title: Transcriptomic analysis of DNA damage response in zebrafish embryos under simulated microgravity

abstract: Space is an extremely hostile environment. Traveling to space has numerous effects on the body of the astronauts at a molecular level. As space agencies prepare for long-term missions, it becomes important to investigate these problems and address them. Thus, in the current study, we assessed the impact of simulated microgravity on the basic pathways especially DNA damage response. We identified a total of 7542 significantly differentially expressed genes. Out of these genes, 4504 were found to be up-regulated while 3038 were down-regulated in the simulated microgravity group in comparison to controls. Pathway enrichment analysis revealed that simulated microgravity has an effect on vital basic biological processes like DNA damage repair, peptide transport, and metabolism. To explore if the same pathways were also altered in humans, we explored the NASA twin study data and found that DDR was also significantly affected in the astronaut but due to ionizing radiation. Upon further investigation, we found that 62 genes belonging to the DDR pathway were mutually differentially expressed in Scott Kelly and the zebrafish embryos. However, there were 29 significantly differentially expressed genes belonging to the DDR pathway in zebrafish embryos that were not found to be differentially expressed in Scott Kelly. Out of these 29 genes, 14 were specific to zebrafish. Upon further investigation, we found that the DDR pathway is affected differently in simulated microgravity as compared to ionizing radiation. These observations provide a preliminary look into the difference in DDR induced by the different factors of spaceflight.


title: SomaticSiMu: a mutational signature simulator

abstract:
SummarySomaticSiMu is an in silico simulator of single and double base substitutions, and single base insertions and deletions in an input genomic sequence to mimic mutational signatures. SomaticSiMu outputs simulated DNA sequences and mutational catalogues with imposed mutational signatures. The tool is the first mutational signature simulator featuring a graphical user interface, control of mutation rates, and built-in visualization tools of the simulated mutations. Simulated datasets are useful as a ground truth to test the accuracy and sensitivity of DNA sequence classification tools and mutational signature extraction tools under different experimental scenarios. The reliability of SomaticSiMu was affirmed by 1) supervised machine learning classification of simulated sequences with different mutation types and burdens, and 2) mutational signature extraction from simulated mutational catalogs.

Availability and ImplementationSomaticSiMu is written in Python 3.8.3. The open-source code, documentation, and tutorials are available at https://github.com/HillLab/SomaticSiMu under the terms of the Creative Commons Attribution 4.0 International License.

Contactkhill22@uwo.ca

Supplementary informationSupplementary data are available at Bioinformatics online.

category: bioinformatics


title: Morphological control of receptor-mediated endocytosis

abstract: Receptor-mediated endocytosis is the primary process for nanoparticle uptake in cells and one of the main entry mechanisms for viral infection. The cell membrane adheres to the particle (nanoparticle or virus) and then wraps it to form a vesicle delivered to the cytosol. Previous findings identified a minimum radius for a spherical particle below which endocytosis cannot occur. This is due to the insufficient driving force, from receptor-ligand affinity, to overcome the energy barrier created by membrane bending. In this paper, we extend this result to the case of clathrin-mediated endocytosis, which is the most common pathway for virus entry. Moreover, we investigate the effect of ligand inhibitors on the particle surface, motivated by viral antibodies, peptides or phage capsids nanoparticles. We determine the necessary conditions for endocytosis by considering the additional energy barrier due to the membrane bending to wrap such inhibiting protrusions. We find that the density and size of inhibitors determine the size range of internalized particles, and endocytosis is completely blocked above critical thresholds. The assembly of a clathrin coat with a spontaneous curvature increases the energy barrier and sets a maximum particle size (in agreement with experimental observations on smooth particles). Our investigation suggests that morphological considerations can inform the optimal design of neutralizing viral antibodies and new strategies for targeted nanomedicine.

category: biophysics


title: Characterization of Cell-Induced Astigmatism in High-Resolution Imaging.

abstract: High-resolution and super-resolution techniques become more frequently used in thick, inhomogeneous samples. In particular for imaging life cells and tissue in which one wishes to observe a biological process at minimal interference and in the natural environment, sample inhomogeneities are unavoidable. Yet sample-inhomogeneities are paralleled by refractive index variations, for example between the cell organelles and the surrounding medium, that will result in the refraction of light, and therefore lead to sample-induced astigmatism. Astigmatism in turn will result in positional inaccuracies of observations that are at the heart of all super-resolution techniques. Here we introduce a simple model and define a figure-of-merit that allows one to quickly assess the importance of astigmatism for a given experimental setting. We found that astigmatism caused by the cells nucleus can easily lead to aberrations up to hundreds of nanometers, well beyond the accuracy of all super-resolution techniques. The astigmatism generated by small objects, like bacteria or vesicles, appear to be small enough to be of any significance in typical super-resolution experimentation.


title: Partitioning of ribonucleoprotein complexes from the cellular actin cortex

abstract:
The cell cortex plays a crucial role in cell mechanics, signaling, and development. However, little is known about the influence of the cortical meshwork on the spatial distribution of cytoplasmic biomolecules. Here, we describe a new fluorescence microscopy method to infer the intracellular distribution of labeled biomolecules with sub-resolution accuracy. Unexpectedly, we find that RNA-binding proteins are partially excluded from the cytoplasmic volume adjacent to the plasma membrane that corresponds to the actin cortex. Complementary diffusion measurements of RNA-protein complexes suggest that a rudimentary model based on excluded volume interactions can explain this partitioning effect. Our results suggest the actin cortex meshwork may play a role in regulating the biomolecular content of the volume immediately adjacent to the plasma membrane.

TeaserA novel microscopy technique reveals partial exclusion of RNA-protein complexes from the actin cortex due to their large hydrodynamic size.


title: Generation of a molecular interactome of the glioblastoma perivascular niche reveals Integrin Binding Sialoprotein as a key mediator of tumor cell migration

abstract: Glioblastoma (GBM) is characterized by extensive microvascular hyperproliferation. In addition to supplying blood to the tumor, GBM vessels also provide trophic support to glioma cells and serve as conduits for migration into the surrounding brain promoting recurrence. Here, we enriched CD31-expressing glioma vascular cells (GVC) and A2B5-expressing glioma tumor cells (GTC) from primary GBM and utilized RNA sequencing to create a comprehensive interaction map of the secreted and extracellular factors elaborated by GVC that can interact with receptors and membrane molecules on GTC. To validate our findings, we utilized functional assays, including a novel hydrogel-based migration assay and in vivo mouse models to demonstrate that one identified factor, the little-studied integrin binding sialoprotein (IBSP) enhances tumor growth and promotes the migration of GTC along the vasculature. This perivascular niche interactome will serve a resource to the research community in defining the potential functions of the GBM vasculature.

category: cancer biology


title: THE SPLICING FACTOR PTBP1 REPRESSES TP63 γ ISOFORM PRODUCTION IN SQUAMOUS CELL CARCINOMA

abstract: The TP63 gene encodes the transcription factor p63. It is frequently amplified or overexpressed in squamous cell carcinomas. Owing to alternative splicing, p63 has multiple isoforms called , {beta}, {gamma} and {delta}. The regulatory functions of p63 may be isoform-specific. The  isoform inhibits the epithelial to mesenchymal transition (EMT) and controls apoptosis, while the {gamma} isoform promotes EMT. Here, we observed in TCGA data that a high ratio of the TP63{gamma} isoform to the other isoforms is a pejorative factor for the survival of patients with head and neck squamous cell carcinoma (HNSCC). We therefore addressed the regulation of the {gamma} isoform. In several tissues (GTEX data), the expression of the RNA-binding protein PTBP1 (polypyrimidine tract binding protein 1) is negatively correlated with the abundance of TP63{gamma}. Accordingly, we demonstrated that PTBP1 depletion in HNSCC cell lines leads to an increase in abundance of the {gamma} isoform. By RNA immunoprecipitation and in vitro interaction assays, we showed that PTBP1 directly binds to TP63 pre-mRNA in close proximity to the TP63{gamma}-specific exon. The region around the TP63{gamma}-specific exon was sufficient to elicit a PTBP1-dependent regulation of alternative splicing in a splice reporter minigene assay. Finally, we demonstrated that the regulation of TP63{gamma} production by PTBP1 is conserved in amphibians, revealing that it encounters a strong evolutionary pressure. Together, these results identify TP63{gamma} as a prognostic marker in HNSCC, and identify PTBP1 as a direct negative regulator of its production.


title: Human Satellite III long non-coding RNA imparts survival benefits to cancer cells

abstract: Long non-coding RNAs are heterogeneous group of transcripts that lack coding potential and have crucial roles in gene regulations. Recent days have seen an increasing association of non-coding RNAs with human diseases, especially cancers. Satellite III (SatIII) lncRNAs are transcribed from pericentromeric heterochromatic region of the human chromosome. Though transcriptionally silent in normal conditions, SatIII is actively transcribed under condition of stress, mainly heat shock. SatII repeat, another component of pericentromeric region of human chromosome, has been associated with wide variety of epithelial cancer. Overexpression of Satellite RNAs induces breast cancer in mice. Though much is known about Satellite RNAs, which includes alpha satellites and SatII repeats, however little is known about SatIII in human cancers. Hence we directed our study to understand the role of human Satellite III repeats in cancerous conditions. In the present study, we show that colon and breast cancer cells transcribe SatIII independent of heat shock, in an HSF1-independent manner. Our study also reveals that, overexpression of SatIII RNA favours cancer cell survival by overriding chemo drug-induced cell death. Knockdown of SatIII sensitizes cells towards chemotherapeutic drugs. SatIII transcript knockdown restores the expression of p53 protein, which in turn facilitates cell death. Heat shock however helps SatIII to continue with its pro-cell survival function. Our results, therefore suggest SatIII to be an important regulator of human cancers. Induction of SatIII is not only a response to the oncogenic stress but also facilitates cancer progression by a distinct pathway that is different from heat stress pathway.


title: Predicting the Effects of Per- and Polyfluoroalkyl Substance Mixtures on Peroxisome Proliferator-Activated Receptor Alpha Activity in Vitro

abstract:
Human exposure to per- and polyfluoroalkyl substances (PFAS) is ubiquitous, with mixtures of PFAS detected in drinking water, food, household dust, and other exposure sources. Animal toxicity studies and human epidemiology indicate that PFAS may act through shared mechanisms including activation of peroxisome proliferator activated receptor  (PPAR). However, the effect of PFAS mixtures on human relevant molecular initiating events remains an important data gap in the PFAS literature. Here, we tested the ability of modeling approaches to predict the effect of diverse PPAR ligands on receptor activity using Cos7 cells transiently transfected with a full length human PPAR (hPPAR) expression construct and a peroxisome proliferator response element-driven luciferase reporter. Cells were treated for 24 hours with two full hPPAR agonists (pemafibrate and GW7647), a full and a partial hPPAR agonist (pemafibrate and mono(2-ethylhexyl) phthalate), or a full hPPAR agonist and a competitive antagonist (pemafibrate and GW6471). Receptor activity was modeled with three additive approaches: effect summation, relative potency factors (RPF), and generalized concentration addition (GCA). While RPF and GCA accurately predicted activity for mixtures of full hPPAR agonists, only GCA predicted activity for full and partial hPPAR agonists and a full agonist and antagonist. We then generated concentration response curves for seven PFAS, which were well-fit with three-parameter Hill functions. The four perfluorinated carboxylic acids (PFCA) tended to act as full hPPAR agonists while the three perfluorinated sulfonic acids (PFSA) tended to act as partial agonists that varied in efficacy between 28-67% of the full agonist, positive control level. GCA and RPF performed equally well at predicting the effects of mixtures with three PFCAs, but only GCA predicted experimental activity with mixtures of PFSAs and a mixture of PFCAs and PFSAs at ratios found in the general population. We conclude that of the three approaches, GCA most accurately models the effect of PFAS mixtures on hPPAR activity in vitro.

HighlightsO_LIPerfluorinated carboxylic acids are full human PPAR agonists
C_LIO_LIPerfluorinated sulfonic acids are partial human PPAR agonists
C_LIO_LIGCA predicts human PPAR activity for mixtures of full and partial agonists
C_LIO_LIGCA predicts human PPAR activity for mixtures of agonists and competitive antagonists
C_LIO_LIGCA accurately predicts human PPAR activity in response to PFAS mixtures
C_LI

category: pharmacology and toxicology


title: Structure-Functional-Selectivity Relationship Studies on A-86929 Analogs and Small Fragments toward Discovery of D1 agonists

abstract: Dopamine regulates normal functions such as movement, reinforcement learning, and cognition, and its dysfunction has been implicated in multiple psychiatric and neurological disorders. Dopamine acts through the D1- (D1R and D5R) and D2-class (D2R, D3R and D4R) of seven transmembrane receptors, and activates both G-protein- and {beta}-arrestin-dependent signaling pathways, to mediate its physiological effects. Current dopamine receptor-based therapies are used to ameliorate motor deficits in Parkinsons disease, or as antipsychotic medications for schizophrenia. These drugs show efficacy for ameliorating only some symptoms caused by dopamine dysfunction and are plagued by debilitating side-effects. Studies in primates and rodents have shown that shifting the balance of dopamine receptor signaling towards the arrestin pathway can be beneficial for inducing normal movement, while reducing motor side-effects such as dyskinesias, and can be efficacious at enhancing cognitive function compared to balanced agonists. Several structure-activity-relationship (SAR) studies have embarked on discovering {beta}-arrestin-biased dopamine agonists, focused on D2 partial agonists, non-catechol D1 agonists, and mixed D1/D2R dopamine receptor agonists. Here, we describe an SAR study to identify novel D1R {beta}-arrestin biased ligands using A-86929, a high-affinity D1R catechol agonist, as a core scaffold. Previously described and novel analogs of A-86929 were synthesized and screened in vitro for structure-functional-selectivity relationships (SFSR) studies to identify chemical motifs responsible for {beta}-arrestin biased activity at both D1 and D2Rs. Most of the A-86929 analogs screened were G protein biased but none of them were exclusively arrestin-biased. Additionally, various catechol aryl fragments were designed and synthesized. Other compounds surveyed included hydroxyl and chloro analogs of dopamine to test for hydrogen bonding and ionic interactions. Some of these small molecular probes displayed weak bias towards the {beta}-arrestin pathway. Continued in-depth SFSR studies informed by structure determination, molecular modeling, and mutagenesis studies will facilitate discovery of potent and efficacious arrestin-biased dopamine receptor ligands.


title: Structure-Functional-Selectivity Relationship Studies on A-86929 Analogs and Small biaryl Fragments toward Discovery of biased D1 agonists


title: The role of parasympathetic mechanisms in the infarct-limiting effect of SGLT2 inhibitor ertugliflozin

abstract:
IntroductionBased on data that outcome in patients with acute myocardial infarction is predicted by final infarct size (IS), reducing IS is of paramount importance. Recent experimental studies have demonstrated a strong infarct-sparing effect of SGLT2 inhibitors - a class of drugs which have proved to be safe and beneficial in patients with heart failure. Repurposing SGLT2 inhibitors for the benefit of patients presenting with acute myocardial infarction should be preceded by investigation of the underlying mechanisms of this infarct limitation. Experimental and clinical data indicate a potential role for autonomic modulation in these mechanisms, specifically sympatho-inhibition. The aim of this study was to investigate the role of the parasympathetic mechanism in the infarct-limiting effect of SGLT2 inhibition.

MethodsFortyeight Sprague Dawley male rats were fed with a standard diet containing either the SGLT2 inhibitor ertugliflozin or vehicle, for 5-7 days. Myocardial ischaemia/reperfusion injury was initiated by a 40-min occlusion of the left anterior descending coronary artery followed by a 2hr period of reperfusion under isoflurane anaesthesia. Bilateral cervical vagotomy was performed 10min prior to myocardial ischaemia. Alternatively, muscarinic receptors were blocked systemically with the non-selective blocker atropine sulphate (2 mg/kg bolus, then 1 mg/kg/h) or the M3-selective blocker 4-DAMP (2 mg/kg bolus).

ResultsPre-treatment with ertugliflozin reduced IS in comparison with the vehicle-treated controls (p<0.001). Bilateral vagotomy, atropine sulphate and 4-DAMP all abolished this infarct-limiting effect (IS 35{+/-}10%, 44{+/-}8%, and 35{+/-}4% respectively; P<0.01 vs. Ertu for vagotomy, P<0.001 vs. Ertu for both atropine sulphate and 4-DAMP).

ConclusionThese results suggest that the Infarct-limiting effect of the SGLT2 inhibitor ertugliflozin, may be mediated via activation of the vagus nerve and M3-cholinoreceptors.

category: physiology


title: Identification and expression analysis of Shaker K+ channel genes in millet (Setaria italica) under abiotic stresses

abstract: Potassium (K+) is one of the essential nutrients for plant, which is involved in plant growth and development and abiotic stress tolerance. The absorption and transport of K+ depends on Shaker K+ channels. Foxtail millet is a Poaceae crop with strong drought stress-tolerant. In this study, we identified ten Shaker K+ channel genes in foxtail millet. Phylogenetic analysis, prediction of conserved motif, and gene structure analysis classified these genes into five groups. The transcription level of these genes under different abiotic stress treatments (cold, heat, NaCl, PEG) and ABA treatment were analyzed by quantitative real-time PCR. Each gene displayed its own regulation pattern under different treatments, suggests these channels play important role in plant adaptation to different environment conditions.

category: plant biology


title: Time-series Multi-spectral Imaging in Soybean for Improving Biomass and Genomic Prediction Accuracy

abstract: Multi-spectral (MS) imaging enables the measurement of characteristics important for increasing the prediction accuracy of genotypic and phenotypic values for yield-related traits. In this study, we evaluated the potential application of temporal MS imaging for the prediction of above-ground biomass (AGB) and determined which developmental stages should be used for accurate prediction in soybean. Field experiments with 198 accessions of soybean were conducted with four different irrigation levels. Five vegetation indices (VIs) were calculated using MS images from soybean canopies from early to late growth stages. To predict the genotypic values of AGB, VIs at the different growth stages were used as secondary traits in a multi-trait genomic prediction. The accuracy of the prediction model increased starting at an early stage of growth (31 days after sowing). To predict phenotypic values of AGB, we employed multi-kernel genomic prediction. Consequently, the prediction accuracy of phenotypic values reached a maximum at a relatively early growth stage (38 days after sowing). Hence, the optimal timing for MS imaging may depend on the irrigation levels.


title: Induction of autotetraploidy and microbiome associations mediate differential responses to pathogens

abstract: It has become increasingly clear that the microbiome plays a critical role in shaping the host organisms response to disease. There also exists mounting evidence that an organisms ploidy level is important in their response to pathogens and parasites. However, no study has determined if or how these two factors influence one another. We investigate the effect of whole-genome duplication in Arabidopsis thaliana on their above-ground (phyllosphere) microbiome, and determine the interacting impacts of ploidy and the microbiome on disease outcome. Using seven independently derived synthetic auto-tetraploid Arabidopsis accessions, a synthetic leaf-associated bacterial community, and the model pathogen Pseudomonas syringae pv. Tomato DC3000, we confirm that polyploids are generally more resistant to pathogens, but illustrate that this resistance may be in part due to a decrease in the reliance on beneficial bacteria. Polyploids fare better against the pathogen than diploids regardless of microbial inoculation, while we observed that diploids harboring an intact microbiome have lower pathogen densities than those without. We then use RNA sequencing to show that diploids have many more differentially expressed defense-related genes in the presence of their phyllosphere microbiota, while polyploids exhibit constitutively activated defenses regardless of exposure to the synthetic community. These results imply that whole-genome duplication can disrupt historical host-microbiome associations, and suggest that a potential cause or consequence of disruption is a heightened capacity for pathogen defense that is less impacted by the microbiome.


title: Characterization of wheat lacking B-type starch granules

abstract: The physicochemical and agronomic properties of a new form of bread wheat, lacking B-type starch granules (BlessT) were assessed. Three BlessT mutant lines, made by combining homoeologous deletions of BGC1, a gene responsible for the control of B-granule content were compared with two sibling lines with normal starch phenotype and the parent line, cv. Paragon. Quantification of starch granule size and number in developing grain confirmed the lack of small, B-type starch granules throughout development in BlessT. Most starch, flour, grain and loaf characteristics did not vary between BlessT and the wild type sibling controls. However, BlessT starches had higher water absorption, reduced grain hardness and higher protein content, and dough made from BlessT flour required more water and had increased elasticity. Despite the lack of B-granules, BlessT lines do not display a significant decrease in total starch content suggesting that it should be possible to produce commercial wheat varieties that lack B-type starch granules without compromising yield. These findings support the potential utility of this novel type of wheat as a specialist crop in applications ranging from bread making and alcohol production to improved industrial starch products.


title: Keeping students connected and engaged in a wet-lab research experience during a time of social distancing via mobile devices and video conferencing software

abstract: Two major COVID-19 pandemic challenges presented for in-person instruction included adhering to social distancing guidelines and accommodating remote learners who were temporarily isolated or permanently participating from afar. At Binghamton University, our First-year Research Immersion (FRI) program was challenged with providing students with a wet lab course-based undergraduate research experience (CURE), an intense hands-on experience that emphasized student teamwork, lab protocol development, iteration, troubleshooting and other elements of the process of science that could not be replicated in a fully remote environment. We developed an innovative technology approach to maximize all students connection to the lab research experience utilizing dedicated mobile devices (iPod Touch) and video conferencing software (Zoom) to synchronously connect remote learners to in-person learners, peer mentors and instructors in our FRI research labs. In this way, despite limited lab capacities and fluctuating remote learning populations, we were able to connect remote learners to their peers and mentors in real-time and give them responsibilities that allowed them to be engaged and feel like meaningful participants in the research process. Although our students reported a preference for in-person labs, they noted this hybrid model was better than other traditionally employed remote-learning lab options. We believe lessons learned here can be applied to improve access to research in all situations and allow us to be prepared for other catastrophic disruptions to the educational system.

category: scientific communication and education


title: Autoregulation of switching behavior by cellular compartment size

abstract: Many kinds of cellular compartments comprise decision making mechanisms that control growth and shrinkage of the compartment in response to external signals. Key examples include synaptic plasticity mechanisms that regulate the size and strength of synapses in the nervous system. However, when synaptic compartments and postsynaptic densities are small such mechanisms operate in a regime where chemical reactions are discrete and stochastic due to low copy numbers of the species involved. In this regime, fluctuations are large relative to mean concentrations, and inherent discreteness leads to breakdown of mass action kinetics. Understanding how synapses and other small compartments achieve reliable switching in the low copy number limit thus remains a key open problem. We propose a novel self regulating signaling motif that exploits the breakdown of mass action kinetics to generate a reliable size-regulated switch. We demonstrate this in simple two and three-species chemical reaction systems and uncover a key role for inhibitory loops among species in generating switching behavior. This provides an elementary motif that could allow size dependent regulation in more complex reaction pathways and may explain discrepant experimental results on well-studied biochemical pathways.

category: systems biology


title: Classifying Literature Extracted Events for Automated Model Extension

abstract:
1In this study, we investigate the integration of three previously developed tools: FLUTE, VIOLIN, and CLARINET. We show how using these tools together adds additional capabilities in extending models from relevant research literature. We illustrate how we plan to address current modeling pitfalls with these tools (such as machine reading errors and literature volume), and how we plan to use these tools as the foundation for an automated model extension framework. Documentation and links to our tools can be found at: violin-tool.readthedocs.io
clarinet-docs.readthedocs.io
flute.readthedocs.io


title: The generalized spatial representation in the prefrontal cortex is inherited from the hippocampus

abstract: Hippocampal and neocortical neural activity is modulated by the position of the individual in space. While hippocampal neurons provide the basis for a spatial map, prefrontal cortical neurons generalize over environmental features. Whether these generalized representations result from a bidirectional interaction with, or are mainly derived from hippocampal spatial representations is not known. By examining simultaneously recorded hippocampal and medial prefrontal neurons, we observed that prefrontal spatial representations show a delayed coherence with hippocampal ones. We also identified subpopulations of cells in the hippocampus and medial prefrontal cortex that formed functional cross-area couplings; these resembled the optimal connections predicted by a probabilistic model of spatial information transfer and generalization. Moreover, cross-area couplings were strongest and had the shortest delay preceding spatial decision-making. Our results suggest that generalized spatial coding in the medial prefrontal cortex is inherited from spatial representations in the hippocampus, and that the routing of information can change dynamically with behavioral demands.

category: neuroscience


title: Capillary stall quantification from optical coherence tomography angiogram maximum intensity projections

abstract: Optical coherence tomography (OCT) is applicable to the study of cerebral microvasculature in vivo. Optimised acquisition schemes enable the generation of three-dimensional OCT angiograms, i.e., volumetric images of red blood cell flux in capillary networks, currently at a repetition rate of up to 1/10 seconds. This makes testable a new class of hypotheses that strive to bridge the gap between microscopic phenomena occurring at the spatial scale of neurons, and less invasive but crude techniques to measure macroscopic blood flow dynamics. Here we present a method for quantifying the occurrence of transient capillary stalls in OCT angiograms, i.e., events during which blood flow through a capillary branch is temporarily occluded. By making the assumption that information on such events is present predominantly in the imaging plane, we implemented a pipeline that automatically segments a network of interconnected capillaries from the maximum intensity projections (MIP) of a series of 3D angiograms. We then developed tools enabling rapid manual assessment of the binary flow status (open/stalled) of hundreds of capillary segments based on the intensity profile of each segment across time. The entire pipeline is optimized to run on a standard laptop computer, requiring no high-performance, low-availability resources, despite very large data volumes. To further reduce the threshold of adoption, and ultimately to support the development of reproducible research methods in the young field, we provide the documented code for scrutiny and re-use under a permissive open-source license.


title: An internal expectation guides Drosophila egg-laying decisions

abstract: When presented with two egg-laying substrates, Drosophila lay most of their eggs on the option with higher relative value. How do flies make this relative-value decision? Might the strategy they use allow them to choose the best option even when they experience substrates with a more complex spatiotemporal profile than in canonical two-choice paradigms? We measured Drosophila egg-laying behavior in diverse substrate environments. In all cases, we found that flies dynamically increase or decrease their egg-laying rates as they explore substrates for a deposition site so as to target eggs to the best, recently visited option. Visiting the best option typically led to a strong reduction in egg laying on other substrates for several minutes, with this timescale varying across fly strains. Our data support a model in which flies compare the value of the current substrate with an internally constructed expectation on the value of available options to regulate the likelihood of laying an egg. We show that dopamine-neuron activity is critical for learning and/or expressing this expectation, similar to its role in certain tasks in vertebrates. Integrating sensory experiences over minutes to generate an internal sense of the quality of available options, i.e., forming an expectation, allows flies to use a dynamic reference point for judging the current substrate and might be a general way in which decisions are made, even beyond flies and egg laying.


title: Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue

abstract: Direct activation of cell-surface receptors is highly desirable for elucidating the physiological roles of receptors. However, subtype-selective ligands are very limited because of the high homology among receptor subtypes. A potential approach for selective activation of a receptor subtype is chemogenetics, in which both point mutagenesis of the receptors and designed ligands are used. However, ligand-binding properties are affected in most current methods. Here, we developed a chemogenetic method for direct activation of metabotropic glutamate receptor 1 (mGlu1), which plays essential roles in cerebellar functions in the brain. Our screening identified a mGlu1 mutant, mGlu1(N264H), that was directly activated by palladium complexes. Notably, a palladium complex showing low cytotoxicity successfully activated mGlu1 in mGlu1(N264H) knock-in mice, revealing that activation of endogenous mGlu1 is sufficient to evoke the critical cellular mechanism of synaptic plasticity, a basis of motor learning in the cerebellum.


title: Powassan Viruses Spread Cell to Cell During Direct Isolation from IxodesTicks and Persistently Infect Human Brain Endothelial Cells and Pericytes

abstract:
Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the Northeastern U.S., with a 2% prevalence in Long Island (LI) deer ticks (Ixodes scapularis). POWVs are transmitted in as little as 15 minutes of a tick bite, and enter the CNS to cause encephalitis (10% fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodes ticks were isolated by directly inoculating VeroE6 cells with tick homogenates and detecting POWV infected cells by immunoperoxidase staining. Inoculated POWV-LI9 and LI41 were exclusively present in infected cell foci, indicative of spread cell to cell, despite growth in liquid culture without an overlay. Cloning and sequencing establish POWV-LI9 as a phylogenetically distinct lineage II POWV strain circulating in LI deer ticks. Primary human brain microvascular endothelial cells (hBMECs) and pericytes form a neurovascular complex that restricts entry into the CNS. We found that POWV-LI9, -LI41 and Lineage I POWV-LB, productively infect hBMECs and pericytes and that POWVs were basolaterally transmitted from hBMECs to lower chamber pericytes without permeabilizing polarized hBMECs. Synchronous POWV-LI9 infection of hBMECs and pericytes induced proinflammatory chemokines, interferon-{beta} (IFN{beta}) and IFN-stimulated genes, with delayed IFN{beta} secretion by infected pericytes. IFN inhibited POWV infection, but despite IFN secretion a subset of POWV infected hBMECs and pericytes remained persistently infected. These findings suggest a potential mechanism for POWVs (LI9/LI41 and LB) to infect hBMECs, spread basolaterally to pericytes and enter the CNS. hBMEC and pericyte responses to POWV infection suggest a role for immunopathology in POWV neurovirulence and potential therapeutic targets for preventing POWV spread to neuronal compartments.

ImportanceWe isolated POWVs from LI deer ticks (I. scapularis) directly in VeroE6 cells and sequencing revealed POWV-LI9 as a distinct lineage II POWV strain. Remarkably, inoculating VeroE6 cells with POWV containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell to cell spread. POWV-LI9, -LI41, and Lineage I POWV-LB strains infected hBMECs and pericytes that comprise neurovascular complexes. POWVs were nonlytically transmitted basolaterally from infected hBMECs to lower chamber pericytes, suggesting a mechanism for POWV transmission across BBB. POWV-LI9 elicited inflammatory responses from infected hBMEC and pericytes that may contribute to immune cell recruitment and neuropathogenesis. This study reveals a potential mechanism for POWVs to enter the CNS by infecting hBMECs and spreading basolaterally to abluminal pericytes. Our findings reveal that POWV-LI9 persists in cells that form a neurovascular complex spanning the BBB, and suggest potential therapeutic targets for preventing POWV spread to neuronal compartments.

category: microbiology


title: RASopathy Mutations Demonstrate the Critical Function of the Cysteine-rich Domain in Maintaining BRAF in an Autoinhibited State

abstract: The RAF kinases are essential effectors of the RAS-GTPases, and germline mutations in BRAF or CRAF are disease drivers in the RASopathy developmental syndromes, with mutations in the cysteine-rich domain (CRD) often observed in BRAF but not CRAF. Although the CRD is known for roles in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the interplay of these activities and their impact on BRAF function in normal and disease states is unclear. Here, we analyze a panel of BRAF-CRD RASopathy mutations and show that they all increase the biological activity of BRAF by relieving autoinhibition and/or enhancing PS/membrane binding. Strikingly, we find that relief of autoinhibition is the predominant factor determining the severity of the mutations. Comparison of the BRAF and CRAF CRDs further indicates that the BRAF-CRD is a stronger mediator of both autoinhibition and PS binding, and we identify residues contributing to these differences. Moreover, given the increased catalytic activity of BRAF versus CRAF, our studies reveal a more critical role for the CRD and autoinhibition in BRAF regulation and demonstrate that CRD-mediated autoinhibition is required to prevent aberrant BRAF signaling, a finding consistent with the high frequency of mutations that disrupt this function in the RASopathies.

category: cell biology


title: RASopathy Mutations Demonstrate the Critical Function of the Cysteine-rich Domain in Maintaining BRAF in an Autoinhibited State to Prevent Aberrant Signaling


title: De novo birth of functional, human-specific microproteins

abstract: We now have a growing understanding that functional short proteins can be translated out of small Open Reading Frames (sORF). Such "microproteins" can perform crucial biological tasks and can have considerable phenotypic consequences. However, their size makes them less amenable to genomic analysis, and their evolutionary origins and conservation are poorly understood. Given their short length it is plausible that some of these functional microproteins have recently originated entirely de novo from non-coding sequence. Here we test the possibility that de novo gene birth can produce microproteins that are functional "out-of-the-box". We reconstructed the evolutionary origins of human microproteins previously found to have measurable, statistically significant fitness effects. By tracing the appearance of each ORF and its transcriptional activation, we were able to show that, indeed, novel small proteins with significant phenotypic effects have emerged de novo throughout animal evolution, including many after the human-chimpanzee split. We show that traditional methods for assessing the coding potential of such sequences often fall short, due to the high variability present in the alignments and the absence of telltale evolutionary signatures that are not yet measurable. Thus we provide evidence that the functional potential intrinsic to sORFs can be rapidly, and frequently realised through de novo gene birth.

category: evolutionary biology


title: Balancing quality and quantity of social relationships in volatile social environments

abstract: Cooperation does not occur in a vacuum: interactions develop over time in social groups that undergo demographic changes. Intuition suggests that stable social environments favour developing few but strong reciprocal relationships (a  focused strategy), while volatile social environments favour the opposite: more but weaker social relationships (a  diversifying strategy). We model reciprocal investments under a quality-quantity tradeoff for social relationships. We find that volatility, counterintuitively, can favour a focused strategy. This result becomes explicable through applying the theory of antagonistic pleiotropy, originally developed for senescence, to social life. Diversifying strategies show superior performance later in life, but with costs paid at young ages while the social network is slowly being built. Under volatile environments, many individuals die before reaching sufficiently old ages to reap the benefits. Social strategies that do well early in life are then favoured: a focused strategy leads individuals to form their first few social bonds quickly and to make strong use of existing bonds. Our model highlights the importance of pleiotropy and population age structure for the evolution of cooperative strategies and other social traits, and shows that it is not sufficient to reflect on the fate of survivors only, when evaluating the benefits of social strategies.


title: Volatile social environments can favour investments in quality over quantity of social relationships


title: BinChecker: a new algorithm for quality assessment of microbial draft genomes

abstract: In the reconstruction of microbial genomes from metagenomic sequence data, the estimation of the final completeness and possible contamination is crucial for quality control. In metagenomics candidate genomes are usually obtained from a metagenome assembly and a subsequent binning of the assembled contigs. BinChecker provides a novel approach to quality assessment that is based on a fast protein domain search and a clustering approach for identification of marker domain ("feature") sets. The feature sets that are used for estimation are not pre-computed for a given database of reference genomes, but are individually found for each bin by adaptive clustering and feature selection. In particular, the adaptivity facilitates the creation and extension of the underlying database, which just requires to add protein feature profiles of reference genomes. Tests with simulated bins indicate that the prediction accuracy of BinChecker meets the current state of the art while providing significant advantages in terms of speed and flexibility.


title: Encounter networks from collective mitochondrial dynamics support the emergence of effective mtDNA genomes in plant cells

abstract: Mitochondria in plant cells form strikingly dynamic populations of largely individual organelles. Each mitochondrion contains on average less than a full copy of the mitochondrial DNA (mtDNA) genome. Here, we asked whether mitochondrial dynamics may allow individual mitochondria to  collect a full copy of the mtDNA genome over time, by facilitating exchange between individuals. Akin to trade on a social network, exchange of mtDNA fragments across organelles may lead to the emergence of full  effective genomes in individuals over time. We characterise the collective dynamics of mitochondria in Arabidopsis thaliana hypocotyl cells using a recent approach combining single-cell timelapse microscopy, video analysis, and network science. We then use a quantitative model to predict the capacity for the sharing and accumulation of genetic information through the networks of encounters between mitochondria. We find that biological encounter networks are strikingly well predisposed to support the collection of full genomes over time, outperforming a range of other networks generated from theory and simulation. Using results from the coupon collectors problem, we show that the upper tail of the degree distribution is a key determinant of an encounter networks performance at this task and discuss how features of mitochondrial dynamics observed in biology facilitate the emergence of full effective genomes.


title: The immunomodulatory properties of the HDAC6 inhibitor ACY241 supports robust anti-tumor response in NSCLC when coupled with the chemotherapy drug Oxaliplatin

abstract:
BackgroundDurable treatments that benefit a wide pool of patients remain elusive for Non-small cell lung cancer (NSCLC). The success of immunotherapy in a subset of NSCLC patients highlights the potential contribution of immune response to anti-tumor immunity while underscoring a need for broadly applicable therapeutic strategies. HDAC inhibitors are a promising class of drugs whose immunomodulatory properties are now being appreciated. In the present study, we evaluated the effects of the HDAC6 inhibitor, ACY241 on lung tumor immune compartment with the goal of understanding the scope of its immunomodulatory properties and its therapeutic potential in combination with Oxaliplatin.

MethodsLung adenocarcinoma-bearing mice were treated with ACY241 or vehicle after which the proportions and phenotype of tumor-associated T cells and macrophages were evaluated by comprehensive flow cytometric analysis. Bulk RNA-sequencing was also conducted on both cellular subsets to interrogate the transcriptomic changes associated with ACY241 treatment relative to vehicle controls. In vivo drug efficacy study was performed by administration of ACY241 and/or Oxaliplatin and assessing tumor growth and survival of tumor-bearing mice. Ex vivo functional studies was performed to assess tumor-associated T cell effector function as it correlates with measured outcomes.

ResultsWe demonstrate that ACY241 promotes increased presence of T and NK cells in the lung tumors of treated mice. The tumor-associated T cells under ACY241 treatment displayed enhanced activation, proliferation, and effector profile. In addition, tumor-associated macrophages exhibited increased expression of MHC and co-stimulatory molecules while expression of inhibitory ligands were reduced. RNA-sequencing of both tumor-associated T cells and macrophages revealed significant genomic changes in both subsets that is consistent with ACY241-mediated enhancement of immune priming. These broad immunomodulatory properties of ACY241 were associated with significantly enhanced tumor-associated T cell effector functionality, robust anti-tumor response, and significantly prolonged survival of NSCLC-bearing mice when combined with the chemotherapy drug Oxaliplatin.

ConclusionCollectively, our studies highlight the broad immunomodulatory effect of ACY241 as a promising HDAC6 inhibitor which coupled with Oxaliplatin promotes robust therapeutic outcomes in a pre-clinical model of NSCLC, providing compelling rationale for the clinical testing of this novel combinatorial regimen in NSCLC.

category: immunology


title: Short '1.2x genome' infectious clone initiates deltavirus replication in Boa constrictor cells

abstract:
Human hepatitis D virus (HDV), discovered in 1977, represented the sole known deltavirus for decades. The dependence on hepatitis B virus (HBV) co-infection and its glycoproteins for infectious particle formation led to the assumption that deltaviruses are human-only pathogens. However, since 2018, several reports have described identification of HDV-like agents from various hosts but without co-infecting hepadnaviruses. Indeed, we demonstrated that Swiss snake colony virus 1 (SwSCV-1) uses arenaviruses as the helper for infectious particle formation, thus shaking the dogmatic alliance with hepadnaviruses for completing deltavirus life cycle. In vitro systems enabling helper virus-independent replication are key for studying the newly discovered deltaviruses. Others and we have successfully used constructs containing multimers of the deltavirus genome for the replication of various deltaviruses via transfection in cell culture. Here, we report the establishment of deltavirus infectious clones with 1.2x genome inserts bearing two copies of the genomic and antigenomic ribozymes. We used SwSCV-1 as the model to compare the ability of the previously reported "2x genome" and the "1.2x genome" plasmid constructs/infectious clones to initiate replication in cell culture. Using immunofluorescence, qRT-PCR, immuno- and northern blotting, we found the 2x and 1.2x genome clones to similarly initiate deltavirus replication in vitro and both induced a persistent infection of snake cells. We hypothesize that duplicating the ribozymes facilitates the cleavage of genome multimers into unit-length pieces during the initial round of replication. The 1.2x genome constructs enable easier introduction of modifications required for studying deltavirus replication and cellular interactions.

IMPORTANCEHepatitis D virus (HDV) is a satellite virus infecting humans with strict association to hepatitis B virus (HBV) co-infection because HBV glycoproteins can mediate infectious HDV particle formation. For decades, HDV was the sole representative of deltaviruses, which had led to hypotheses suggesting that it evolved in humans, the only known natural host. Recent sequencing studies have led to the discovery of HDV-like sequences across a wide range of species, representing a paradigm shift in deltavirus evolution. Molecular biology tools such as infectious clones, which enable initiation of deltavirus infection without helper virus, are key to demonstrate that the recently found deltaviruses are capable of independent replication. Such tools will enable identification of the potential helper viruses. Here, we report a 1.2x genome copy strategy for designing plasmid-based infectious clones to study deltaviruses and to demonstrate that plasmid delivery into cultured snake cells sufficiently initiates replication of different deltaviruses.

category: molecular biology


title: Mechanical Compression Creates a Quiescent Muscle Stem Cell Niche

abstract: Skeletal muscles can regenerate throughout life time from resident Pax7-expressing (Pax7+) muscle stem cells (MuSCs)1-3. Pax7+ MuSCs are normally quiescent and localized at a niche in which they are attached to the extracellular matrix basally and compressed against the myofiber apically3-5. Upon muscle injury, MuSCs lose apical contact with the myofiber and re-enter cell cycle to initiate regeneration. Prior studies on the physical niche of MuSCs focused on basal elasticity6,7, and significance of the apical force exerted on MuSCs remains unaddressed. Here we simulate MuSCs mechanical environment in vivo by applying physical compression to MuSCs apical surface. We demonstrate that compression drives activated MuSCs back to a quiescent stem cell state, even when seeded on different basal elasticities. By mathematical modeling and manipulating cell tension, we conclude that low overall tension combined with high edge tension generated by compression lead to MuSC quiescence. We further show that apical compression results in up-regulation of Notch downstream genes, accompanied by increased levels of nuclear Notch. The compression-induced nuclear Notch is ligand-independent, as it does not require the canonical S2-cleavage of Notch by ADAM10/17. Our results fill the knowledge gap on the role of apical tension for MuSC fate. Implications to how stem cell fate and activity are interlocked with the mechanical integrity of its resident tissue are discussed.


title: Molecular insights into phosphorylation-induced allosteric conformational changes in β2-adrenergic receptor

abstract: The large conformational flexibility of G protein-coupled receptors (GPCRs) has been a puzzle in structural and pharmacological studies for the past few decades. Apart from structural rearrangements induced by ligands, enzymatic phosphorylations by GPCR kinases (GRKs) at the carboxy-terminal tail (C-tail) of a GPCR also makes conformational alterations to the transmembrane helices and facilitates the binding of one of its transducer proteins named {beta}-arrestin. Phosphorylation-induced conformational transition of the receptor that causes specific binding to {beta}-arrestin but prevents the association of other transducers such as G proteins lacks atomistic understanding and is elusive to experimental studies. Using microseconds of all-atom conventional and Gaussian accelerated molecular dynamics (GaMD) simulations, we investigate the allosteric mechanism of phosphorylation induced-conformational changes in {beta}2-adrenergic receptor, a well-characterized GPCR model system. Free energy profiles reveal that the phosphorylated receptor samples a new conformational state in addition to the canonical active state corroborating with recent nuclear magnetic resonance experimental findings. The new state has a smaller intracellular cavity that is likely to accommodate {beta}-arrestin better than G protein. Using contact map and inter-residue interaction energy calculations, we found the phosphorylated C-tail adheres to the cytosolic surface of the transmembrane domain of the receptor. Transfer entropy calculations show that the C-tail residues drive the correlated motions of TM residues, and the allosteric signal is relayed via several residues at the cytosolic surface. Our results also illustrate how the redistribution of inter-residue nonbonding interaction couples with the allosteric communication from the phosphorylated C-tail to the transmembrane. Atomistic insight into phosphorylation-induced {beta}-arrestin specific conformation is therapeutically important to design drugs with higher efficacy and fewer side effects. Our results therefore open novel opportunities to fine-tune {beta}-arrestin bias in GPCR signaling.


title: Membrane remodeling due to mixture of multiple curvature proteins: Insights from mesoscopic continuum modeling

abstract:
We present an extension of the Monte Carlo based mesoscopic membrane model, where the membrane is represented as a dynamically triangulated surface and the proteins are modeled as anisotropic inclusions formulated as in-plane nematic field variables adhering to the deformable elastic sheet. In the extended model, we have augmented the Hamiltonian to study membrane deformation due to a mixture of multiple types of curvature generating proteins. This feature opens the door for understanding how multiple kinds of curvature-generating proteins may be working in a coordinated manner to induce desired membrane morphologies. For example, among other things, we study membrane deformations and tubulation due to a mixture of positive and negative curvature proteins as mimics of various proteins from BAR domain family working together for curvature formation and stabilization. We also study the effect of membrane anisotropy, which manifests as membrane localization and differential binding affinity of a given curvature protein, leading to insights into the tightly regulated cargo sorting and transport processes. Our simulation results show different morphology of deformed vesicles that depend on the curvatures and densities of the participating proteins as well as on the protein-protein and membrane-proteins interactions.

SignificanceMembrane remodeling requires highly orchestrated interactions between several types of lipids and curvature proteins. Experimentally probing the membrane deformation intermediates is non-trivial. For example, it is not known exactly how two or more different kinds of BAR domain proteins work in concert to induce and stabilize certain membrane curvatures. In this work, we use mesoscopic continuum modeling and explore the factors that induce and stabilize a range of membrane deformation due to a mixture of positive and negative curvature proteins.


title: Membrane remodeling due to mixture of multiple curvature proteins


title: Non-conserved metabolic regulation by LKB1 distinguishes human and mouse lung adenocarcinoma

abstract: KRAS is the most frequently mutated oncogene in human lung adenocarcinomas (hLUAD) and activating mutations in KRAS frequently co-occur with loss-of-function mutations in the tumor suppressor genes, TP53 or STK11/LKB1. However, mutation of all three genes is rarely observed in hLUAD, even though engineered mutations of all three genes produces a highly aggressive lung adenocarcinoma in mice (mLUAD). Here we provide an explanation of this difference between hLUAD and mLUAD by uncovering an evolutionary divergence in regulation of the glycolytic enzyme triosephosphate isomerase (TPI1). Using KRAS/TP53 mutant hLUAD cell lines, we show that TPI1 enzymatic activity can be altered via phosphorylation at Ser21 by the Salt Inducible Kinases (SIKs) in an LKB1-dependent manner; this allows modulation of glycolytic flux between completion of glycolysis and production of glycerol lipids. This metabolic flexibility appears to be critical in rapidly growing cells with KRAS and TP53 mutations, explaining why loss of LKB1 creates a metabolic liability in these tumors. In mice, the amino acid at position 21 of TPI1 is a Cys residue which can be oxidized to alter TPI1 activity, allowing regulation of glycolytic flux balance without a need for SIK kinases or LKB1. Our findings reveal an unexpected role for TPI1 in metabolic reprogramming and suggest that LKB1 and SIK family kinases are potential targets for treating KRAS/TP53 mutant hLUAD. Our data also provide a cautionary example of the limits of genetically engineered murine models as tools to study human diseases such as cancers.


title: Mechanistic Model of Telomere Length Homeostasisin Yeast

abstract: Cells maintain homeostatic telomere length, and this homeostatic disruption leads to various types of diseases. Presently, it is not clear how telomeres achieve homeostasis. One of the prevailing hypotheses is a protein-counting model with a built-in sensor mechanism that counts proteins that directly regulate the telomeric length. However, it does not explain telomere length regulation at the mechanistic level. Here, we present a mathematical model based on the underlying molecular mechanisms of length regulation needed to establish telomere length homeostasis in yeast. We perform both deterministic and stochastic simulations to validate the models with the experimental data of Teixeira et al., rate-balance plot, and phase plane analysis to understand the nature of dynamics exhibited by the models. For global analysis, we constructed bifurcation diagrams. The model explains the role of negative and positive feedback loops and a delay between telomerase and telomere-bound proteins, leading to oscillations in telomere length. We map these in-silico results to Teixeiras proposition of telomeres making a transition between extendible and non-extendible states.


title: Expression and Neurotransmitter Association of the Synaptic Calcium Sensor Synaptotagmin in the Avian Auditory Brain Stem

abstract: In the avian auditory brain stem, acoustic timing and intensity cues are processed in separate, parallel pathways via the two division of the cochlear nucleus, nucleus angularis (NA) and nucleus magnocellularis (NM). Differences in excitatory and inhibitory synaptic properties, such as release probability and short-term plasticity, contribute to differential processing of the auditory nerve inputs. We investigated the distribution of synaptotagmin, a putative calcium sensor for exocytosis, via immunohistochemistry and double immunofluorescence in the embryonic and hatchling chick brain stem (Gallus gallus). We found that the two major isoforms, synaptotagmin 1 (Syt1) and synaptotagmin 2 (Syt2), showed differential expression. In the NM, anti-Syt2 label was strong and resembled the endbulb terminals of the auditory nerve inputs, while anti-Syt1 label was weaker and more punctate. In NA, both isoforms were intensely expressed throughout the neuropil. A third isoform, synaptotagmin 7 (Syt7), was largely absent from the cochlear nuclei. In nucleus laminaris (NL, the target nucleus of NM), anti-Syt2 and anti-Syt7 strongly labeled the dendritic lamina. These patterns were established by embryonic day 18 and persisted to postnatal day 7. Double labeling immunofluorescence showed Syt1 and Syt2 were associated with Vesicular Glutamate Transporter 2 (VGluT2), but not Vesicular GABA Transporter (VGAT), suggesting these Syt isoforms were localized to excitatory, but not inhibitory, terminals. These results suggest that Syt2 is the major calcium binding protein underlying excitatory neurotransmission in the timing pathway comprising NM and NL, while Syt2 and Syt1 regulate excitatory transmission in the parallel intensity pathway via cochlear nucleus NA.


title: Polyamine elevation and nitrogen stress are toxic hallmarks of chronic sleep loss in Drosophila melanogaster

abstract: Chronic sleep loss profoundly impacts health in ways coupled to metabolism; however, much existing literature links sleep and metabolism only on acute timescales. To explore the impact of chronically reduced sleep, we conducted unbiased metabolomics on heads from three Drosophila short-sleeping mutants. Common features included elevated ornithine and polyamines; and lipid, acyl-carnitine, and TCA cycle changes suggesting mitochondrial dysfunction. Biochemical studies of overall, circulating, and excreted nitrogen in sleep mutants demonstrate a specific defect in eliminating nitrogen, suggesting that elevated polyamines may function as a nitrogen sink. Both supplementing polyamines and inhibiting their synthesis with RNAi regulated sleep in control flies. Finally, both polyamine-supplemented food and high-protein feeding were highly toxic to sleep mutants, suggesting their altered nitrogen metabolism is maladaptive. Together, our results suggest polyamine accumulation specifically, and nitrogen stress in general, as potential mechanisms linking chronic sleep loss to adverse health outcomes.


title: Ataxin-2 Disordered Region Promote Huntingtin Protein Aggregation And Neurodegeneration In Drosophila Models Of Huntington's Disease.

abstract: The Ataxin-2 (Atx2) protein contributes to the progression of neurodegenerative phenotypes in animal models of amyotrophic lateral sclerosis (ALS), type 2 spinocerebellar ataxia (SCA-2), Parkinsons Disease (PD) and Huntingtons Disease (HD). However, because the Atx2 protein contains multiple separable activities, deeper understanding requires experiments to address the exact mechanisms by which Atx2 modulates neurodegeneration (ND) progression. Recent work on two ALS models, C9ORF72 and FUS, in Drosophila has shown that a C-terminal intrinsically disordered region (cIDR) of Atx2 protein, required for assembly of ribonucleoprotein (RNP) granules, is essential for the progression of neurodegenerative phenotypes as well as for accumulation of protein inclusions associated with these ALS models. Here we show that the Atx2-cIDR also similarly contributes to the progression of degenerative phenotypes and accumulation of Huntingtin protein aggregates in Drosophila models of HD. Because Huntingtin is not an established component of RNP granules, these observations support a recently hypothesised, unexpected protein-handling function for RNP granules, which could contribute to the progression of Huntingtons disease and, potentially, other proteinopathies.


title: Dual medial prefrontal cortex and hippocampus projecting neurons in the paraventricular nucleus of the thalamus

abstract: The paraventricular nucleus (PVT) of the midline thalamus is a critical higher-order cortico-thalamo-cortical integration site that plays a critical role in various behaviors including reward seeking, cue saliency, and emotional memory. Anatomical studies have shown that PVT projects to both medial prefrontal cortex (mPFC) and hippocampus (HC). However, dual mPFC-HC projecting neurons which could serve a role in synchronizing mPFC and HC activity during PVT-dependent behaviors, have not been explored. Here we used a dual retrograde adenoassociated virus (AAV) tracing approach to characterize the location and proportion of different projection populations that send collaterals to mPFC and/or ventral hippocampus (vHC). Additionally, we examined the distribution of calcium binding proteins calretinin (CR) and calbindin (CB) with respect to these projection populations PVT. We found that PVT contains separate populations of cells that project to mPFC, vHC, and those that innervate both regions. Interestingly, dual mPFC-HC projecting cells expressed neither CR or CB. Topographically, mPFC- and vHC-projecting CB+ and CR+ cells clustered around dual projecting neurons in PVT. These results are consistent with the features of dual mPFC-vHC projecting cells in the nucleus reuniens (RE) and suggestive of a functional mPFC-PVT-vHC system that may support mPFC-vHC interactions in PVT-dependent behaviors.


title: Oral and injected tamoxifen alter adult hippocampal neurogenesis in female and male mice

abstract: Inducible Cre recombinase facilitates temporal control of genetic recombination in numerous transgenic model systems, a feature which has made it a popular tool for studies of adult neurogenesis. One of the most common forms of inducible Cre, CreERT2, requires activation by the synthetic estrogen tamoxifen (TAM) to initiate recombination of LoxP-flanked sequences. To date, most studies deliver TAM via intraperitoneal injection. But the introduction of TAM-infused commercial chows has recently expanded the possible modes of TAM delivery. Despite the widespread use of TAM-inducible genetic models in adult neurogenesis research, the comparative efficiency and off-target effects of TAM administration protocols is surprisingly infrequently studied. Here we compare a standard, 5 day TAM injection regimen with voluntary consumption of TAM-infused chow. First, we used adult NestinCreERT2;Rosa-LoxP-STOP-LoxP-EYFP reporter mice to show that 2 weeks of TAM chow and 5 days of injections led to LoxP recombination in a similar phenotypic population of neural stem and progenitor cells in the adult dentate gyrus. However, TAM chow resulted in substantially less overall recombination than injections. TAM administration also altered adult neurogenesis, but in different ways depending on administration route: TAM injection disrupted neural progenitor cell proliferation 3 weeks after TAM, whereas TAM chow increased neuronal differentiation of cells generated during the diet period. These findings provide guidance for selection of TAM administration route and appropriate controls in adult neurogenesis studies using TAM-inducible Cre mice. They also highlight the need for better understanding of off-target effects of TAM in other neurological processes and organ systems.


title: Systematic phenotyping and characterization of the 3xTg-AD mouse model of Alzheimer's Disease

abstract:
Animal models of disease are valuable resources for investigating pathogenic mechanisms and potential therapeutic interventions. However, for complex disorders such as Alzheimers disease (AD), the generation and availability of innumerous distinct animal models present unique challenges to AD researchers and hinder the success of useful therapies. Here, we conducted an in-depth analysis of the 3xTg-AD mouse model of AD across its lifespan to better inform the field of the various pathologies that appear at specific ages, and comment on drift that has occurred in the development of pathology in this line since its development 20 years ago. This modern characterization of the 3xTg-AD model includes an assessment of impairments in behavior, cognition, and long-term potentiation followed by quantification of amyloid beta (A{beta}) plaque burden and neurofibrillary tau tangles, biochemical levels of A{beta} and tau protein, and neuropathological markers such as gliosis and accumulation of dystrophic neurites. We also present a novel comparison of the 3xTg-AD model with the 5xFAD model using the same deep-phenotyping characterization pipeline. The results from these analyses are freely available via the AD Knowledge Portal (https://admodelexplorer.synapse.org). Our work demonstrates the utility of a characterization pipeline that generates robust and standardized information relevant to investigating and comparing disease etiologies of current and future models of AD.

Contribution to the Field StatementAlzheimers Disease (AD) is an age-related neurodegenerative disorder characterized by progressive memory impairments and affects more than 30 million individuals worldwide. Using animal models of AD, researchers have elucidated disease progression and hallmark pathologies that may underpin the memory impairments seen in patients. However, therapeutic targets have failed to translate successfully from animal studies to human clinical trials, prompting a reassessment of the development, use, and interpretation of data acquired using the innumerous AD animal models available to researchers. To address these shortcomings, we have developed a robust and reproducible modern characterization of pathologies within current and future animal models of AD to better assess distinct pathologies that arise at specific brain regions and ages of different models. Using the popular 3xTg-AD mouse, we demonstrate the utility of these deep-phenotyping analyses and highlight the drift that affected development of pathologies in this line over the past two decades. Utilizing this same systematic characterization, we also perform a direct comparison with 5xFAD mice, another popular animal model of AD. The robust and standardized data generated from these systematic deep-phenotyping analyses are available for broad use by the AD research community to assess, compare, and determine appropriate animal models of AD.


title: Dynamic DNA methylation changes in the COMT gene promoter region in response to mental stress and its modulation by transcranial direct current stimulation

abstract: Changes in epigenetic modifications present a mechanism how environmental factors like the experience of stress can alter gene regulation. While stress-related disorders have consistently been associated with differential DNA methylation, little is known about the time scale in which these alterations emerge. We investigated dynamic DNA methylation changes in whole blood of 42 healthy male individuals in response to a stressful cognitive task, its association with concentration changes in cortisol and its modulation by transcranial direct current stimulation (tDCS). We observed a continuous increase in COMT promotor DNA methylation which correlated with higher saliva cortisol levels and was still detectable one week later. However, this lasting effect was suppressed by concurrent activity-enhancing anodal tDCS to the dorsolateral prefrontal cortex. Our findings support the significance of gene-specific DNA methylation in whole blood as potential biomarkers for stress-related disorders. Moreover, they suggest alternative molecular mechanisms possibly involved in lasting behavioral effects of tDCS.


title: Pyruvate dehydrogenase kinase supports macrophage NLRP3 inflammasome activation during acute inflammation

abstract: Activating macrophage NLRP3 inflammasome can promote excessive inflammation, leading to severe cell and tissue damage and organ dysfunction. Here, we showed that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuated macrophage NLRP3 inflammasome activation. Broad rewiring of intracellular metabolism and enhanced autophagic flux occurred in inflammasome-activated macrophages, but neither was necessary for the PDHK-regulated reduction of NLRP3 inflammasome activity. PDHK inhibition protected against inflammation-induced mitochondrial fragmentation and cristae remodeling and improved mitochondrial function by repurposing mitochondria from ROS production to ATP generation. Inhibition of PDHK increased the expression of the mitochondrial fusion protein optic atrophy-1 (OPA1). Suppression of OPA1 partially reversed the effect of PDHK inhibition on NLRP3 inflammasome activation. In conclusion, our study suggests that inhibition of PDHK dampens macrophage NLRP3 inflammasome activation during acute inflammation by ameliorating mitochondrial damage in a mechanism separate from its canonical role as a metabolic regulator.


title: Identification of Thermotolerant Rice Genotypes with Allele Coding at Seedling Stage

abstract: Rice-The most important plant in the world to ensure food security. Heat is one of the main factors that greatly limit rice production. With the increasing global warming, industrialization there is a great effect on climate change which requires us to see various alternatives for strains that are more tolerant to heat so that some techniques are developed to filter a large number of genotypes for high temperature tolerance. Here we report the standardization of Temperature Induction Response (TIR) technique to identify thermotolerant rice genotypes. The phenotypic characteristics of Rice due to high temperature is calculated with germination (%), growth of the seedling and molecular analysis is also considered. The heat stress is provided to the plants with the help of TIR protocol with the adjustment of temperature to lethal (55{degrees}C) and sub-lethal levels (38-55{degrees}C) in a TIR chamber with alterations in humidity. Of the 74 genotypes screened, 14 showed thermo tolerance caused by high temperatures. Both tolerant and sensitive genotypes were separated based on their survival percentages. The tolerant class are selected based on the growth and development of genotypes having high survival percentage and also their shoot and root lengths, fresh and dry weights are compared to the heat tolerant checks N22, Dular and Nipponbare. These genotypes have intrinsic heat tolerance and thus can be explored as a source of donors in breeding programs intended for global warming. The molecular markers which are identified to be linked with heat tolerant class through allele code are quite helpful and can be used in marker assisted breeding approach to attain heat tolerance in cultivated varieties.


title: Compound Screening with Deep Learning for Neglected Diseases: Leishmaniasis

abstract: AO_SCPLOWBSTRACTC_SCPLOWDeep learning provides a tool for improving screening of candidates for drug re-purposing to treat neglected diseases. We show how a new pipeline can be developed to address the needs of repurposing for Leishmaniasis. In combination with traditional molecular docking techniques, this allows top candidates to be selected and analyzed, including for molecular descriptor similarity.


title: A general theoretical framework for trait-based eco-evolutionary dynamics: population structure, intraspecific variation, and community assembly

abstract: To understand how functional traits shape ecological communities it is necessary to understand both how traits across the community affect its functioning and how eco-evolutionary dynamics within the community change the traits over time. Of particular interest are so-called evolutionarily stable communities (ESCs), since these are the end points of eco-evolutionary dynamics and can persist over long time scales. One theoretical framework that has successfully been used for assembling ESCs is adaptive dynamics. However, this framework cannot account for intraspecific variation-- neither locally nor across structured populations. On the other hand, in moment-based approaches, intraspecific variation is accommodated, but community assembly has been neglected. This is unfortunate as some questions regarding for example local adaptation vis-a-vis diversification into multiple species requires both facets. In this paper we develop a general theoretical framework that bridges the gap between these two approaches. We showcase how ESCs can be assembled using the framework, and illustrate various aspects of the framework using two simple models of resource competition. We believe this unifying framework could be of great use to address questions regarding the role of functional traits in communities where population structure, intraspecific variation, and eco-evolutionary dynamics are all important.

category: ecology


title: Sperm of more colorful males are better adapted to ovarian fluids in lake char (Salmonidae)

abstract: Fish often spawn eggs with ovarian fluids that have been hypothesized to support sperm of some males over others (cryptic female choice). Alternatively, sperm reactions to ovarian fluids could reveal male strategies linked to their likely roles during spawning. Sperm of males who would usually be close to females during spawning are then expected to be better adapted to the presence of ovarian fluids than to water only, while the reverse would be expected for males that typically spawn at larger distance to the females. We tested these predictions with gametes and ovarian fluids from wild-caught lake char (Salvelinus umbla). We found that sperm of more colorful males showed increased sperm velocity in diluted ovarian fluids while sperm of paler males were fastest in water only. We then let equal numbers of sperm compete for fertilizations in the presence or absence of ovarian fluids and used microsatellite markers to assign in total 1,464 embryos (from 70 experimental trials) to their fathers. Overall, sperm of more colorful males reached higher fertilization success than sperm of pale males. This difference was enhanced by the presence of ovarian fluids and best explained by the increased sperm velocity. Sperm competitiveness was not enhanced with decreasing genetic distance to a given female, although parallel stress tests on embryos had revealed that females would profit more from mating with least related males rather than most colored ones. We conclude that sperm of more colorful males are best adapted to ovarian fluids, and that the observed reaction norms reveal male strategies rather than cryptic female choice.


title: Anther cones increase pollen release in buzz-pollinated Solanum flowers

abstract: The widespread evolution of tube-like anthers releasing pollen from apical pores is associated with buzz pollination, in which bees vibrate flowers to remove pollen. The mechanical connection among anthers in buzz-pollinated species varies from loosely held conformations, to anthers tightly held together with trichomes or bio-adhesives forming a functionally joined conical structure (anther cone). Joined anther cones in buzz-pollinated species have evolved independently across plant families and via different genetic mechanisms, yet their functional significance remains mostly untested. We used experimental manipulations to compare vibrational and functional (pollen release) consequences of joined anther cones in three buzz-pollinated species of Solanum (Solanaceae). We applied bee-like vibrations to focal anthers in flowers with ("joined") and without ("free") experimentally created joined anther cones, and characterised vibrations transmitted to other anthers and the amount of pollen released. We found that joined anther architectures cause non-focal anthers to vibrate at higher amplitudes than free architectures. Moreover, in the two species with naturally loosely held anthers, anther fusion increases pollen release, while in the species with a free but naturally compact architecture it does not. We discuss hypotheses for the adaptive significance of the convergent evolution of joined anther cones.


title: Loss of Protein Kinase D2 Activity Protects Against Bleomycin-induced Dermal Fibrosis in Mice

abstract:
BackgroundDermal fibrosis occurs in many human diseases, particularly systemic sclerosis (SSc) where persistent inflammation leads to collagen deposition and fiber formation in skin and multiple organs. The family of protein kinase D (PKD) has been linked to inflammatory responses in various pathological conditions, however, its role in inflammation-induced dermal fibrosis has not been well defined. Here, using a murine fibrosis model that gives rise to dermal lesions similar to those in SSc, we investigated the role of PKD in dermal fibrosis in mice lacking PKD2 activity.

MethodsHomozygous kinase-dead PKD2 knock-in mice (PKD2SSAA/SSAA-KI) were obtained through intercrossing mice heterozygous for PKD2S707A/S711A (PKD2SSAA). The wild-type and KI mice were subjected to repeated subcutaneous injection of bleomycin (BLM) to induce dermal inflammation and fibrosis. As controls, mice were injected with PBS. At the end of the experiment, mouse skin at the injection site was dissected, stained, and analyzed for morphological changes and expression of inflammatory and fibrotic markers. PKD-regulated signaling pathways were examined by real-time RT-qPCR and Western blotting. In a separate experiment, BLM-treated mice were administered with or without a PKD inhibitor, CRT0066101 (CRT). The effects of CRT on dermal fibrosis were analyzed similarly. The identity of the PKD expressing cells were probed using myeloid lineage markers CD45, CD68 in BLM-treated mouse tissues.

ResultsDermal thickness and collagen fibers of kinase-dead PKD2-KI mice were significantly reduced in response to BLM treatment as compared to the wild-type mice. These mice also exhibited reduced -smooth muscle actin (-SMA) and collagen expression. At molecular levels, both transforming growth factor {beta}1 (TGF-{beta}1) and interleukin-6 (IL-6) mRNAs were decreased in the KI mice treated with BLM as compared to those in the wild-type mice. Similarly, CRT significantly blocked BLM-induced dermal fibrosis and inhibited the expression of -SMA, collagen, and IL-6 expression. Further analysis indicated that PKD2 was mainly expressed in CD45+/CD68+ myeloid cells that could be recruited to the lesional sites to promote the fibrotic process of the skin in response to BLM.

ConclusionsKnock-in of the kinase-dead PKD2 or inhibition of PKD activity in mice protected against BLM-induced dermal fibrosis by reducing dermis thickness and expression of fibrotic biomarkers including -SMA, collagen, and inflammatory/fibrotic mediators including TGF-{beta}1 and IL-6. PKD2 does this potentially through modulating the recruitment and function of myeloid cells in skin of BLM-treated mice. Overall, our study demonstrated a potential critical role of PKD catalytic activity in inflammation-induced dermal fibrosis.

category: genetics


title: Effects of chronic dietary hexavalent chromium on bioaccumulation and immune responses in the sea cucumber Apostichopus japonicus

abstract: Sea cucumbers Apostichopus japonicus (3.54 {+/-} 0.01 g of wet weight) were exposed to five concentrations of dietary hexavalent chromium [0 (control), 100, 200, 400, and 800 mg Cr6+/kg dry weight] amended with K2Cr2O7 for 30 days. The bioaccumulation and immune responses [antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT); hydrolytic enzymes: acid phosphatase (ACP) and alkaline phosphatase (AKP)] of sea cucumbers were subsequently evaluated. This study found that the order of Cr accumulation in the experimental tissues was respiratory tree > intestine > body wall. Significantly lower SOD activities occurred in the 400 mg/kg group compared to that in the control group. Higher dietary Cr6+ exposure (400 and 800 mg Cr6+ /kg dry weight) did not negatively alter the CAT activities, but significantly inhibited CAT activities in 100 mg/kg group, compared to control group. ACP activities in groups 200, 400 and 800 mg/kg were significantly lower than those in control group, while no significant differences occurred in AKP activities among groups. The present study provides important information into the bioaccumulation and immune responses of the sea cucumber A. japonicus in response to chronic dietary Cr6+ exposure.


title: It is the Locus Coeruleus! Or... is it? : A proposition for analyses and reporting standards for structural and functional magnetic resonance imaging of the noradrenergic Locus Coeruleus

abstract:
The noradrenergic locus coeruleus (LC) in the brainstem shows early signs of protein pathologies in neurodegenerative diseases such as Alzheimers and Parkinsons disease. As the LCs small size (approximately 2.5 mm in width) presents a challenge for molecular imaging, the past decade has seen a steep rise in structural and functional Magnetic Resonance (MR) studies aiming to characterise the LCs changes in ageing and neurodegeneration. However, given its position in the brainstem and small volume, great care must be taken to yield methodologically reliable MR results as spatial deviations in transformations can greatly reduce the statistical power of the analyses at the group level. Here, we suggest a spatial transformation procedure and a set of quality assessment methods which allow LC researchers to achieve the spatial precision necessary for investigating this small but potentially impactful brain structure.

Using a combination of available toolboxes (SPM12, ANTs, FSL, FreeSurfer), individual structural and functional 3T LC scans are transformed into MNI space via a study-specific anatomical template. Following this, the precision of spatial alignment in individual MNI-transformed images is quantified using in-plane distance measures based on slice-specific centroids of structural LC segmentations and based on landmarks of salient anatomical features in mean functional images, respectively.

Median in-plane distance of all landmarks on the transformed structural as well as functional LC imaging data were below 2 mm, thereby falling below the typical LC width of 2.5 mm suggested by post-mortem data.

With the set of spatial post-processing steps outlined in this paper and available for download, we hope to give readers interested in LC imaging a starting point for a reliable analysis of structural and functional MR data of the LC and to have also taken a first step towards establishing reporting standards of LC imaging data.


title: A Budding Yeast Model and Screen to Define the Functional Consequences of Oncogenic Histone Missense Mutations

abstract: Somatic missense mutations in histone genes turn these essential proteins into oncohistones, which can drive oncogenesis. Understanding how missense mutations alter histone function is challenging in mammals as mutations occur in a single histone gene. For example, described oncohistone mutations predominantly occur in the histone H3.3 gene, despite the human genome encoding 15 H3 genes. To understand how oncogenic histone missense mutations alter histone function, we leveraged the budding yeast model, which contains only two H3 genes, to explore the functional consequences of oncohistones H3K36M, H3G34W, H3G34L, H3G34R, and H3G34V. Analysis of cells that express each of these variants as the sole copy of H3 reveals that H3K36 mutants show different drug sensitivities compared to H3G34 mutants. This finding suggests that changes to proximal amino acids in the H3 N-terminal tail alter distinct biological pathways. We exploited the caffeine sensitive growth of H3K36 mutant cells to perform a high copy suppressor screen. This screen identified genes linked to histone function and transcriptional regulation, including Esa1, a histone H4/H2A acetyltransferase; Tos4, a forkhead-associated domain-containing gene expression regulator; Pho92, an N6-methyladenosine RNA binding protein and Sgv1/Bur1, a cyclin-dependent kinase. We show that the Esa1 lysine acetyltransferase activity is critical for suppression of the caffeine sensitive growth of H3K36R mutant cells while the previously characterized binding interactions of Tos4 and Pho92 are not required for suppression. This screen identifies pathways that could be altered by oncohistone mutations and highlights the value of yeast genetics to identify pathways altered by such mutations.


title: Contrast Solution Properties and Scan Parameters Influence the Apparent Diffusivity of Computed Tomography Contrast Agents in Articular Cartilage

abstract: The inability to detect early degenerative changes to the articular cartilage surface that commonly precede bulk osteoarthritic degradation is an obstacle to early disease detection for research or clinical diagnosis. Leveraging a known artifact that blurs tissue boundaries in clinical arthrograms, contrast agent diffusivity can be derived from computed tomography arthrography (CTa) scans. We combined experimental and computational approaches to study protocol variations that may alter the CTa-derived apparent diffusivity. In experimental studies on bovine cartilage explants, we examined how contrast agent dilution and transport direction (absorption vs. desorption) influence the apparent diffusivity of untreated and enzymatically digested cartilage. Using multiphysics simulations, we examined mechanisms underlying experimental observations and the effects of image resolution, scan interval and early scan termination. The apparent diffusivity during absorption decreased with increasing contrast agent concentration by an amount similar to the increase induced by tissue digestion. Models indicated that osmotically induced fluid efflux strongly contributed to the concentration effect. Simulated changes to spatial resolution, scan spacing and total scan time all influenced the apparent diffusivity, indicating the importance of consistent protocols. With careful control of imaging protocols and interpretations guided by transport models, CTa-derived diffusivity offers promise as a biomarker for early degenerative changes.

category: bioengineering


title: An Information Theoretic Framework for Protein Activity Measurement

abstract: Nonparametric analytical Rank-based Enrichment Analysis (NaRnEA) is a novel gene set analysis method which leverages an analytical null model derived under the Principle of Maximum Entropy. NaRnEA critically improves over two widely used methods - Gene Set Enrichment Analysis (GSEA) and analytical Rank-based Enrichment Analysis (aREA) - as shown by differential activity measurement of ~2,500 transcriptional regulatory proteins across three cohorts in The Cancer Genome Atlas (TCGA) based on the enrichment of their transcriptional targets in differentially expressed genes. Phenotype-matched proteomic data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) was used to evaluate measurement accuracy. We show that the sample-shuffling empirical null models leveraged by GSEA and aREA are overly conservative, a shortcoming that is critically addressed by NaRnEAs optimal analytical null model.


title: Identification and validation of a non-genetically encoded vulnerability to XPO1 inhibition in malignant rhabdoid tumors - expanding patient-driven discovery beyond the Nof1.

abstract:
Malignant rhabdoid tumors (MRTs) are rare, aggressive pediatric solid tumors, characterized by a 22q11 deletion that inactivates the SMARCB1 gene. Outcomes remain poor despite multimodality treatment. MRTs are among the most genomically stable cancers and lack therapeutically targetable genetic mutations. We utilized metaVIPER, an extension of the Virtual Inference of Protein-activity by Enriched Regulon (VIPER) algorithm, to computationally infer activated druggable proteins in the tumor of an eight month old patient and then expanded the analysis to TCGA and TARGET cohorts. In vitro studies were performed on a panel of MRT and atypical teratoid/rhabdoid tumor cell lines. Two patient-derived xenograft (PDX) mouse models of MRT were used for in vivo efficacy studies. MetaVIPER analysis from the patients tumor identified significantly high inferred activity of nuclear export protein Exportin-1 (XPO1). Expanded metaVIPER analysis of TCGA and TARGET cohorts revealed consistent elevations in XPO1 inferred activity in MRTs compared to other cancer types. All MRT cell lines demonstrated baseline activation of XPO1. MRT cell lines demonstrated in vitro sensitivity to the XPO1 inhibitor, selinexor which led to cell cycle arrest and induction of apoptosis. Targeted inhibition of XPO1 in patient-derived xenograft models of MRT using selinexor resulted in abrogation of tumor growth. Selinexor demonstrates efficacy in preclinical models of MRT. These results support investigation of selinexor in a phase II study in children with MRT and illustrate the importance of an N-of-1 approach in driving discovery beyond the single patient.

Statement of Translational RelevanceWe describe the patient-driven discovery of XPO1 activation as a non-genetically encoded vulnerability in MRTs. The application of metaVIPER analysis to tumors lacking actionable oncogenic alterations represents a novel approach for identifying potential therapeutic targets and biomarkers of response. Our preclinical validation of selinexor confirms XPO1 inhibition as a promising therapeutic strategy for the treatment of MRT.


title: Finding and Keeping the Beat: Neural Mechanisms Differ as Beat Perception Unfolds

abstract: Perception of a regular beat is essential to our ability to synchronize movements to music in an anticipatory fashion. Beat perception requires multiple, distinct neural functions, corresponding to the perceptual stages that occur over time, including 1) detection that regularity is present (beat finding), 2) prediction of future regular events to enable anticipation (beat continuation), and 3) dynamic adjustment of predictions as the rhythmic stimulus changes (beat adjustment). The striatum has been shown to be crucial for beat perception generally, although it is unclear how, or whether, distinct regions of the striatum contribute to these different stages of beat perception. Here, we used fMRI to investigate the activity of striatal subregions during the different stages of beat perception. Participants listened to pairs of rhythms (polyrhythms) whose temporal structure induced distinct perceptual stages--finding, continuation, and adjustment of the beat. Dorsal putamen was preferentially active during beat finding, whereas the ventral putamen was preferentially active during beat adjustment. We also observed that anterior insula activity was sensitive to metrical structure (greater when polyrhythms were metrically incongruent than when they were congruent). These data implicate the dorsal putamen in the detection of regularity, possibly by detection of coincidences between cortical oscillations, and the ventral putamen in the adjustment of regularity perception, possibly by integration of prediction errors in ongoing beat predictions. Additionally, activity in the supramarginal and superior temporal gyri correlated with beat tapping performance, and activity in the superior temporal gyrus correlated with beat perception (performance on the Beat Alignment Test).


title: Anatomical and functional connectivity support the existence of a salience network node within the caudal ventrolateral prefrontal cortex

abstract: Three large-scale brain networks are considered essential to cognitive flexibility: the ventral and dorsal attention (VAN and DAN) and salience (SN) networks. The ventrolateral prefrontal cortex (vlPFC) is a known component of the VAN and DAN, but there is an important gap in the current knowledge regarding its involvement in the SN. In this study, we used a translational and multimodal approach to fulfill this gap and demonstrate the existence of a SN node within the vlPFC. First, we used tract-tracing methods in non-human primates (NHP) to quantify the anatomic connectivity strength between the different vlPFC areas and the frontal and insular cortices. The strongest connections with the dorsal anterior cingulate cortex (dACC) and anterior insula (AI) locations comprising the two main cortical SN nodes were derived from the caudal area 47/12. This location also has strong axonal projections to subcortical structures of the salience network, including the dorsomedial thalamus, hypothalamus, sublenticular extended amygdala, and periaqueductal gray. Second, we used a seed-based functional connectivity analysis in NHP resting-state functional MRI (rsfMRI) data to validate the caudal area 47/12 as an SN node. Third, we used the same approach in human rsfMRI data to identify a homologous structure in caudal area 47/12, also showing strong connections with the SN cortical nodes, thus confirming the caudal area 47/12 as the SN node in the vlPFC. Taken together, the vlPFC contains nodes for all three cognitive networks, the VAN, DAN, and SN. Thus, the vlPFC is in a position to switch between these three cognitive networks, pointing to a key role as an attentional hub. Its tight additional connections to the orbitofrontal, dorsolateral, and ventral premotor cortices, places the vlPFC at the center for switching behaviors based on environmental stimuli, computing value and cognitive control.


title: Context-dependent selectivity to natural scenes in the retina

abstract: Retina ganglion cells extract specific features from natural scenes and send this information to the brain. In particular, they respond to local light increase (ON responses), and/or decrease (OFF). However, it is unclear if this ON-OFF selectivity, characterized with synthetic stimuli, is maintained when they are stimulated with natural scenes. Here we recorded the responses of ganglion cells of mice and axolotls to stimuli composed of natural images slightly perturbed by patterns of random noise to determine their selectivity during natural stimulation. The ON-OFF selectivity strongly depended on the natural image. A single ganglion cell can signal luminance increase for one natural image, and luminance decrease for another. Modeling and experiments showed that this was due to the non-linear combination of different pathways of the retinal circuit. Despite the versatility of the ON-OFF selectivity, a systematic analysis demonstrated that contrast was reliably encoded in these responses. Our perturbative approach thus uncovers the selectivity of retinal ganglion cells to more complex features than initially thought during natural scene stimulation.