of resource availability, probability of reproduction with resource, and damage costs. We found that agents following the self-assessment with damage strategy were prevalent in scenarios with no probability of reproduction without the resource, independently of other variables. We also found that agents following the non-aggressive strategy occurred in all scenarios. However, agents using the non-aggressive strategy were prevalent only in scenarios with probability of reproduction with the resource. Finally, we observed that agents using mutual-assessment occurred only in a scenario with high risk of damage, low availability of resources, and with probability of reproduction without the resource. These results indicate that agents following the self-assessment with damage and non-aggressive strategies may be able to stay at most scenarios.

category: animal behavior and cognition


title: Game of theories: an agent-based model of how variation in contest cost-benefits determines the evolution of contest resolution rules


title: The consequence of fiber orientation downsampling on the computation of white matter tract-related deformation

abstract:
Incorporating neuroimaging-revealed structural details into finite element (FE) head models opens vast new opportunities to better understand brain injury mechanisms. Recently, growing efforts have been made to integrate fiber orientation from diffusion tensor imaging (DTI) into FE models to predict white matter (WM) tract-related deformation that is biomechanically characterized by tract-related strains. Commonly used approaches often downsample the spatially enriched fiber orientation to match the FE resolution with one orientation per element (i.e., element-wise orientation implementation). However, the validity of such downsampling operation and corresponding influences on the computed tract-related strains remain elusive. To address this, the current study proposed a new approach to integrate voxel-wise fiber orientation from one DTI atlas (isotropic resolution of 1 mm3) into FE models by embedding orientations from multiple voxels within one element (i.e., voxel-wise orientation implementation). By setting the responses revealed by the newly proposed voxel-wise orientation implementation as the reference, we evaluated the reliability of two previous downsampling approaches by examining the downsampled fiber orientation and the computationally predicted tract-related strains secondary to one concussive impact. Two FE models with varying element sizes (i.e., 6.37 {+/-} 1.60 mm and 1.28 {+/-} 0.55 mm, respectively) were incorporated. The results showed that, for the model with a large voxel-mesh resolution mismatch, the downsampled element-wise fiber orientation, with respect to its voxel-wise counterpart, exhibited an absolute deviation over 30{degrees} across the WM/gray matter interface and the pons regions. Accordingly, this orientation deviation compromised the computation of tract-related strains with normalized root-mean-square errors up to 30% and underestimated the peak tract-related strains up to 10%. For the other FE model with finer meshes, the downsampling-induced effects were lower, both on the fiber orientation and tract-related strains. Taken together, the voxel-wise orientation implementation is recommended in future studies as it leverages the DTI-delineated fiber orientation to a larger extent than the element-wise orientation implementation. Thus, this study yields novel insights on integrating neuroimaging-revealed fiber orientation into FE models and may better inform the computation of WM tract-related deformation, which are crucial for advancing the etiological understanding and computational predictability of brain injury.

Graphic abstract

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category: bioengineering


title: Fiber orientation downsampling compromises the computation of white matter tract-related deformation


title: Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells

abstract: Selection of the target site is an inherent question for any project aiming for directed transgene integration. Genomic safe harbour (GSH) loci have been proposed as safe sites in the human genome for transgene integration. Although several sites have been characterised for transgene integration in the literature, most of these do not meet criteria set out for a GSH and the limited set that do have not been characterised extensively. Here, we conducted a computational analysis using publicly available data to identify 25 unique putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression and minimal disruption of the native transcriptome in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.


title: HSV-1 and influenza infection induce linear and circular splicing of the long NEAT1 isoform

abstract: The herpes simplex virus 1 (HSV-1) virion host shut-off (vhs) protein cleaves both cellular and viral mRNAs but not circular RNAs (circRNAs) without an internal ribosome entry site. Here, we show that vhs activity leads to an accumulation of circRNAs relative to linear mRNAs during HSV-1 infection. Strikingly, we found that circular splicing of the long isoform (NEAT1_2) of the nuclear paraspeckle assembly transcript 1 (NEAT1) was massively induced during HSV-1 infection in a vhs-independent manner while NEAT1_2 was still bound to the chromatin. This was associated with induction of linear splicing of NEAT1_2 both within and downstream of the circRNA. NEAT1_2 splicing was absent in uninfected cells but can be induced by ectopic co-expression of the HSV-1 immediate-early proteins ICP22 and ICP27. Interestingly, NEAT1_2 circular and linear splicing was also up-regulated in influenza infection but absent in stress conditions, which disrupt transcription termination similar to but not in the same manner as HSV-1 and influenza infection. Finally, large-scale analysis of published RNA-seq data uncovered induction of NEAT1_2 splicing in cancer cells upon inhibition or knockdown of cyclin-dependent kinase 7 (CDK7) or the MED1 subunit of the Mediator complex phosphorylated by CDK7. Interestingly, CDK7 inhibition also disrupted transcription termination, highlighting a possible link between disruption of transcription termination and NEAT1_2 splicing.

category: bioinformatics


title: Large Scale Discovery of Microbial Fibrillar Adhesins and Identification of Novel Adhesive Domain Families

abstract:
Fibrillar adhesins are bacterial cell surface proteins that mediate interactions with the environment including host cells during colonisation or other bacteria during biofilm formation. These proteins are characterised by a stalk that projects the adhesive domain closer to the binding target. Fibrillar adhesins evolve quickly and thus can be difficult to computationally identify, yet they represent an important component for understanding bacterial host interactions.

To detect novel fibrillar adhesins we developed a random forest prediction approach based on common characteristics we identified for this protein class. We applied this approach to Firmicute and Actinobacterial proteomes, yielding over 6,500 confidently predicted fibrillar adhesins. To verify the approach we investigated predicted fibrillar adhesins that lacked a known adhesive domain. Based on these proteins, we identified 24 sequence clusters representing potential novel members of adhesive domain families. We used AlphaFold to verify that 15 clusters showed structural similarity to known adhesive domains such as the TED domain.

Overall our study has made a significant contribution to the number of known fibrillar adhesins and has enabled us to identify novel members of adhesive domain families involved in the bacterial pathogenesis.

ImportanceFibrillar adhesins are a class of bacterial cell surface proteins that enable bacteria to interact with their environment. We developed a Machine Learning approach to identify fibrillar adhesins and applied this classification approach on the Firmicutes and Actinobacteria Reference Proteomes. This method allowed us to detect a high number of novel fibrillar adhesins, and also novel members of adhesive domain families. To confirm our predictions of these potential adhesin protein domains, we predicted their structure using the AlphaFold tool.


title: Large Scale Discovery of Microbial Fibrillar Adhesins and Identification of Novel Members of Adhesive Domain Families


title: Semi-automated computational assessment of cancer organoid viability using rapid live-cell microscopy

abstract: The creation of patient-derived cancer organoids represents a key advance in preclinical modeling and has recently been applied to a variety of human solid tumor types. However, conventional methods used to assess cellular viability in tissue specimens are poorly suited for the evaluation of cancer organoids because they are time-intensive and involve tissue destruction. To address this issue, we established a suite of 3-dimensional patient-derived glioma organoids, treated them with chemoradiotherapy, stained organoids with non-toxic cell dyes, and imaged them using a rapid laser scanning confocal microscopy method termed "Apex Imaging". We then developed and tested a fragmentation algorithm to quantify heterogeneity in the topography of the organoids as a potential surrogate marker of viability. This algorithm, SSDquant, provides a 3-dimensional visual representation of the organoid surface and a numerical measurement of the sum-squared distance (SSD) from the derived mass center of the organoid. We tested whether SSD scores correlate with traditional immunohistochemistry-derived cell viability markers (cellularity and cleaved caspase 3 expression) and observed statistically significant associations between them using linear regression analysis. Our work describes a quantitative, non-invasive approach for the serial measurement of patient-derived cancer organoid viability, thus opening new avenues for the application of these models to studies of cancer biology and therapy.


title: What's past is past, mostly: Brassicaceae host plants mask the feedback from the previous year's soil history on bacterial communities, except when the Brassicaceae hosts experience drought

abstract: Previous soil history and the current plant hosts are two plant-soil feedbacks that operate at different time-scales to influence the structure soil bacterial communities. In this study, we used a MiSeq metabarcoding strategy to describe the impact of five Brassicaceae host plant species, and three different soil histories, on the structure of their bacterial root and rhizosphere communities at full flower. We found that the Brassicaceae host plants were consistently significant in structuring the bacterial communities. Four host plants (Sinapis alba, Brassica napus, B. juncea, B. carinata) formed nearly the same bacterial communities, regardless of soil history. Camelina sativa host plants structured phylogenetically distinct bacterial communities compared to the other hosts, particularly in their roots. Soil history established the previous year was only a significant factor for bacterial community structure when the feedback of the Brassicaceae host plants was weakened, potentially due to limited soil moisture during a dry year. Understanding how plant-soil feedbacks operate at different time-scales and are involved in how microbial communities are structured is a pre-requisite for employing microbiome technologies in improving agricultural systems.

category: plant biology


title: Small transcriptional differences lead to distinct NF-κB dynamics in quasi-identical cells

abstract: Transcription factor dynamics is fundamental to determine the activation of accurate transcriptional programs and yet is heterogeneous at single-cell level. The source of this dynamic variability is not completely understood. Here we focus on the nuclear factor{kappa} B (NF-{kappa}B), whose dynamics have been reported to cover a wide spectrum ranging from oscillatory to non-oscillatory. We show that clonal populations of immortalized fibroblasts derived from a single mouse embryo (that can hence be considered quasi-identical) display robustly distinct dynamics upon tumor necrosis [a] (TNF-[a]) stimulation. Combining transcriptomics, data-constrained mathematical modelling, and mRNA interference we show that small differences in the expression of genes belonging to the NF-{kappa}B regulatory circuit are predictive of the distinct responses to inflammatory stimuli observed among the clones. We propose that this transcriptional fine-tuning can be a general mechanism to produce cell subpopulations with distinct dynamic responses to stimuli within homogeneous cell populations.

category: systems biology


title: Using coexpression to explore cell-type diversity with the fcoex package

abstract:
Here, we present the fcoex package, which infers coexpression from scRNA-seq data and yields multiple, overlapping classes of cells based on coexpression modules. The tool extends the current scRNA-seq toolbox, providing a multi-hierarchy view on cell functionality and enabling the development of more complete cell atlases. Single-cell RNA sequencing (scRNA-seq) captures details of the cellular landscape, basing a fine-grained view on biological processes. Current pipelines, however, are restricted to single-label perspectives, missing details of the classification landscape. In the pbmc3k blood cell dataset, fcoex detects known classes, like antigen-presenting cells and a new theoretical group of cells, marked by the expression of FCGR3A (CD16). Fcoex extends the current scRNA-seq toolbox, providing a multi-hierarchy view on cell functions as a tool to develop complete cell type atlases.

Availability and ImplementationFcoex is written in R and openly available in Bioconductor (https://bioconductor.org/packages/fcoex/).

Supplementary informationSupplementary data is available at the end of the manuscript. Source code for analysis is available at https://github.com/csbl-inovausp/fcoex_analysis;


title: Transparent and Conformal Microcoil Arrays for Spatially Selective Neuronal Activation

abstract: Magnetic stimulation represents a compelling modality for achieving neuronal activation with high spatial resolution and low toxicity. Stimulation coils can be designed to achieve localized, spatially asymmetric fields that target neurons of a particular orientation. Furthermore, these devices may be encapsulated within biopolymers thereby avoiding direct metal/tissue interfaces that could induce chronic inflammation and glial scarring. Herein, we report a multiplexed microcoil array for localized activation of cortical neurons and retinal ganglion cells. We designed a computational model that related the activation function to the geometry and arrangement of coils, and selected a geometry with a region of activation <50 m wide. We then fabricated SU8/Cu/SU8 tri-layer devices which were flexible, transparent and conformal and featured four individually-addressable microcoil stimulation elements. Interfaced with ex vivo cortex or retina slices from GCaMP6-transfected mice, we observed that individual neurons located within 40 m of the element tip could be activated repeatedly and in a dose (power) dependent fashion. Taken together, these results highlight the potential of magnetic stimulation devices for brain-machine interfaces and could open new routes toward bioelectronic therapies including prosthetic vision devices.

category: neuroscience


title: Neural Oscillatory Characteristics of Feedback Associated Activity in Globus Pallidus Interna

abstract: Neural oscillatory activities in basal ganglia have prominent roles in cognitive processes on local and global scales. However, the characteristics of high frequency oscillatory activities during cognitive tasks have not been extensively explored in human Globus Pallidus internus (GPi). This study aimed to investigate amplitude and interhemispheric coupling of bilateral GPi high gamma bursts in dystonia and Parkinsons Disease (PD) patients, in on and off medication states, after feedback during the Intra-Extra-Dimension shift (IED) task. Bilateral GPi Local Field Potentials (LFP) activity was recorded via externalized DBS electrodes during the IED task. Inter hemisphere phase synchrony was assessed using Inter-Site Phase Clustering (ISPC). Transient high gamma activity ([~]100-150Hz) was observed immediately after feedback in the dystonia patient. Moreover, these bursts were phase synchronous between left and right GPis with an antiphase clustering of phase differences. In contrast, no synchronous high gamma activity was detected in the PD patient with or without dopamine administration. The off-med PD patient displayed enhanced low frequency clusters ameliorated by medication in the on-med state. Furthermore, an increased low frequency activity was observed after feedback of incorrect trials in both disease states. The current study provides a rare report of antiphase homotopic synchrony in human GPi, potentially related to incorporating and processing feedback information. The absence of these activities in off and on-med PD indicates the potential presence of impaired medication independent circuits related to feedback processing. Together, these findings are helpful in pointing to the potential role of GPis synchronized high frequency activity in cognitive tasks and feedback information processing.


title: Intermittent brain network reconfigurations and the resistance to social media influence

abstract:
Since their development, social media has grown as a source of information and has a significant impact on opinion formation. Individuals interact with others and content via social media platforms in a variety of ways but it remains unclear how decision making and associated neural processes are impacted by the online sharing of informational content, from factual to fabricated. Here, we use EEG to estimate dynamic reconfigurations of brain networks and probe the neural changes underlying opinion change (or formation) within individuals interacting with a simulated social media platform. Our findings indicate that the individuals who changed their opinions are characterized by less frequent network reconfigurations while those who did not change their opinions tend to have more flexible brain networks with frequent reconfigurations. The nature of these frequent network configurations suggests a fundamentally different thought process between intervals in which individuals are easily influenced by social media and those in which they are not. We also show that these reconfigurations are distinct to the brain dynamics during an in-person discussion with strangers on the same content. Together, these findings suggest that brain network reconfigurations may not only be diagnostic to the informational context but also the underlying opinion formation.

Author SummaryDistinctive neural underpinnings of opinion formation and change during in-person and online social interactions are not well understood. Here, we analyze EEG recordings of the participants interacting with a simulated social media platform and during an in-person discussion using a network-based analysis approach. We show that the structure of network reconfigurations during these interactions is diagnostic of the opinion change and the context in which information was received.


title: Lysophosphatidylcholine induces heat pain hypersensitivity in obese mice fed with a high-fat diet through activation of peripheral Acid-Sensing Ion Channel 3

abstract: Diet induced obesity is one of the major causes of obesity, which affects 13% of the worlds adult population. Obesity is correlated to chronic pain regardless of other components of the metabolic syndrome. Our study focuses on investigating the effect of high-fat diet induced obesity on peripheral sensory neurons activity and pain perception, followed by deciphering the underlying cellular and molecular mechanisms that involve Acid-Sensing Ion Channel 3 (ASIC3). We show here that heat sensitive C-fibers from mice made obese by consumption of a high-fat diet exhibited an increased activity during baseline and upon heating. Obese mice showed long-lasting heat pain hypersensitivity once obesity was well established, while mechanical sensitivity was not affected. We found that the serum of obese mice was enriched in lysophosphatidylcholine (LPC) species (LPC16:0, LPC18:0 and LPC18:1), which activate ASIC3 channels and increased peripheral neuron excitability. Genetic deletion and in vivo pharmacological inhibition of ASIC3 protected and rescued mice from obesity-induced thermal hypersensitivity. Our results identify ASIC3 channels in DRG neurons and circulating LPC species that activate them as a mechanism contributing to heat pain hypersensitivity associated with high-fat diet induced obesity.


title: Early detection of sublexical and lexical processing in beginning readers: evidence from Steady-State Visual Evoked Potentials (SSVEPs)

abstract:
There are multiple levels of processing relevant to reading that vary in their visual, sublexical and lexical orthographic processing demands. Segregating distinct cortical sources for each of these levels has been challenging in EEG studies of early readers. To address this challenge, we applied recent advances in analyzing high-density EEG using Steady-State Visual Evoked Potentials (SSVEPs) via data-driven Reliable Components Analysis (RCA) in a group of early readers spanning from kindergarten to second grade. Three controlled stimulus contrasts--familiar words versus unfamiliar pseudofonts, familiar words versus orthographically legal pseudowords, and orthographically legal pseudowords versus orthographically illegal nonwords--were used to isolate visual print/letter selectivity, sublexical processing, and lexical processing, respectively. We found robust responses specific to each of these processing levels, even in kindergarteners who have limited knowledge of print. Moreover, comparing amplitudes of these three stimulus contrasts across three reading fluency-based groups and three grade-based groups revealed fluency group and grade group main effects only for lexical contrast (i.e., words versus orthographically legal pseudowords). Furthermore, we found that sublexical orthography-related responses shifted their topographic distribution from the right to left hemisphere from kindergarten to first and second grades. Results suggest that, with more sensitive measures, the sublexical and lexical fine tuning for words--as a bio-marker of reading ability--can be detected at a much earlier stage than previously assumed.

Declarations of interestNone


title: Nonlinear dynamics in auditory cortical activity reveal the neural basis of perceptual warping in speech categorization

abstract: Surrounding context influences speech listening, resulting in dynamic shifts to category percepts. To examine its neural basis, event-related potentials (ERPs) were recorded during vowel identification with continua presented in random, forward, and backward orders to induce perceptual nonlinearities. Behaviorally, sequential order shifted listeners categorical boundary vs. random delivery revealing perceptual warping (biasing) of the heard phonetic category dependent on recent stimulus history. ERPs revealed later ([~]300 ms) activity localized to superior temporal and middle/inferior frontal gyri that predicted listeners hysteresis magnitudes. Findings demonstrate that top-down, stimulus history effects on speech categorization are governed by interactions between frontotemporal brain regions.


title: Non-monotonic effects of GABAergic synaptic inputs on neuronal firing

abstract:
GABA is generally known as the principal inhibitory neurotransmitter in the nervous system, usually acting by hyperpolarizing membrane potential. However, GABAergic currents sometimes exhibit non-inhibitory effects, depending on the brain region, developmental stage or pathological condition. Here, we investigate the diverse effects of GABA on the firing rate of several single neuron models, using both analytical calculations and numerical simulations. We find that GABAergic synaptic conductance and output firing rate exhibit three qualitatively different regimes as a function of GABA reversal potential, EGABA: monotonically decreasing for sufficiently low EGABA (inhibitory), monotonically increasing for EGABA above firing threshold (excitatory); and a non-monotonic region for intermediate values of EGABA. In the non-monotonic regime, small GABA conductances have an excitatory effect while large GABA conductances show an inhibitory effect. We provide a phase diagram of different GABAergic effects as a function of GABA reversal potential and glutamate conductance. We find that noisy inputs increase the range of EGABA for which the non-monotonic effect can be observed. We also construct a micro-circuit model of striatum to explain observed effects of GABAergic fast spiking interneurons on spiny projection neurons, including non-monotonicity, as well as the heterogeneity of the effects. Our work provides a mechanistic explanation of paradoxical effects of GABAergic synaptic inputs, with implications for understanding the effects of GABA in neural computation and development.

Author summaryNeurons in nervous systems mainly communicate at chemical synapses by releasing neurotransmitters from the presynaptic side that bind to receptors on the post-synaptic side, triggering ion flow through ion channels on the cell membrane and changes in the membrane potential of the post-synaptic neuron. Gamma-aminobutyric acid (GABA) is the principal neurotransmitter expressed by inhibitory neurons. Its binding to GABAergic ionotropic receptors mainly causes a flow of chloride ions across the membrane, and typically hyperpolarizes the post-synaptic neuron, resulting in firing suppression. While GABA is canonically viewed as an inhibitory neurotransmitter, non-inhibitory effects have been observed in early stages of development, in stress-related disorders, and in specific parts of brain structures such as cortex, cerebellum and hippocampus [1-4]. Here, we employ analytical and computational approaches on spiking neuronal models to investigate the mechanisms of diverse effects of GABAergic synaptic inputs. We find that in addition to monotonically excitatory or monotonically inhibitory effects, GABAergic inputs show non-monotonic effects, for which the effect depends on the strength of the input. This effect is stronger in the presence of noise, and is observed in different models both at the single cell, and at the network level. Our findings provide a mechanistic explanation of several paradoxical experimental observations, with potential implications for neural network dynamics and computation.


title: Altricial bird early-stage embryos express the molecular "machinery" to respond to maternal thyroid hormone cues

abstract: Maternal hormones, such as thyroid hormones transferred to embryos and eggs, are key signalling pathways to mediate maternal effects. To be able to respond to maternal cues, embryos must express key molecular  machinery of the hormone pathways, such as enzymes and receptors. While altricial birds begin thyroid hormone (TH) production only at/after hatching, experimental evidence suggests that their phenotype can be influenced by maternal THs deposited in the egg. However, it is not understood, how and when altricial birds express genes in the TH-pathway. For the first time, we measured the expression of key TH-pathway genes in altricial embryos, using two common altricial ecological model species (pied flycatcher, Ficedula hypoleuca and blue tit Cyanistes caeruleus). Deiodinase DIO1 gene expression could not be reliably confirmed in either species, but deiodinase enzyme DIO2 and DIO3 genes were expressed in both species. Given that DIO2 coverts T4 to biologically active T3, and DIO3 mostly T3 to inactive forms of thyroid hormones, our results suggest that embryos may modulate maternal signals. Thyroid hormone receptor (THRA and THRB) and monocarboxyl membrane transporter gene (SLC15A2) were also expressed, enabling TH-responses. Our results suggest that early altricial embryos may be able to respond and potentially modulate maternal signals conveyed by thyroid hormones.


title: Spatiotemporal transcriptomics reveals pathogenesis of viral myocarditis

abstract: A significant fraction of sudden death in children and young adults is due to myocarditis, an inflammatory disease of the heart, most often caused by viral infection. Here we used integrated single-cell and spatial transcriptomics to create a high-resolution, spatially resolved map of reovirus-induced myocarditis in neonatal murine hearts. We assayed hearts collected at three timepoints after reovirus infection and studied the temporal, spatial, and cellular heterogeneity of host-virus interactions. We further assayed the intestine, the primary site of reovirus infection to establish a full chronology of molecular events that ultimately lead to myocarditis. We implemented targeted enrichment of viral transcripts to establish the cellular targets of the virus in the intestine and the heart. Our data give insight into the cell-type specificity of innate immune responses, and into the transcriptional states of inflamed cardiac cells that recruit circulating immune cells, including cytotoxic T cells which induce pyroptosis in the myocarditic tissue. Analyses of spatially restricted gene expression in myocarditic regions and the border zone around those regions identified immune-mediated cell-type specific injury and stress responses. Overall, we observe a dynamic and complex network of cellular phenotypes and cell-cell interactions associated with viral myocarditis.

category: genomics


title: Attentive deep learning-based tumor-only somatic mutation classifier achieves high accuracy agnostic of tissue type and capture kit.

abstract: In precision oncology, reliable identification of tumor-specific DNA mutations requires sequencing tumor DNA and non-tumor DNA (so-called "matched normal") from the same patient. The normal sample allows researchers to distinguish acquired (somatic) and hereditary (germline) variants. The ability to distinguish somatic and germline variants facilitates estimation of tumor mutation burden (TMB), which is a recently FDA-approved pan-cancer marker for highly successful cancer immunotherapies; in tumor-only variant calling (i.e., without a matched normal), the difficulty in discriminating germline and somatic variants results in inflated and unreliable TMB estimates. We apply machine learning to the task of somatic vs germline classification in tumor-only samples using TabNet, a recently developed attentive deep learning model for tabular data that has achieved state of the art performance in multiple classification tasks (Arik and Pfister 2019). We constructed a training set for supervised classification using features derived from tumor-only variant calling and drawing somatic and germline truth-labels from an independent pipeline incorporating the patient-matched normal samples. Our trained model achieved state-of-the-art performance on two hold-out test datasets: a TCGA dataset including sarcoma, breast adenocarcinoma, and endometrial carcinoma samples (F1-score: 88.3), and a metastatic melanoma dataset, (F1-score 79.8). Concordance between matched-normal and tumor-only TMB improves from R2 = 0.006 to 0.705 with the addition of our classifier. And importantly, this approach generalizes across tumor tissue types and capture kits and has a call rate of 100%. The interpretable feature masks of the attentive deep learning model explain the reasons for misclassified variants. We reproduce the recent finding that tumor-only TMB estimates for Black patients are extremely inflated relative to that of White patients due to the racial biases of germline databases. We show that our machine learning approach appreciably reduces this racial bias in tumor-only variant-calling.


title: solPredict: Antibody apparent solubility prediction from sequence by transfer learning

abstract: There is growing interest in developing therapeutic mAbs for the route of subcutaneous administration for several reasons, including patient convenience and compliance. This requires identifying mAbs with superior solubility that are amenable for high-concentration formulation development. However, early selection of developable antibodies with optimal high-concentration attributes remains challenging. Since experimental screening is often material and labor intensive, there is significant interest in developing robust in silico tools capable of screening thousands of molecules based on sequence information alone. In this paper, we present a strategy applying protein language modeling, named solPredict, to predict the apparent solubility of mAbs in histidine (pH 6.0) buffer condition. solPredict inputs embeddings extracted from pretrained protein language model from single sequences into a shallow neutral network. A dataset of 220 diverse, in-house mAbs, with extrapolated protein solubility data obtained from PEG-induced precipitation method, were used for model training and hyperparameter tuning through five-fold cross validation. An independent test set of 40 mAbs were used for model evaluation. solPredict achieves high correlation with experimental data (Spearman correlation coefficient = 0.86, Pearson correlation coefficient = 0.84, R2 = 0.69, and RMSE = 4.40). The output from solPredict directly corresponds to experimental solubility measurements (PEG %) and enables quantitative interpretation of results. This approach eliminates the need of 3D structure modeling of mAbs, descriptor computation, and expert-crafted input features. The minimal computational expense of solPredict enables rapid, large-scale, and high-throughput screening of mAbs during early antibody discovery.


title: AttentionSiteDTI: Attention Based Model for Predicting Drug-Target Interaction Using 3D Structure of Protein Binding Sites

abstract: In this study, we introduce and implement an interpretable graph-based deep learning prediction model, which utilizes protein binding sites along with self-attention to learn which protein binding sites interact with a given ligand. Our proposed model enables interpretability by identifying the protein binding sites that contribute the most towards the Drug-Target Interaction. Results on three benchmark datasets show improved performance compared to previous graph-based models. More significantly, unlike previous studies our model performance remains close to the optimal performance when tested with new proteins (ie., high generalizablity). Through multidisciplinary collaboration, we further experimentally evaluate the practical potential of our proposed approach. To achieve this, we first computationally predict binding interaction of some candidate compounds with a target protein, then experimentally validate the binding interactions for these pairs in the laboratory. The high agreement between the computationally-predicted and experimentally-observed (measured) DTIs illustrates the potential of our method as an effective pre-screening tool in drug re-purposing applications.


title: Interpretable and Generalizable Attention-Based Model for Predicting Drug-Target Interaction Using 3D Structure of Protein Binding Sites: SARS-CoV-2 Case Study and in-Lab Validation


title: Predicting the Toxicity of Druggable Proteins to Human Tissues

abstract: Assessing in vivo tissue toxicity of therapeutic targets remains a major challenge in drug development and drug safety research. We developed TissueTox, an algorithm that learns from multi-omic features of a target protein and predicts toxicity in human body systems and tissues. Predicted TissueTox scores accurately differentiate drugs that failed clinical trials from those that succeeded, and, importantly, can be used to identify the tissues where toxic events occurred.


title: Consequence of adjustments for demographic or clinical covariates and a recommended solution in genome-wide association studies

abstract: Genome-wide association study (GWAS) is fundamentally designed to detect disease-causing genes. To reduce spurious associations or improve statistical power, about 80% of GWASs arbitrarily adjusted for demographic or clinical covariates. However, adjustment strategies in GWASs have not achieved consistent conclusions. Given the initial aim of GWAS that is to identify the causal association between a specific causal single-nucleotide polymorphism (SNP) and disease trait, we summarized all complex relationships of the target SNP, covariate and disease trait into 15 causal diagrams according to various roles of the covariate. Following each causal diagram, we conducted a series of theoretical justifications and statistical simulations. Our results demonstrate that it is unadvisable to adjust for any demographic or clinical covariates. We illustrate our point by applying GWASs for body mass index (BMI) and breast cancer, including adjusting and non-adjusting for age and smoking status. Genetic effects and P values might vary across different strategies. Instead, adjustments for SNPs (G') should be strongly recommended when G' are in linkage disequilibrium with the target SNP, and correlated with disease trait conditional on the target SNP. Specifically, adjustment for such G' can block all the confounding paths between the target SNP and disease trait, and avoid over-adjusting for colliders or intermediaries.


title: Revealing a hidden intermediate of rotatory catalysis with X-ray crystallography and Molecular simulations

abstract: The mechanism of rotatory catalysis in ATP-hydrolyzing molecular motors remain an unresolved puzzle in biological energy transfer. Notwith standing the wealth of available biochemical and structural information inferred from years of experiments, knowledge on how the coupling between the chemical and mechanical steps within motors enforces directional rotatory movements remains fragmentary. Even more contentious is to pinpoint the rate-limiting step of a multi-step rotation process. Here, using Vacuolar or V1-type hexameric ATPase as an exemplary rotational motor, we present a model of the complete 4-step conformational cycle involved in rotatory catalysis. First, using X-ray crystallography a new intermediate or  dwell is identified, which enables the release of an inorganic phosphate (or Pi) after ATP hydrolysis. Using molecular dynamics simulations, this new dwell is placed in a sequence with three other crystal structures to derive a putative cyclic rotation path. Free-energy simulations are employed to estimate the rate of the hexameric protein transformations, and delineate allosteric effects that allow new reactant ATP entry only after hydrolysis product exit. An analysis of transfer entropy brings to light how the sidechain-level interactions transcend into larger-scale reorganizations, highlighting the role of the ubiquitous arginine-finger residues in coupling chemical and mechanical information. Inspection of all known rates encompassing the 4-step rotation mechanism implicates overcoming of the ADP interactions with V1-ATPase to be the rate-limiting step of motor action.

category: biophysics


title: Differential Histone-DNA Interactions Dictate Nucleosome Recognition of the Pioneer Transcription Factor Sox

abstract: Pioneer transcription factors (PTFs) have the remarkable ability to directly bind to chromatin for stimulating vital cellular processes. In this work, we dissect the universal binding mode of Sox PTF by combining extensive molecular simulations and DNA footprinting techniques. As a result, we show that when Sox consensus DNA is located at the solvent-facing DNA strand, Sox binds to the compact nucleosome without imposing any significant conformational changes. We also reveal that the base-specific Sox:DNA interactions (base reading) and the Sox-induced DNA changes (shape reading) are concurrently required for sequence-specific DNA recognition. Among different nucleosomal positions, such a specific reading mechanism is satisfied solely at superhelical location 2 (SHL2). While SHL2 acts transparently to Sox binding, SHL4 permits only shape reading, and SHL0 (dyad) allows no reading mechanism. These findings demonstrate for the first time that Sox-based nucleosome recognition is essentially guided by the distinct histone-DNA interactions, permitting varying degrees of DNA flexibility.


title: Scalable Adaptive Protein Ensemble Refinement Integrating Flexible Fitting

abstract: Recent advances in cryo-electron microscopy (cryo-EM) has enabled modeling macro-molecular complexes that are essential components of life. The density maps obtained from cryo-EM experiments is often integrated with ab - initio, knowledge-driven or first principles-based computational methods to build, fit and refine protein structures inside the electron density maps. Going beyond a single stationary-structure determination scheme, it is becoming more common to interpret the experimental data with a set of multiple physical models all of which contributes to the average observation seen by the experiment. Hence, there is a need to decide on the quality of an ensemble of protein structures on-the-fly, while refining them against the density maps. In this work, we demonstrate such adaptive decision making capabilities during flexible fitting of biomolecules. Our solution uses RADICAL tools (RCT) and we test this new implementation in exascale high performance computing environment for two proteins, Adenylate Kinase (ADK) and Carbon Monoxide Dehydrogenase (CODH). Our results indicate that using multiple replicas in flexible fitting with adaptive decision making improves the overall quality of fit and model by 40 % improvement when compared against the traditional flexible fitting approach. These advances are agnostic to system-size and computing environments.


title: High genetic diversity of bivalve transmissible neoplasia in the blue mussel Mytilus trossulus Gould from the subarctic Sea of Okhotsk

abstract: There are increasing findings of the bivalve transmissible neoplasia derived from the Pacific mussel Mytilus trossulus (MtrBTN) in populations of different Mytilus species worldwide. The Subarctic is an area where this disease has not yet been sought despite the fact that Mytilus spp. are widespread there, and M. trossulus itself is a boreal species. We used cytological and histological techniques to diagnose disseminated neoplasia in a sample of M. trossulus from Magadan in the subarctic Sea of Okhotsk. Neoplasia was identified in 11 of 214 mussels studied. Using mtDNA COI sequencing, we revealed genotypes identical or nearly identical to known MtrBTN ones in the hemolymph of most of the diseased mussels. Both MtrBTN evolutionary lineages have been identified, the widespread MtrBTN2, and MtrBTN1, so far only known from M. trossulus in British Columbia on the other side of the Pacific from Magadan. In addition, MtrBTN2 was represented by two common diverged mtDNA haplolineages. These conclusions have been confirmed for selected cancerous mussels by molecular cloning of COI and additional nuclear and mtDNA genes. On the background of high genetic diversity, different cancers were similar in terms of ploidy (range 4.0 - 5.8n) and nuclear to cell ratio. Our study provides the first description of neoplasia and MtrBTN in mussels from the Sea of Okhotsk and from the Subarctic, of both MtrBTN1 and MtrBTN2 in the same mussel population, and the first direct comparison between these transmissible cancers.

category: cancer biology


title: Compromised CDK12 activity causes dependency on the non-essential spliceosome components

abstract:
Prostate cancer (PC) is the most common cancer in men and after development of the castration-resistant PC (CRPC), there are no curative treatment options. Inactivating mutations in cyclin-dependent kinase 12 (CDK12) define an aggressive sub-type of CRPC. We hypothesized that compromised CDK12 activity leads to significant rewiring of the CRPC cells, and that this rewiring results in actionable synthetic lethal interactions.

MethodsWe used combinatorial lethal screening, ChIP-seq data, RNA-seq data, global alternative splicing analysis, and comprehensive mass spectrometry (MS) profiling to understand how the compromised CDK12 activity rewires the CRPC cells. In addition, we used DepMap-, PC- and CRPC-datasets as a strategy to identify factors that are selectively required by the CDK12-mutant cells.

ResultsWe show that inhibition of O-GlcNAc transferase (OGT) and CDK12 induces cancer cell-selective growth-defect. OGT catalyzes all nucleocytoplasmic O-GlcNAcylation, and we use unbiased MS-profiling to show that the short-term CDK12 inhibition induces hyper-O-GlcNAcylation of the spliceosome-machinery in PC and CRPC cells. Integration of DepMap- and a small scale-drug screen data reveled that depletion of CDK12 activity causes addiction to non-essential spliceosome components (CLK1/4 and SRPK1). CDK12-mutant tumors overexpress CLK1/4 and SRPK1. Finally, we show that the genomes of the CDK12-mutant tumors have lower DNA methylation, and that CDK12 inhibition induces the expression of the genes marked by DNA methylation.

ConclusionsCompromised CDK12 activity rewires DNA methylation, transcription and splicing, and this rewiring renders the affected cells addicted on the non-essential spliceosome components. We propose that inactivation of CDK12 is a biomarker for sensitivity against inhibitors of the non-essential spliceosome components just entering the clinical trials.

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title: Physiological activation of the nephron central command drives endogenous kidney tissue regeneration

abstract: Tissue regeneration is limited in several organs including the kidney, contributing to the high prevalence of kidney disease globally. However, evolutionary and physiological adaptive responses and the presence of renal progenitor cells suggest existing remodeling capacity. This study uncovered a novel endogenous tissue remodeling mechanism in the kidney that is activated by the loss of body fluid and salt and involves a unique niche of chief cells called macula densa (MD) that control resident progenitor cells via secreted angiogenic, growth and extracellular matrix remodeling factors, cytokines and chemokines. Serial intravital imaging, MD Wnt mouse models and transcriptome analysis provide functional and molecular characterization of this newly identified MD program for kidney regeneration complemented with human and therapeutic translation. The concept that chief cells responding to organ-specific physiological inputs control local progenitors and direct them to remodel or repair tissues may be applicable to other organs and diverse tissue regenerative therapeutic strategies.

category: physiology


title: GOLDEN TANGERINE TOMATO ROOTS SHOW INCREASED ACCUMULATION OF ACYCLIC CAROTENOIDS, LESS ABSCISIC ACID, DROUGHT SENSITIVITY, AND IMPAIRED ENDOMYCORRHIZAL COLONIZATION

abstract:
Heirloom golden tomato fruit varieties are highly nutritious as they accumulate tetra-cis-lycopene, which has a higher bioavailability and recognised health benefits in treating anti-inflammatory diseases compared to all-trans-lycopene isomers found in red tomatoes. We investigated if photoisomerization of tetra-cis-lycopene occurs in roots of the golden tangerine Micro-Tom variety (tangmic), and how this affects root to shoot biomass, mycorrhizal colonization, abscisic acid accumulation, and responses to drought. tangmic plants grown in soil under glasshouse conditions displayed a reduction in height, number of flowers, fruit yield, and root length compared to wild type (WT). Soil inoculation with Rhizophagus irregularis revealed fewer arbuscules and other fungal structures in the endodermal cells of roots in tangmic relative to WT. The roots of tangmic hyperaccumulated acyclic cis-carotenes, while only trace levels of xanthophylls and abscisic acid were detected. In response to a water deficit, leaves from the tangmic plants displayed a rapid decline in maximum quantum yield of photosystem II compared to WT, indicating a defective root to shoot signalling response to drought. The lack of xanthophylls biosynthesis in tangmic roots reduced abscisic acid levels, thereby likely impairing endomycorrhiza colonisation and drought-induced root to shoot signalling.

Research HighlightsO_LIPhotoisomerization of prolycopene to lycopene is limited in root plastids.
C_LIO_LIRoots of tangerine reveal an important tissue sink to store micronutrients such as prolycopene.
C_LIO_LIRoots of tangerine lack ABA and show impaired mycorrhizal colonization.
C_LIO_LIThe tangerine plant is drought sensitive and has a smaller biomass as well as reduced yield.
C_LI


title: Recut: a Concurrent Framework for Sparse Reconstruction of Neuronal Morphology

abstract: Advancement in modern neuroscience is bottlenecked by neural reconstruction, a process that extracts 3D neuron morphology (typically in tree structures) from image volumes at the scale of hundreds of GBs. We introduce Recut, an automated and accelerated neural reconstruction pipeline, which provides a unified, and domain specific sparse data representation with 79x reduction in the memory footprint. Recuts reconstruction can process 111 Kneurons/day or 79 TB/day on a 24-core workstation, placing the throughput bottleneck back on microscopic imaging time. Recut allows the full brain of a mouse to be processed in memory on a single server, at 89.5x higher throughput over existing I/O-bounded methods. Recut is also the first fully parallelized end-to-end automated reconstruction pipeline for light microscopy, yielding tree morphologies closer to ground truth than the state-of-the-art while removing involved manual steps and disk I/O overheads. We also optimized pipeline stages to linear algorithmic complexity for scalability in dense settings and allow the most timing-critical stages to optionally run on accelerated hardware.


title: Early stage differentiation of glia in human and mice.

abstract: Central nervous system macroglia (astrocytes and oligodendrocytes) are required for normal brain development and function, and are among the last cells to emerge during neurodevelopment. Many questions remain about their emergence in the brain and spinal cord, including how early glial fates are specified during development or differentiation, and similarly when subtypes of glia are specified. Here, we used single-cell RNA sequencing (scRNAseq) to analyze [~]90,000 cells across multiple timepoints during the differentiation of astrocytes and oligodendrocytes from human induced pluripotent stem cells and mouse embryonic stem cells. Using time series analysis of gene expression, we uncovered multiple genes involved in fate specification of glial subtypes in both species. We examined gene expression changes during intermediate states of glial specification, and were able to identify genes that were correlated with the choice between neuron versus glia in both species. Using our scRNAseq data we optimized previous mouse astrocyte differentiation protocols by highlighting and removing non-required transition states and decreasing the overall protocol from 3 weeks to less than 12 days. Our data will be useful for researchers interested in optimizing glial differentiations in either species, and provide a window into human glial differentiation, which is difficult to study given its lateness in development.


title: Reconfigurations in brain networks upon awakening from slow wave sleep: Interventions and implications in neural communication

abstract:
Sleep inertia is the brief period of impaired alertness and performance experienced immediately after waking. While the neurobehavioral symptoms of sleep inertia are well-described, less is known about the neural mechanisms underlying this phenomenon. A better understanding of the neural processes during sleep inertia may offer insight into the cognitive impairments observed and the awakening process generally. We observed brain activity following abrupt awakening from slow wave sleep during the biological night. Using electroencephalography (EEG) and a network science approach, we evaluated power, clustering coefficient, and path length across frequency bands under both a control condition and a blue-enriched light intervention condition in a within-subject design. We found that under control conditions, the awakening brain is typified by an immediate reduction in global theta, alpha, and beta power. Simultaneously, we observed a decrease in the clustering coefficient and an increase in path length within the delta band. Exposure to blue-enriched light immediately after awakening ameliorated these changes, but only for clustering. Our results suggest that long-range network communication within the brain is crucial to the waking process and that the brain may prioritize these long-range connections during this transitional state. Our study highlights a novel neurophysiological signature of the awakening brain and provides a potential mechanistic explanation for the effect of light in improving performance after waking.

One sentence summaryBlue-enriched light partially accelerates the rapid prioritization of long-range communication within the human brain that characterizes sleep inertia


title: High-Speed Low-Light In Vivo Two-Photon Voltage Imaging of Large Neuronal Populations

abstract: Monitoring spiking activity across large neuronal populations at behaviorally relevant timescales is critical for understanding neural circuit function. Unlike calcium imaging, voltage imaging requires kilohertz sampling rates which reduces fluorescence detection to near shot noise levels. High-photon flux excitation can overcome photon-limited shot noise but photo-bleaching and photo-damage restricts the number and duration of simultaneously imaged neurons. We investigated an alternative approach aimed at low two-photon flux, voltage imaging below the shot noise limit. This framework involved developing: a positive-going voltage indicator with improved spike detection (SpikeyGi); an ultra-fast two-photon microscope for kilohertz frame-rate imaging across a 0.4x0.4mm2 field of view, and; a self-supervised denoising algorithm (DeepVID) for inferring fluorescence from shot-noise limited signals. Through these combined advances, we achieved simultaneous high-speed, deep-tissue imaging of more than one hundred densely-labeled neurons over one hour in awake behaving mice. This demonstrates a scalable approach for voltage imaging across increasing neuronal populations.


title: Linker histone H1FOO is required for bovine preimplantation development by regulating lineage specification and nucleosome assembly

abstract: Linker histone H1 binds to the nucleosome and is implicated in the regulation of the chromatin structure and function. The H1 variant H1FOO is heavily expressed in oocytes and early embryos. However, given the poor homology of H1FOO among mammals, the functional role of H1FOO during early embryonic development remains largely unknown, especially in domestic animals. Here, we find that H1FOO is not only expressed in oocytes and early embryos but granulosa cells and spermatids in cattle. We then demonstrate that the interference of H1FOO results in early embryonic developmental arrest in cattle using either RNA editing or Trim-Away approach. H1FOO depletion leads to compromised expression of critical lineage-specific genes at the morula stage and affects the establishment of cell polarity. Interestingly, H1FOO depletion causes a significant increase in expression genes encoding other linker H1 and core histones. Concurrently, there is an increase of H3K9me3 and H3K27me3, two markers of repressive chromatin and a decrease of H4K16ac, a marker of open chromatin. Importantly, overexpression of bovine H1FOO results in severe embryonic developmental defects. In sum, we propose that H1FOO controls the proper chromatin structure that is crucial for the fidelity of cell polarization and lineage specification during bovine early development.


title: Severe disturbance overflows the stabilizing buffer of variable biotic interactions

abstract: Recent studies have uncovered that biotic interaction strength varies over time in real ecosystems intrinsically and/or responding to anthropogenic disturbances. Little is known, however, about whether such interaction variability strengthens or weakens community resistance against disturbances. Here, we examine how the change in interaction strength after pesticide application mediates disturbance impacts on a freshwater community using outdoor mesocosms. We show that the change in interaction strength buffered the disturbance impact but amplified it once the disturbance severity exceeded a certain threshold. Importantly, we also show that interactions fluctuating more temporally under no disturbances were more changeable in response to pesticide applications. Our findings suggest that a severe disturbance may have a surprise impact on a biological community amplified by their own interaction variability, but the possibility still remains that we can predict the consequences of the disturbance by measuring the interaction variability before the disturbance occurs.

category: ecology


title: Trailing-edge zombie forests can increase population persistence in the face of climate change

abstract: Climate-driven habitat shifts pose challenges for dispersal-limited, late-maturing taxa such as trees. Older trees are often the most reproductive individuals in the population, but as habitats shift, these individuals can be left behind in the trailing range edge, generating "zombie forests" that may persist long after the suitable habitat has shifted. Are these zombie forests vestiges of ecosystems past or do they play an ecological role? To understand how zombie forests affect population persistence, we developed a spatially explicit, stage-structured model of tree populations occupying a shifting habitat. Our model shows that seed dispersal from zombie forests to the range core can considerably increase the maximum rate of climate change that a population can withstand. Moreover, the entire core population can ultimately descend from recruitment-limited zombie forests, highlighting their demographic value. Our results suggest that preserving trailing-edge zombie forests can greatly increase population persistence in the face of climate change.


title: BSImp: imputing partially observed methylation patterns for evaluating methylation heterogeneity

abstract: DNA methylation is one of the most studied epigenetic modifications that has applications ranging from transcriptional regulation to aging, and can be assessed by bisulfite sequencing (BS-seq) at single base-pair resolution. The permutations of methylation statuses at bisulfite converted reads reflect the methylation patterns of individual cells. These patterns at specific genomic locations are sought to be indicative of cellular heterogeneity within a cellular population, which are predictive of developments and diseases; therefore, methylation heterogeneity has potentials in early detection of these changes. Computational methods have been developed to assess methylation heterogeneity using methylation patterns formed by four CpGs, but the nature of shotgun sequencing often give partially observed patterns, which makes very limited data available for downstream analysis. While many programs are developed to impute methylation levels genomewide, currently there is only one method developed for recovering partially observed methylation patterns; however, the program needs lots of data to train and cannot be used directly; therefore, we developed a probabilistic-based imputation method that uses information from neighbouring sites to recover partially observed methylation patterns speedily. It is demonstrated to allow for the evaluation of methylation heterogeneity at three times more regions genome-wide with high accuracy for data with moderate depth. To make it more user-friendly we also provide a computational pipeline for genome-screening, which can be used in both evaluating methylation levels and profiling methylation patterns genomewide for all cytosine contexts, which is the first of its kind. Our method allows for accurate estimation of methylation levels and makes evaluating methylation heterogeneity available for much more data with reasonable coverage, which has important implications in using methylation heterogeneity for monitoring changes within the cellular populations that were impossible to detect for the assessment of development and diseases.


title: Structural mechanism for bi-directional actin crosslinking by T-plastin

abstract:
To fulfill the cytoskeletons diverse functions in cell mechanics and motility, actin networks with specialized architectures are built by crosslinking proteins, which bridge filaments to control micron-scale network geometry through nanoscale binding interactions via poorly defined structural mechanisms. Here, we introduce a machine-learning enabled cryo-EM pipeline for visualizing active crosslinkers, which we use to analyze human T-plastin, a member of the evolutionarily ancient plastin/fimbrin family of tandem calponin-homology domain (CHD) proteins. We define a sequential bundling mechanism which enables T-plastin to bridge filaments in both parallel and anti-parallel orientations. Our structural, biochemical, and cell biological data highlight inter-CHD linkers as key structural elements underlying flexible but stable crosslinking which are likely to be disrupted by mutations causing hereditary bone diseases. Beyond revealing how plastins are evolutionary optimized to crosslink dense actin networks with mixed polarity, our cryo-EM workflow will broadly enable analysis of the structural mechanisms underlying cytoskeletal network construction.

One sentence summaryCryo-EM, biochemical, and cellular studies reveal how the crosslinking protein T-plastin bridges actin filaments in two opposing orientations.


title: The eukaryotic translation initiation factor eIF4E reprogrammes the splicing machinery anddrives alternative splicing

abstract: Aberrant RNA splicing contributes to the pathogenesis of many malignancies including Acute Myeloid Leukemia (AML). While mutation is the best described mechanism underpinning aberrant splicing, recent studies show that predictions based on mutations alone likely underestimate the extent of this dysregulation. Here, we show that elevation of the eukaryotic translation initiation factor eIF4E reprogrammes splicing of nearly a thousand RNAs in model cell lines. In AML patient specimens which did not harbour known splice factor mutations, ~4000 transcripts were differentially spliced based on eIF4E levels and this was associated with poor prognosis. Inhibition of eIF4E in cell lines reverted the eIF4E-dependent splice events examined. Splicing targets of eIF4E act in biological processes consistent with its role in malignancy. This altered splicing program likely arose from eIF4E-dependnet increases in the production of many components of the spliceosome including SF3B1 and U2AF1 which are frequently mutated in AML. Notably, eIF4E did not drive mutation of these factors, only their production. eIF4E also physically associated with many splice factors including SF3B1, U2AF1, and UsnRNAs. Importantly, many eIF4E-dependent splice events differed from those arising from SF3B1 mutation, and were more extensive highlighting that these splicing profiles arise from distinct mechanisms. In all, our studies provide a paradigm for how dysregulation of a single factor, eIF4E, can alter splicing.


title: Novel Fusarium Wilt Resistance Genes Uncovered in the Wild Progenitors and Heirloom Cultivars of Strawberry

abstract:
Fusarium wilt, a soilborne disease caused by Fusarium oxysporum f. sp. fragariae, poses a significant threat to strawberry (Fragaria x ananassa) production in many parts of the world. This pathogen causes wilting, collapse, and death in susceptible genotypes. We previously identified a dominant gene (FW1) on chromosome 2B that confers resistance to race 1 of the pathogen and hypothesized that gene-for-gene resistance to Fusarium wilt was widespread in strawberry. To explore this, a genetically diverse collection of heirloom and modern cultivars and wild octoploid ecotypes were screened for resistance to Fusarium wilt races 1 and 2. Here we show that resistance to both races is widespread and that resistance to race 1 is mediated by dominant genes (FW1, FW2, FW3, FW4, and FW5) on three non-homoeologous chromosomes (1A, 2B, and 6B). The resistance proteins encoded by these genes are not yet known; however, plausible candidates were identified that encode pattern recognition receptor or other proteins known to mediate gene-for-gene resistance in plants.

High-throughput genotyping assays for SNPs in linkage disequilibrium with FW1-FW5 were developed to facilitate marker-assisted selection and accelerate the development of race 1 resistant cultivars. This study laid the foundation for identifying the genes encoded by FW1-FW5, in addition to exploring the genetics of resistance to race 2 and other races of the pathogen, as a precaution to averting a Fusarium wilt pandemic.

Key MessageSeveral race-specific resistance genes were identified and rapidly deployed via marker-assisted selection to develop strawberry cultivars resistant to Fusarium wilt, a devastating soil-borne disease.


title: Structural basis for substrate recognition, ligation and activation by a hyperactive Asn peptide ligase from Viola yedoensis

abstract:
Peptide asparaginyl ligases (PALs) belong to a limited class of enzymes from cyclotide-producing plants, that perform site-specific ligation reactions after a target peptide Asx (Asn/Asp) binds to the ligase active site. How PALs specifically recognize their polypeptide substrates has remained elusive especially at the prime binding side of the enzyme. Here we captured VyPAL2, a catalytically efficient PAL from Viola yedoensis, in an activated state, with and without a bound substrate. The bound structure shows one ligase with the N-terminal polypeptide tail from another ligase molecule trapped at its active site, revealing how Asx inserts in the enzymes S1 pocket and why a hydrophobic residue is required at the substrate P2 position. Beside illustrating the role played by P1 and P2 residues as primary anchors for the enzyme reaction, these results provide a mechanistic explanation for the role of the "Gatekeeper" residue at the surface of the S2 pocket, in shifting the non-prime portion of the substrate and, as a result, the activity towards either ligation or hydrolysis. These results detail the molecular events that occur during proenzyme maturation in the plant vacuolar compartment, suggest a mechanism for ligation, and will inform the design of peptide ligases with tailored specificities.

One sentence summaryWe captured VyPAL2, a catalytically efficient plant peptide ligase with a bound substrate, providing the molecular basis for substrate recognition and ligation.

category: biochemistry


title: Allosteric regulation of the proteasomes catalytic sites by the proteasome activator PA28gamma/REGgamma

abstract:
Proteasome Activator 28{gamma} (PA28{gamma}) is a member of the 11S family of proteasomal regulators that is constitutively expressed in the nucleus and is implicated in certain cancers, lupus, rheumatoid arthritis, and Poly-glutamine neurodegenerative diseases. However, how PA28{gamma} functions in protein degradation remains unclear. Though PA28{gamma}s mechanism has been investigated for some time, many alternative hypotheses have not been tested: e.g. 1) substrate selection, 2) allosteric upregulation of the Trypsin-like catalytic site, 3) allosteric inhibition of the Chymotrypsin- and Caspase-like catalytic sites, 4) conversion of the Chymotrypsin- or Caspase-like sites to new Trypsin-like catalytic sites, and 5) gate-opening in combination with these. The purpose of this study was to conclusively determine how PA28{gamma} regulates proteasome function. Here, we rigorously and definitively show that PA28{gamma} uses an allosteric mechanism to upregulate the proteolytic activity of the 20S proteasomes Trypsin-like catalytic site. Using a constitutively open channel proteasome, we were able to dissociate gating affects from catalytic affects demonstrating that the PA28{gamma}-increases the affinity (Km) and Vmax for Trypsin-like peptide substrates. Mutagenesis of PA28{gamma} also reveals that it does not select for (i.e. filter) peptide substrates, and does not change the specificity of the other active sites to trypsin-like. Further, using Cryo-EM we were able to visualize the C7 symmetric PA28{gamma}-20S proteasome complex at 4.4[A] validating its expected 11S-like quaternary structure and proteasome binding mode. The results of this study provide unambiguous evidence that PA28{gamma} functions by allosterically upregulating the T-L like site in the 20S proteasome.

Significance StatementThis study rigorously demonstrates that PA28g allosterically activates the b-2 proteolytic site of the 20S proteasome directly without affecting 20S gating. Further, we generated the first human 11S-human 20S proteasome cryo-EM structure of the PA28g-20S complex showing that, despite its different affects on 20S activity, it has a similar quaternary structure as the other 11S family members. The significance of these findings is paramount as the b-2 site is responsible for post-basic cleavage and suggests that PA28g is specialized to degrade positively charged DNA binding proteins. Further, b-2 upregulation via PA28g could provide a protective effect against poly-glutamine expanded proteins, like Huntingtin. This work provides a framework for PA28g drug development to treat PA28g addicted cancers and Huntingtons Disease.


title: MeasureIce: Accessible on-the-fly measurement of ice thickness in cryo-electron microscopy

abstract: Ice thickness is arguably one of the most important factors limiting the resolution of protein structures determined by cryo electron microscopy. The amorphous atomic structure of the ice that stabilizes and protects biological samples in cryo-EM grids also imprints some addition noise in the TEM images. Ice that is too thick jeopardizes the success of particle picking and reconstruction of the biomolecule in the worst case and, at best, deteriorates eventual map resolution. Minimizing the thickness of the ice layer and thus the magnitude of its noise contribution is thus imperative in cryo-EM grid preparation. In this paper we introduce MeasureIce, a simple, easy to use ice thickness measurement tool for screening and selecting acquisition areas of cryo-EM grids. We show that it is possible to simulate thickness-image intensity look-up tables using elementary scattering physics and thereby adapt the tool to any microscope without time consuming experimental calibration. We benchmark our approach using two alternative techniques: the "ice-channel" technique and tilt-series tomography. We also demonstrate the utility of ice thickness measurement for selecting holes in gold grids containing an Equine apoferritin sample, achieving a 1.88 [A]ngstrom resolution in subsequent refinement of the atomic map.


title: Long Noncoding RNA RROL Provides Chromatin Scaffold for MYC-WDR82 Interaction to Impact Lipid Metabolism and Tumor Cell Growth in Multiple Myeloma

abstract: Long noncoding RNAs (lncRNA) can drive the tumorigenesis and be susceptible to therapeutic intervention. To define the landscape of therapeutically actionable lncRNA dependencies in multiple myeloma (MM), we coupled our extensive lncRNA transcriptomic profile with lncRNA targeted CRISPR interference viability screen and identified RNA Regulator of Lipogenesis (RROL) as a leading lncRNA dependency in MM. RROL shares its origin with the microRNA locus MIR17HG, however supports the proliferation and survival of MM cells in a microRNA- and DROSHA- independent manner. We found that RROL provides a chromatin scaffold for the functional interaction between c-MYC and WDR82 to promote the regulation of the lipogenic pathways via the transcriptional control of the rate-limiting enzyme ACC1 in MM cells. Inhibition of RROL with clinically applicable antisense molecules disrupts its transcriptional and functional activities causing potent anti-tumor effects both in vitro and in vivo in two pre-clinical animal models. This study establishes lncRNA RROL as a therapeutically actionable dependency with a unique mechanism of action in support of myeloma cell growth.


title: EIF4EBP1 is transcriptionally upregulated by MYCN and associates with poor prognosis in neuroblastoma

abstract:
BackgroundNeuroblastoma (NB) accounts for 15% of cancer-related deaths in childhood despite considerable therapeutic improvements. While several risk factors, including MYCN amplification and alterations in RAS and p53 pathway genes, have been defined in NB, the clinical outcome is very variable and difficult to predict. Since genes of the mTOR pathway are up-regulated in MYCN-amplified NB, we aimed to define the predictive value of the mTOR substrate-encoding gene eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1) expression in NB patients.

MethodsSeveral independent NB patient cohorts with corresponding mRNA expression data were analyzed for EIF4EBP1 expression. An institutional NB cohort consisting of 69 prospectively collected tumors was employed to immunohistochemically analyze expression of EIF4EBP1-encoded protein (4EBP1). In addition, we performed an in vitro luciferase reporter gene assay with an episomal EIF4EBP1 promoter and genetically modulated MYCN expression in NB cells.

FindingsEIF4EBP1 mRNA expression was positively correlated with MYCN expression and elevated in stage 4 and high-risk NB patients. High EIF4EBP1 mRNA expression was associated with reduced overall and event-free survival in the entire group of NB patients in three cohorts, as well as in stage 4 and high-risk patients. High levels of 4EBP1 were significantly associated with prognostically unfavorable NB histology. Functional analyses in vitro revealed that EIF4EBP1 expression is transcriptionally controlled by MYCN binding to the EIF4EBP1 promoter.

InterpretationHigh EIF4EBP1 expression is associated with poor prognosis in NB patients and may serve to stratify patients with high-risk NB.

FundingG.L. was supported by funding from the Elterninitiative Dusseldorf e.V., the Research Commission of the Medical Faculty of Heinrich Heine University, the Deutsche Forschungsgemeinschaft (Grant LE 3751/2-1), and the German Cancer Aid (Grant 70112624). The laboratory of T.G.P.G. is supported by the Barbara und Wilfried Mohr Foundation. BR is supported by the Israel Science Foundation (grant No. 1436/19).

RESEARCH IN CONTEXT Evidence before this studyNB represents a particularly heterogeneous cancer entity, with 5-year event-free survival rate ranging from 50% to 98% depending on the patients risk group. While genes of the nutrient-sensing mTOR pathway were found to be up-regulated in MYCN-amplified NB tumors, their clinical relevance and prognostic value in NB patients remain unclear. In particular, the mTOR substrate-encoding gene EIF4EBP1 was studied in NB by three different groups and high EIF4EBP1 mRNA expression was observed in MYCN-amplified or contradictorily in more favorable stages 1 and 2 patients. Also, EIF4EBP1 was included in a prognostic gene signature for poor overall survival in NB. However, the prognostic value of EIF4EBP1 alone was not determined in NB and the expression of EIF4EBP1 encoded protein, 4EBP1, was not analyzed in NB tumor tissues and not correlated with clinicopathological features such as histological subtypes. Additionally, the transcriptional regulation of the EIF4EBP1 promoter by MYCN was not characterized.

Added value of this studyThis study uncovers the prognostic potential of EIF4EBP1 at the mRNA and protein levels in NB patients. We report that high EIF4EBP1 expression is correlated with poor survival in three independent cohorts and that high 4EBP1 levels is associated with a prognostically unfavorable histological subtype. High EIF4EBP1 expression is also a factor of poor prognosis in stage 4 and high-risk patient groups. Finally, we found that MYCN activates the human EIF4EBP1 promoter through binding at three binding motifs.

Implications of all the available evidenceEIF4EBP1 mRNA and 4EBP1 protein expression have prognostic value in NB, especially to stratify patients with advanced and more aggressive NB, such as patients with stage 4 disease and high-risk patients including those with unfavorable histological subtype NB. Enhanced EIF4EBP1 mRNA and 4EBP1 protein expression in NB are driven by direct transcriptional activation of EIF4EBP1 by MYCN.


title: Stress-impaired reward pathway promotes distinct feeding behavior patterns

abstract:
Psychosocial stress can impact feeding behavior outcomes. Although many studies have examined alterations to food intake, little is known about how stress affects feeding behavior patterns. To determine the impact of psychological stress on feeding behavior patterns, mice were subjected to various psychosocial stressors (social isolation, intermittent high-fat-diet, or physical restraint) prior to timed observations in a feeding arena that incorporated multiple bait loci. In addition, in vivo microdialysis was used to assess the effects of stressors on the reward system by measuring dopamine levels in the nucleus accumbens (NAcc) shell. Impaired feeding behavior patterns characterized by significant deviations in bait selection (i.e. fixated feeding) and prolonged periods of eating (i.e. protracted feeding) were observed in stressed mice relative to non-stressed controls. In addition to clear behavioral effects, the stressors also negatively impacted dopamine levels at the nucleus accumbens shell. Normalization of dopamine reversed the fixated feeding behavior, whereas specifically inhibiting neuronal activity in the dopaminergic neurons of the ventral tegmental area that project to the nucleus accumbens shell caused similar impairments in feeding. Given that the deviations were not consistently accompanied by changes in the amount of bait consumed, body weight, or metabolic factors, the qualitative effects of psychosocial stressors on feeding behavior likely reflect perturbations to a critical pathway in the mesolimbic dopamine system. These findings provide compelling evidence that aberrations in feeding behavior patterns can be developed as sensitive biomarkers of psychosocial stress and possibly a prodromal state of neuropsychiatric diseases.

Significance StatementFeeding behavior can be affected by neuropsychiatric disorders including psychosocial stressors, and the evaluation of eating behavior was mainly based on food intake. However, it is speculated that not only food intake but also feeding behavior patterns can be affected in such disorders. The biological processes underlying the feeding behavior patterns have not been clarified yet. We found that aberrant feeding behaviors in mice characterized by fixated feeding were provoked by psychosocial stressors. The qualitative effects of psychosocial stressors on feeding behavior reflect perturbations in the mesolimbic dopamine system. These findings provide compelling evidence that aberrations in feeding behavior patterns can be developed as sensitive biomarkers of psychosocial stress and possibly a prodromal state of neuropsychiatric diseases.


title: Neurexins in serotonergic neurons regulate serotonin transmission and complex mouse behaviors

abstract: Extensive serotonin (5-HT) innervation throughout the brain corroborates 5-HTs modulatory role in numerous cognitive activities. Volume transmission is the major mode for 5-HT transmission but mechanisms underlying 5-HT signaling are still largely unknown. Abnormal brain 5-HT levels and function have been implicated in autism spectrum disorder (ASD). Neurexin (Nrxn) genes encode presynaptic cell adhesion molecules important for the regulation of synaptic neurotransmitter release, notably glutamatergic and GABAergic transmission. Mutations in Nrxn genes are associated with neurodevelopmental disorders including ASD. However, the role of Nrxn genes in the 5-HT system is poorly understood. Here, we generated a mouse model with all three Nrxn genes disrupted specifically in 5-HT neurons to study how Nrxns affect 5-HT transmission. Loss of Nrxns in 5-HT neurons impaired 5-HT release in the dorsal raphe nucleus and dorsal hippocampus and decreased serotonin transporter distribution in specific brain areas. Furthermore, 5-HT neuron-specific Nrxn knockout reduced sociability and increased depressive-like behavior. Our results highlight functional roles for Nrxns in 5-HT neurotransmission and the execution of complex behaviors.


title: Role of anterior insula cortex in context-induced relapse of nicotine-seeking

abstract: Tobacco use is the leading cause of preventable death worldwide, and relapse during abstinence remains the key barrier to successful treatment of tobacco addiction. During abstinence, environmental contexts associated with nicotine use can induce craving and contribute to relapse. The insular cortex (IC) is thought to be a critical substrate of nicotine addiction and relapse. However, its specific role in context-induced relapse of nicotine-seeking is not fully known. In this study, we report a novel rodent model of context-induced relapse to nicotine-seeking after punishment-imposed abstinence, which models self-imposed abstinence through increasing negative consequences of excessive drug use. Using the neuronal activity marker Fos we find that the anterior (aIC), but not the middle or posterior IC, shows increased activity during context-induced relapse. Combining Fos with retrograde labelling of aIC inputs, we show projections to aIC from contralateral aIC and basolateral amygdala exhibit increased activity during context-induced relapse. Next, we used fiber photometry in aIC and observed phasic increases in aIC activity around nicotine-seeking responses during self-administration, punishment, and the context-induced relapse tests. Next, we used chemogenetic inhibition in both male and female rats to determine whether activity in aIC is necessary for context-induced relapse. We found that chemogenetic inhibition of aIC decreased context-induced nicotine-seeking after either punishment- or extinction-imposed abstinence. These findings highlight the critical role nicotine-associated contexts play in promoting relapse, and they show that aIC activity is critical for this context-induced relapse following both punishment and extinction imposed abstinence.


title: Drosophila gustatory projections are segregated by taste modality and connectivity

abstract: Gustatory sensory neurons detect caloric and harmful compounds in potential food and convey this information to the brain to inform feeding decisions. To examine the signals that gustatory neurons transmit and receive, we reconstructed gustatory axons and their synaptic sites in the adult Drosophila melanogaster brain, utilizing a whole-brain electron microscopy volume. We reconstructed 87 gustatory projections from the proboscis labellum in the right hemisphere and 57 from the left, representing the majority of labellar gustatory axons. Gustatory neurons contain a nearly equal number of interspersed pre-and post-synaptic sites, with extensive synaptic connectivity among gustatory axons. Morphology- and connectivity-based clustering revealed six distinct groups, likely representing neurons recognizing different taste modalities. The vast majority of synaptic connections are between neurons of the same group. This study resolves the anatomy of labellar gustatory projections, reveals that gustatory projections are segregated based on taste modality, and uncovers synaptic connections that may alter the transmission of gustatory signals.


title: Drosulfakinin signaling modulates female sexual receptivity in Drosophila

abstract: Female sexual behavior as an innate behavior is of prominent biological importance for survival and reproduction. However, molecular and circuit mechanisms underlying female sexual behavior is not well understood. Here, we identify the Cholecystokinin-like peptide Drosulfakinin (DSK) promotes female sexual behavior in Drosophila. Manipulation both Dsk and DSK neuronal activity impact female sexual receptivity. In addition, we reveal that Dsk-expressing neurons receive input signal from R71G01GAL4 neurons to promote female sexual receptivity. Based on intersectional technique, we further found the regulation of female sexual behavior relies mainly on medial DSK neurons rather than lateral DSK neurons, and medial DSK neurons modulate female sexual behavior by acting on its receptor CCKLR-17D3. Thus, we characterized DSK/CCKLR-17D3 as R71G01GAL4 neurons downstream signaling to regulate female sexual behavior.


title: A striatal circuit balances learned fear in the presence and absence of sensory cues

abstract: During fear learning, defensive behaviors need to be finely balanced, to allow animals to return to normal behaviors after the termination of threat-indicating sensory cues. Nevertheless, the circuits underlying such balancing are largely unknown. Here, we investigate the role of direct (D1R+) - and indirect (Adora+) pathway neurons of the amygdala-striatal transition zone (AStria) in fear learning. In-vivo Ca2+ imaging revealed that fear learning increased the responses of D1R+ AStria neurons to an auditory CS, given that the animal moved. In Adora+ neurons, fear learning also induced a differential activity during freezing and movement, albeit with little influence of the CS. In-vivo optogenetic silencing during the training day showed that plasticity in D1R+ AStria neurons contributes to auditory-cued fear memories, whereas Adora+ neurons suppressed learned freezing when no CS was present. Circuit tracing experiments identified cortical input structures to the AStria, and recording of optogenetically-evoked EPSCs at the corresponding projection revealed different forms of long-term plasticity at synapses onto D1R+ and Adora+ AStria neurons. Taken together, direct- and indirect pathways neurons of the AStria show differential signs of in-vivo and ex-vivo plasticity after fear learning, and balance defensive behaviors in the presence and absence of aversively motivated sensory cues.


title: Escape Steering by Cholecystokinin Peptidergic Signaling

abstract: Escape is an evolutionarily conserved and essential avoidance response. Considered to be innate, most studies on escape responses focused on hard-wired circuits. We report here that peptidergic signaling is an integral and necessary component of the Caenorhabditis elegans escape circuit. Combining genetic screening, electrophysiology and calcium imaging, we reveal that a neuropeptide NLP-18 and its cholecystokinin receptor CKR-1 enable the escape circuit to execute a full omega ({Omega}) turn, the last motor step where the animal robustly steers away from its original trajectory. We demonstrate in vivo and in vitro that CKR-1 is a Gq protein coupled receptor for NLP-18. in vivo, NLP-18 is mainly secreted by the gustatory sensory neuron (ASI) to activate CKR-1 in the head motor neuron (SMD) and the turn-initiating interneuron (AIB). Removal of NLP-18, removal of CKR-1, or specific knockdown of CKR-1 in SMD or AIB neurons lead to shallower turns hence less robust escape steering. Consistently, elevation of head motor neuron (SMD)s Ca2+ transients during escape steering is attenuated upon the removal of NLP-18 or CKR-1. in vitro, synthetic NLP-18 directly evokes CKR-1-dependent currents in oocytes and CKR-1-dependent Ca2+ transients in SMD. Thus, cholecystokinin signaling modulates an escape circuit to generate robust escape steering.


title: Manganese is a Physiologically Relevant TORC1 Activator in Yeast and Mammals.

abstract:
The essential biometal manganese (Mn) functions as a cofactor for several enzymatic activities that are critical for the prevention of human diseases. Whether intracellular Mn levels may also modulate signaling events has so far remained largely unexplored. The target of rapamycin complex 1 (TORC1, mTORC1 in mammals) is a conserved protein kinase complex that requires metal co-factors to phosphorylate its downstream effectors as part of a central, homeostatic process that coordinates cell growth and metabolism in response to nutrient and/or growth factor availability. Using genetic and biochemical approaches, we show here that TORC1 activity is exquisitely sensitive to stimulation by Mn both in vivo and in vitro. Mn-mediated control of TORC1 depends on Smf1 and Smf2, two members of the family of natural resistance-associated macrophage protein (NRAMP) metal ion transporters, the turnover of which is subjected to feedback control by TORC1 activity. Notably, increased Mn levels and consequent activation of TORC1 cause retrograde dysregulation and antagonize the rapamycin-induced gene expression and autophagy programs in yeast. Because Mn also activates mTORC1 signaling in aminoacid starved human cells, our data indicate that intracellular Mn levels may constitute an evolutionary conserved physiological cue that modulates eukaryotic TORC1/mTORC1 signaling. Our findings therefore reveal a hitherto elusive connection between intracellular Mn levels, mTORC1 activity, and human diseases.

Highlights- Mn acts as a primordial activator of TORC1
- NRAMP transporters control TORC1 through cytoplasmic Mn levels
- TORC1 regulates Mn levels through feedback circuits impinging on NRAMP transporters
- Mn-mediated TORC1 activation modulates retrograde response, autophagy, and mitophagy

category: cell biology


title: DRP1 regulates endoplasmic reticulum sheets to control mitochondrial DNA replication and segregation

abstract: Mitochondria are multi-faceted organelles crucial for cellular homeostasis that contain their own genome. Mitochondrial DNA (mtDNA) codes for several essential components of the electron transport chain, and mtDNA maintenance defects lead to mitochondrial diseases. mtDNA replication occurs at endoplasmic reticulum (ER)-mitochondria contact sites and is regulated by mitochondrial dynamics. Specifically, mitochondrial fusion is essential for mtDNA maintenance. In contrast, while loss of mitochondrial fission causes the aggregation of nucleoids (mtDNA-protein complexes), its role in nucleoid distribution remains unclear. Here, we show that the mitochondrial fission protein DRP1 regulates nucleoid segregation by altering ER sheets, the ER structure associated with protein synthesis. Specifically, DRP1 loss or mutation leads to altered ER sheets that enhance physical interaction with mitobulbs, mitochondrial structures containing aggregated nucleoids. Importantly, nucleoid distribution and mtDNA replication were rescued by expressing the ER sheet protein CLIMP63. Thus, our work identifies a novel fission-independent mechanism by which DRP1 regulates mtDNA replication and distribution.


title: Inference for epidemic models with time varying infection rates: tracking the dynamics of oak processionary moth in the UK

abstract:
O_LIInvasive pests pose a great threat to forest, woodland and urban tree ecosystems. The oak processionary moth (OPM) is a destructive pest of oak trees, first reported in the UK in 2006. Despite great efforts to contain the outbreak within the original infested area of South-East England, OPM continues to spread.
C_LIO_LIHere we analyse data of the numbers of OPM nests removed each year from two parks in London between 2013 and 2020. Using a state-of-the-art Bayesian inference scheme we estimate the parameters for a stochastic compartmental SIR (susceptible, infested, removed) model with a time varying infestation rate to describe the spread of OPM.
C_LIO_LIWe find that the infestation rate and subsequent basic reproduction number have remained constant since 2013 (with R0 between one and two). This shows further controls must be taken to reduce R0 below one and stop the advance of OPM into other areas of England.
C_LIO_LISynthesis. Our findings demonstrate the applicability of the SIR model to describing OPM spread and show that further controls are needed to reduce the infestation rate. The proposed statistical methodology is a powerful tool to explore the nature of a time varying infestation rate, applicable to other partially observed time series epidemic data.
C_LI