title: Locomotor strategies inside the blind walks: freely moving previsual rat pups in the open field test

abstract:
Blind walks of previsual rat pups in the open field test were considered as a supposedly random process. At this age, immature rats should rely only on non-visual modalities for sampling a novel space and constructing putative cognitive maps. Spontaneous locomotion of outbred Wistar rat infants (n=51) on their 13th postnatal day was tracked for 2 minutes and analyzed offline for the presence of random components and/or strategies of locomotion. Three distinct patterns were observed. A large portion of the pups (n=22) performed sub-diffusive localized walks in the center. A smaller cohort (n=9) undertook almost immediate quasi-linear raids in a random direction, thus succeeded in reaching a shelter (i.e., walls vicinity). The rest (n=20) demonstrated a mixed strategy: localized walks interspersed with quasi-linear raids. An algorithm for automated segmentation of the open field trajectories into the localized walks and quasi-linear fragments was developed; the fragments were analyzed separately.

Statistical analysis shows the localized walks to be diffusive (i.e., Brownian) only within 2-3 seconds, but essentially sub-diffusive for longer time scales. The autocorrelation function shows that sub-diffusion was caused by the trajectories re-attraction. Self-odor traces can be a physical cue ensuring the effect.

Gender or body weights were not significant predictors for any locomotor parameters. The localized walks can be considered as a primary level of idiothetically-cued path integration in previsual pups, whereas the run trials can be a form of an active escape response. The applied method of space potentials avails the automated functional segmentation of locomotor tracks and thus brings advantages of big data analysis for studying non-visual navigation in animals.


title: A simple DLP-bioprinting strategy produces cell-laden crypt-villous structures for an advanced 3D gut model

abstract:
The intestine is a complex tissue with a characteristic three-dimensional (3D) crypt-villous architecture, which plays a key role in the intestinal function. This function is also regulated by the intestinal stroma that actively supports the intestinal epithelium, maintaining homeostasis. Efforts to account for the 3D complex structure of the intestinal tissue have been focused mainly in mimicking the epithelial barrier, while solutions to include the stromal compartment are scarce and unpractical to be used in routine experiments. Here we demonstrate that by employing an optimized bioink formulation and the suitable printing parameters it is possible to produce fibroblast-laden crypt-villous structures by means of digital light processing (DLP) stereolithography. This process provides excellent cell viability, accurate spatial resolution and high printing throughput, resulting in a robust biofabrication approach that yields functional gut mucosa tissues compatible with conventional testing techniques.

Teaser3D bioprinting approach for the direct fabrication of advanced cell-laden tissue constructs by means of visible-light photopolymerization.

category: bioengineering


title: Superparamagnetic iron oxide enclosed hollow gold nanostructure with tunable surface plasmon resonances to promote near-infrared photothermal conversion

abstract: In this study, to enhance deep tissue penetration by near-infrared (NIR) light, a novel superparamagnetic iron oxide enclosed hollow gold nanoshell (SPIO-HGNS) structure with tunable size and surface plasmon resonance (SPR) in the NIR range was designed and synthesized through a 2-step template-enabled galvanic replacement reaction. Here, Ag coated SPIO (SPIO-Ag) was prepared as a template with tunable outer diameters by way of adjusting the Ag content. SPIO-HGNS with variable hollow gold inner diameters can then be synthesized based on the determined outer diameter of the SPIO-Ag template through a galvanic replacement reaction between HAuCl3 and Ag coating on the SPIO surface. With incrementing amounts of Ag, three SPIO-HGNS structures were synthesized with comparable shell thicknesses around 6.7 nm and an average inner diameter of 38.7, 39.4, and 40.7 nm, respectively, evidenced by TEM and ICP results. The structure of SPIO-HGNS was confirmed by identifying Au111 lattice and the elemental mapping of Fe and Au using energy-dispersive X-ray spectroscopy. The Ultraviolet-Visible-NIR absorption spectra showed red-shifted SPR peaks (820, 855, and 945 nm) with the increasing inner diameters of SPIO-HGNS, which was also supported by an absorption cross-section simulation. The photothermal results showed that the three SPIO-HGNS structures, when exposed to ~30 s of 400 mW laser irradiation, exhibited photothermal temperature rises of 5.9, 4.6, and 2.9 {degrees}C, respectively. This study explored the tuning of SPR properties in NIR-responsive magneto-plasmonic nanoparticles through a facile preparation procedure, paving the way for potential applications in photothermal therapies.


title: Correlative imaging of spatio-angular dynamics of molecular assemblies and cells with multimodal instant polarization microscope

abstract: Biological function depends on the spatio-angular architecture of macromolecules - for example, functions of lipid membrane and cytoskeletal polymers arise from both the spatial and the angular organization of the constituent molecules. Correlative imaging of cellular and molecular architecture is valuable across cell biology and pathology. However, current live imaging methods primarily focus on spatial component of the architecture. Imaging the dynamic angular architecture of cells and organelles requires fast polarization-, depth-, and wavelength-diverse measurement of intrinsic optical properties and fluorophore concentration, but remains challenging with current designs. We report a multimodal instant polarization microscope (miPolScope) that combines a broadband polarization-resolved detector, automation, and reconstruction algorithms to enable label-free imaging of phase, retardance, and orientation, multiplexed with fluorescence imaging of concentration, anisotropy, and orientation of molecules at diffraction-limited resolution and high speed. miPolScope enabled multimodal imaging of myofibril architecture and contractile activity of beating cardiomyocytes, cell and organelle architecture of live HEK293T and U2OS cells, and density and anisotropy of white and grey matter of mouse brain tissue across the visible spectrum. We anticipate these developments in joint quantitative imaging of density and anisotropy to enable new studies in tissue pathology, mechanobiology, and imaging-based screens.


title: Recombination-aware Phylogeographic Inference Using the Structured Coalescent with Ancestral Recombination

abstract: Movement of individuals between populations or demes is often restricted, especially between geographically isolated populations. The structured coalescent provides an elegant theoretical framework for describing how movement between populations shapes the genealogical history of sampled individuals and thereby structures genetic variation within and between populations. However, in the presence of recombination an individual may inherit different regions of their genome from different parents, resulting in a mosaic of genealogical histories across the genome, which can be represented by an Ancestral Recombination Graph (ARG). In this case, different genomic regions may have different ancestral histories and so different histories of movement between populations. Recombination therefore poses an additional challenge to phylogeographic methods that aim to reconstruct the movement of individuals from genealogies, although also a potential benefit in that different loci may contain additional information about movement. Here, we introduce the Structured Coalescent with Ancestral Recombination (SCAR) model, which builds on recent approximations to the structured coalescent by incorporating recombination into the ancestry of sampled individuals. The SCAR model allows us to infer how the migration history of sampled individuals varies across the genome from ARGs, and improves estimation of key population genetic parameters such as population sizes, recombination rates and migration rates. Using the SCAR model, we explore the potential and limitations of phylogeographic inference using full ARGs. We then apply the SCAR to lineages of the recombining fungus Aspergillus flavus sampled across the United States to explore patterns of recombination and migration across the genome.

category: bioinformatics


title: Transformation of alignment files improves performance of variant callers for long-read RNA sequencing data

abstract: Long-read RNA sequencing (lrRNA-seq) produces detailed information about full-length transcripts, including novel and sample-specific isoforms. Furthermore, there is opportunity to call variants encoded in the transcribed regions of genes directly from lrRNA-seq data. However, most state-of-the-art variant callers have been developed for genomic DNA and thus require modifications to call variants from lrRNA-seq data. Here, we benchmark variant callers GATK, DeepVariant, Clair3, and NanoCaller on PacBio lrRNA-seq, or "Iso-Seq", data. In particular, we found that careful processing of alignment files is critical to achieve better calling performance of indels and SNPs using DeepVariant and indels using Clair3.


title: Haplotype-enhanced inference of somatic copy number profiles from single-cell transcriptomes

abstract: Genome instability and aberrant alterations of transcriptional programs both play important roles in cancer. However, their relationship and relative contribution to tumor evolution and therapy resistance are not well-understood. Single-cell RNA sequencing (scRNA-seq) has the potential to investigate both genetic and non-genetic sources of tumor heterogeneity in a single assay. Here we present a computational method, Numbat, that integrates haplotype information obtained from population-based phasing with allele and expression signals to enhance detection of CNVs from scRNA-seq data. To resolve tumor clonal architecture, Numbat exploits the evolutionary relationships between subclones to iteratively infer the single-cell copy number profiles and tumor clonal phylogeny. Analyzing 21 tumor samples composed of multiple myeloma, breast, and thyroid cancers, we show that Numbat can accurately reconstruct the tumor copy number profile and precisely identify malignant cells in the tumor microenvironment. We uncover additional subclonal complexity contributed by allele-specific alterations, and identify genetic subpopulations with transcriptional signatures relevant to tumor progression and therapy resistance. We hope that the increased power to characterize genomic aberrations and tumor subclonal phylogenies provided by Numbat will help delineate contributions of genetic and non-genetic mechanisms in cancer.


title: SPRI: Spatial Pattern Recognition using Information based method for spatial gene expression data

abstract: The rapid development of spatially resolved transcriptomics has made it possible to analyze spatial gene expression patterns in complex biological tissues. To identify such genes, we propose a novel and robust nonparametric information-based approach, SPRI, to recognize their spatial patterns. SPRI directly models spatial transcriptome raw count data without model assumptions, which transforms the problem of spatial expression pattern recognition into the detection of dependencies between spatial coordinate pairs with gene read count as the observed frequencies. SPRI was used to analyze four recent published spatially resolved transcriptome data, and all results showed that SPRI outperforms prior methods, by robustly detecting more genes with significant spatial expression patterns, and revealing biological insights that cannot be identified by other methods.


title: Omics-informed CNV calls reduce false positive rate and improve power for CNV-trait associations

abstract: Copy number variations (CNV) are believed to play an important role in a wide range of complex traits but discovering such associations remains challenging. Whilst whole genome sequencing (WGS) is the gold standard approach for CNV detection, there are several orders of magnitude more samples with available genotyping microarray data. Such array data can be exploited for CNV detection using dedicated software (e.g., PennCNV), however these calls suffer from elevated false positive and negative rates. In this study, we developed a CNV quality score that weights PennCNV calls (pCNV) based on their likelihood of being true positive. First, we established a measure of pCNV reliability by leveraging evidence from multiple omics data (WGS, transcriptomics and methylomics) obtained from the same samples. Next, we built a predictor of omics-confirmed pCNVs, termed omics-informed quality score (OQS), using only PennCNV software output parameters. Promisingly, OQS assigned to pCNVs detected in close family members was up to 35% higher than the OQS of pCNVs not carried by other relatives (P < 3.0-10-90), outperforming other scores. Finally, in an association study of four anthropometric traits in 89,516 Estonian Biobank samples, the use of OQS led to a relative increase in the trait variance explained by CNVs of up to 34% compared to raw pCNVs or previous quality scores. Overall, we put forward a flexible framework to improve any CNV detection method leveraging multi-omics evidence, applied it to improve PennCNV calls and demonstrated its utility by improving the statistical power for downstream association analyses.


title: Growing Phylogenetic Trees Using Hierarchical Clustering

abstract: We compare mitochondrial DNA of different species to build a phylogenetic tree. The main challenge is that the calculation of the Levenshtein distance is very slow for large sequences. We introduce an approximate scheme which uses Levenshtein for lower-level details and a fast distance based on sequence length for high-level attributes. The tree is built using average linkage hierarchical clustering.


title: Resolving single-cell copy number profiling for large datasets

abstract: The advances of single-cell DNA sequencing (scDNA-seq) enable us to characterize the genetic heterogeneity of cancer cells. However, the high noise and low coverage of scDNA-seq impede the estimation of copy number variations (CNVs). In addition, existing tools suffer from intensive execution time and often fail on large datasets. Here, we propose SeCNV, a novel method that leverages structural entropy, to profile the copy numbers. SeCNV adopts a local Gaussian kernel to construct a matrix, depth congruent map, capturing the similarities between any two bins along the genome. Then SeCNV partitions the genome into segments by minimizing the structural entropy from the depth congruent map. With the partition, SeCNV estimates the copy numbers within each segment for cells. We simulate nine datasets with various breakpoint distributions and amplitudes of noise to benchmark SeCNV. SeCNV achieves a robust performance, i.e., the F1-scores are higher than 0.95 for breakpoint detections, significantly outperforming state-of-the-art methods. SeCNV successfully processes large datasets (>50,000 cells) within four minutes while other tools failed to finish within the time limit, i.e., 120 hours. We apply SeCNV to single-nucleus sequencing (SNS) datasets from two breast cancer patients and acoustic cell tagmentation (ACT) sequencing datasets from eight breast cancer patients. SeCNV successfully reproduces the distinct subclones and infers tumor heterogeneity. SeCNV is available at https://github.com/deepomicslab/SeCNV.


title: Highly perturbed genes and hub genes associated with type 2 diabetes in different tissues of adult humans: A bioinformatics analytic workflow

abstract:
IntroductionType 2 diabetes (T2D) has a complex etiology which is not fully elucidated. Identification of gene perturbations and hub genes of T2D may assist in personalizing care.

ObjectivesWe aimed to identify highly perturbed genes and hub genes associated with T2D in different tissues of adult humans via an extensive workflow.

MethodsWorkflow comprised five sequential steps: systematic review of NCBI GEO database; identification and classification of differentially expressed genes (DEG); identification of highly perturbed genes via meta-analysis; identification of hub genes via network analysis; downstream analyses. Three meta-analytic strategies: random effects model (REM); vote counting approach (VC); p-value combining approach (CA), were applied. Nodes having above average betweenness, closeness, and degree in the network were defined as hub genes. Downstream analyses included gene ontologies, Kyoto Encyclopedia of Genes and Genomes pathways, metabolomics, COVID-19 related genes, and Genotype-Tissue Expression profiles.

ResultsAnalysis of 27 eligible microarrays identified 6284 DEG (4592 down-regulated and 1692 up-regulated) within four tissue types. Tissue-specific gene expression was significantly greater than tissue non-specific (shared) gene expression. Meta-analysis of DEG identified 49, 27, and 8 highly perturbed genes via REM, VC, and CA, respectively, producing a compiled set of 79 highly perturbed (41 down-regulated and 38 up-regulated) genes. The 28 hub genes comprised 13 up-regulated, 9 down-regulated, and 6 predicted genes. Downstream analyses identified enrichments of: shared genes with other diabetes phenotypes; insulin synthesis and action related pathways and metabolomics; mechanistic associations with apoptosis and immunity-related pathways, COVID-19 related gene sets; and cell types demonstrating over- and under-expression of marker genes of T2D.

ConclusionsWe identified highly perturbed genes and hub genes of T2D and revealed their associations with other diabetes phenotypes and COVID-19 as well as pathophysiological manifestations such as those related to insulin, immunity, and apoptosis. Broader utility of the proposed pipeline is envisaged.


title: An Analysis of Protein Language Model Embeddings for Fold Prediction

abstract: The identification of the protein fold class is a challenging problem in structural biology. Recent computational methods for fold prediction leverage deep learning techniques to extract protein fold-representative embeddings mainly using evolutionary information in the form of multiple sequence alignment (MSA) as input source. In contrast, protein language models (LM) have reshaped the field thanks to their ability to learn efficient protein representations (protein-LM embeddings) from purely sequential information in a self-supervised manner. In this paper, we analyze a framework for protein fold prediction using pre-trained protein-LM embeddings as input to several fine-tuning neural network models which are supervisedly trained with fold labels. In particular, we compare the performance of six protein-LM embeddings: the LSTM-based UniRep and SeqVec, and the transformer-based ESM-1b, ESM-MSA, ProtBERT, and ProtT5; as well as three neural networks: Multi-Layer Perceptron (MLP), ResCNN-BGRU (RBG), and Light-Attention (LAT). We separately evaluated the pairwise fold recognition (PFR) and direct fold classification (DFC) tasks on well-known benchmark datasets. The results indicate that the combination of transformer-based embeddings, particularly those obtained at amino acid-level, with the RBG and LAT fine-tuning models performs remarkably well in both tasks. To further increase prediction accuracy, we propose several ensemble strategies for PFR and DFC, which provide a significant performance boost over the current state-of-the-art results. All this suggests that moving from traditional protein representations to protein-LM embeddings is a very promising approach to protein fold-related tasks.


title: Clinically driven knowledge distillation for sparsifying high-dimensional multi-omics survival models

abstract: Recently, various methods have been proposed to integrate different heterogeneous high-dimensional genomic data sources to predict cancer survival, often in addition to widely available and highly predictive clinical data. Although clinical applications of survival models have high sparsity requirements, most state-of-the-art models do not naturally exhibit this sparsity, as they are based on random forests or deep learning. We propose to use[l] 1-penalized linear student models within a knowledge distillation framework to sparsify underlying multi-omics black-box teachers. We show that by excluding clinical variables from our[l] 1 penalty, we can effectively guide the knowledge distillation, reaching virtually identical discriminative performance to the teachers while using on average 140 features or less across the 17 cancer datasets from The Cancer Genome Atlas (TCGA) considered in our study.


title: Multiphase condensates from a kinetically arrested phase transition

abstract: The formation of biomolecular condensates through liquid-liquid phase separation from proteins and nucleic acids is emerging as a spatial organisational principle used by living cells. Many such biomolecular condensates are not, however, homogeneous fluids, but contain an internal structure consisting of distinct sub-compartments with different compositions. In many instances, such compartments inside the condensate are depleted in the biopolymers that make up the condensate. Here, we describe that this multiphase structure arises from a kinetically arrested phase transition. The combination of a change in composition coupled with a slow response to this change can lead to the spontaneous formation of multiple emulsions consisting of several inner cores within a polymer-rich middle phase. In the case of liquid-like biomolecular condensates, the slow diffusion of biopolymers causes nucleation of biopolymer-poor liquid inside of the condensate to achieve the new equilibrium composition. This framework shows that multiphase condensates can be a result of kinetic trapping, rather than thermodynamic stability, and provides and avenue to understand and control the internal structure of condensates in vitro and in vivo.

category: biophysics


title: Elasticity generates indissoluble biomolecular condensates

abstract: While biomolecular condensates are often liquid-like, many experiments found that condensates also exhibit solid-like behaviors, which may make them irreversible and indissoluble. Despite the critical biological significance of indissoluble condensates to cellular fitness and diseases, the physical mechanisms underlying the stabilities of solid-like condensates are still unclear. In this work, we study the effects of elasticity on the dissolution of biomolecular condensates. We demonstrate that the bulk stress inside condensates may prevent the condensates from dissolution and obtain the new equilibrium conditions of elastic condensate: the osmotic pressure minus the bulk stress is uniform inside and outside condensates. To verify our theories, we simulate the two-fluid model in which the slow component corresponding to the polymer network generates elastic stress. Our theoretical predictions are nicely confirmed and independent of microscopic details. Moreover, we obtain a phase diagram on the stability of elastic condensates and identify a minimum bulk modulus for the condensates to be indissoluble, both numerically and theoretically. Our results may have implications in developing drugs targeting irreversible condensates.


title: Quantitative live-cell imaging of secretion activity reveals dynamic immune responses

abstract: The measurement of cytokine secretions has contributed to the development of immunology; however, new methods that enable highly sensitive and efficient analysis are required for the precise characterisation of dynamic secretion activity when using rare cells or limited human specimens. Here, we report a new technology for quantitative live-cell imaging of secretion activity (qLCI-S), that enables high-throughput and dual-colour detection of prolonged secretion activity at the single-cell level, followed by transcriptome analysis for individual cells based on their phenotype. The power of the qLCI-S was demonstrated by visualising the individual and longitudinal cytokine secretion patterns of group 2 innate lymphoid cells, which comprised <0.01% human peripheral blood mononuclear cells, and identifying their minor subpopulations. This new technology will provide new insights into the spatiotemporal dynamic nature of various secretory functions and the development of fundamental tools for phenotypic drug discovery and regenerative and precision medicine.


title: Tree of mutually exclusive oncogenes

abstract: Identifying cancer driver genes and their interrelations is critical in understanding cancer progression mechanisms. In this paper, we introduce ToMExO, a probabilistic method to infer cancer driver genes and how they affect each other, using cross-sectional data from cohorts of tumors. We model cancer progression dynamics using a tree with sets of driver genes in the nodes. This model explains the temporal orders among driver mutations and their mutual exclusivity patterns. We introduce a dynamic programming procedure for the likelihood calculation and build an MCMC inference algorithm. Together with our engineered MCMC moves, our efficient likelihood calculations enable us to work with datasets having hundreds of genes and thousands of tumors in the datasets. We demonstrate our methods performance on several synthetic datasets covering various scenarios for cancer progression dynamics. We then present the analyses of several biological datasets using the ToMExO method and validate the results using a set of method-independent metrics.

category: cancer biology


title: Longitudinal examination of perfusion and angiogenesis markers in primary colorectal tumors shows distinct signatures for metronomic and maximum-tolerated dose strategies

abstract: 1Metronomic chemotherapy (MET) has been developed to address the shortcomings of maximum-tolerated chemotherapy (MTD) in regard to toxicity and development of resistance mechanisms in the tumor. In colorectal cancer (CRC), MET is a promising novel strategy to treat locally advanced malignancies when used as neoadjuvant chemotherapy (NAC). However, so far there are no preclinical studies to assess the impact of MET NAC in CRC to assess the benefits and challenges of this approach. Here, we used a primary model of CRC (via azoxymethane) to analyze longitudinal changes in angiogenesis in primary tumors under MET and MTD NAC using a combination of diffuse reflectance spectroscopy and mRNA expression (via qPCR). Our results show that MET and MTD NAC lead to increased mean tissue oxygen saturation (8% and 5%, respectively) and oxyhemoglobin (15% and 10%) between weeks 2 and 5 of NAC, and that such increases are caused by distinct molecular signatures in the angiogenic program. Specifically, we find that in the MET group there is a sustained increase in Hif-1a, Aldoa, and Pgk1 expression, suggesting upregulated glycolysis, whereas MTD NAC causes a significant reduction in the expression of the aforementioned genes and of Vegf, leading to vascular remodeling in MTD-treated tumors. Taken together, this study demonstrates the ability of combined optical and molecular methodologies to provide a holistic picture of tumor response to therapy in CRC in a minimally invasive manner.


title: Testicular Dysgenesis Syndrome-like morphology and gene expression, and activation of Hypoxia Inducible Factor 1 Alpha in juvenile lamb testes following developmental exposure to low-level environmental chemical mixture.

abstract: Current declines in male reproductive health may, in part, be driven by anthropogenic environmental chemical exposure. Using the biosolid treated pasture (BTP) sheep model, this study examined the effects of gestational exposure to a translationally relevant mixture of environmental chemicals. At 8 weeks of age, ram lambs gestationally exposed to BTP were lighter than control, and their testes contained fewer germ cells and had a greater proportion of Sertoli-cell-only seminiferous tubules. Effects of biosolid exposure on the testicular transcriptome correlated with human testicular dysgenesis syndrome (TDS) patient data. Common differentially expressed genes indicated changes in apoptotic and mTOR signalling, which concurs with previous gene expression data from neonatal BTP lambs. Gene expression data and immunohistochemistry indicates increased HIF1 activation and nuclear localisation in BTP exposed animals, known to disrupt testosterone synthesis. Together, these results provide a potential mechanism for the pathogenesis of this phenotype, and TDS in humans.

category: pharmacology and toxicology


title: Using cGMP analogues to modulate photoreceptor light sensitivity: Perspectives for the treatment of retinal degeneration.

abstract:
Cyclic nucleotide-gated (CNG) channels play an essential role within the phototransduction cascade in vertebrates. Although rod and cone light responses are mediated through similar pathways, each photoreceptor type relies on a specific CNG-channel isoform. In many forms of retinal degeneration, increased cGMP levels initiate a pathophysiological rollercoaster, which starts with an enhanced CNG-channel activation, often in rod photoreceptors. This causes cell death of both rods and cones, and eventually leads to complete blindness. Unfortunately, the targeting of the desired channel isoform still constitutes the bottleneck for many therapeutic schemes.

Here, we present a novel strategy, based on cGMP analogues with opposite types of action, which allows for the selective modulation of either rod or cone photoreceptors. A combined treatment with a weak rod-selective CNG-channel inhibitor (Rp-8-Br-PET-cGMPS) and a cone-selective CNG-channel activator (8-pCPT-cGMP) preserved the normal CNG-channel function at physiological and pathological cGMP levels. The effectiveness of this approach was tested and confirmed in explanted mouse retina. Under physiological conditions, the inhibitor silenced the rods selectively and decreased the dependency of cone responses on light intensity. Remarkably, the activator, when applied together with the inhibitor, reinstated only the light responsiveness of cones. Yet, when applied alone, the activator dampened rod responses more strongly than those of cones.

Hence, combinations of cGMP analogues with desired properties may elegantly address the isoform-specificity problem in future pharmacological therapies. Beyond therapies for retinal degeneration diseases, treatments based on this strategy may allow modulation of visual performance in certain light environments or disease conditions.

One Sentence SummaryA combination of cGMP analogues with opposite types of action can selectively modulate either rod or cone photoreceptor function.

category: physiology


title: PDR6-mediated camalexin efflux and disease resistance are regulated through direct phosphorylation by the kinases OXI1 and AGC2-2

abstract: Plant immune signaling largely relies on post-translational modifications to establish a rapid and appropriate defense response to different pathogen types and infection pressure. Specific pleiotropic drug resistance (PDR) transporters can transport secondary metabolites to contribute to pathogen invasion resistance. However, the establishment of the post-translational regulation of PDR transporters that efflux secondary metabolites is unclear. In this study, by detecting the camalexin contents on the leaf surfaces of mutants and overexpression lines, two AGC kinases, namely, OXI1 and its closest homologue AGC2-2, were found to be related to extracellular camalexin secretion. The overexpression of OXI1 or AGC2-2 resulted in an increase in camalexin contents on the leaf surface and a decrease in camalexin contents in the leaf interior. These effects increased the resistance of the transgenic lines to surface-inoculated Pseudomonas syringae and Botrytis cinerea. Through in vitro kinase assay and in vivo phosphorylation level detection, we confirmed that the two kinases were related to the phosphorylation modification of PDR6. Pull-down assays, bimolecular fluorescence complementation, and rapamycin-dependent delocalization assays indicated the existence of direct protein-protein interaction between the two kinases and PDR6. By using LC-MS/MS, we also identified the PDR6 phosphorylation sites that were modified by the two kinases in vitro. Through the expression of the dephosphorylated variants of PDR6 in the mutant background, the action site S31 of OXI1 and the action sites S33 and S827 of AGC2-2 were found to have positive effects on the efflux activity of PDR6. In addition, T832, the action site of OXI1, may contribute to the stability of PDR6 on the plasma membrane.

category: plant biology


title: Eradicating Citrus Greening By Dual Action Targeting of Liberibacter asiaticus and Diaphorina citri.

abstract:
Huanglongbing (citrus greening) is caused by the unculturable, gram negative bacterium Liberibacter asiaticus and transmitted by the Asian Citrus Psyllid (Diaphorina citri). Prior research demonstrated that the rifampicin-derivative TPR-1 as well as Palisades Therapeutic (PT) compounds PT159 and PT160 were effective at inhibiting growth of the L. cresens, the only culturable model for Las, showing 100% inhibition at 0.5 g mL-1, and 80% inhibition at 0.05 g mL-1.

Research with the PT glucocorticoid antagonist compounds demonstrated an inhibition of bacterial growth in L crescens as well, possibly a synergistic addition to TPR-1. A search of the L. asiaticus genome revealed the presence of 3 putative glucocorticoid response elements (GRE). Putative GRE are present in all organisms and may be involved in organism signaling as well as host-pathogen crosstalk.

In Drosophila, the estrogen-related receptor (ERR) transcription factor is a master regulator of larval and pupal maturation. ERR binds to GRE promoter elements in the genome to regulate the transcription of pathways involved in glucose metabolism. Null mutations of ERR fail to leave pupation and demonstrate elevated glucose levels when compared to wild-type. Treatment of Drosophila larvae with PT compounds demonstrated developmental delay disruptions in pupation, including premature pupation and increased lethality. Pupae that had been fed PT compounds as larvae showed elevated glucose levels when compared to controls. Sequences identified in Drosophila ERR showed high homology to 2 proteins in D. citri (ERR-like 1 and ERR-like 2), leading us to hypothesize that a glucocorticoid antagonist may disrupt psyllid development. Because these genes and signaling pathways are highly conserved between these taxa, we propose that Drosophila serves as a good model for screening potential compounds for the control of psyllid populations.

Collectively, we hypothesize that the dual action of these compounds offers a solution to HLB that can salvage the dying citrus industry in Florida and provide the first effective treatment. By simultaneous targeting of both Las and D. citri, it may be possible to both cure infected trees and block psyllid development in adults feeding on those same trees. Disruption of the psyllids ability to carry Las or cure Las from the psyllid gut are also both substantial possibilities.


title: Removal Of Environmental Pollutants (Lead, Chromium And Cadmium) Using Root And Leaf Tissues Of Indian Mustard, Rice And Wheat Plants

abstract: The environment is polluted day by day and it is very much expensive to remediate the environmental pollutants by physicochemical process. Therefore, using plants as a process to remediate pollutants is essential. In this study, phytoremediation of root and leaf tissues of Indian mustard (Brassica juncea), rice (Oryza sativa) and wheat (Triticum aestivum) against three most environment pollutants viz. lead (Pb), chromium (Cr) and cadmium (Cd) of Buriganga riverbank soil of Dhaka city, Bangladesh were assessed. The highest amount of Pb was found in the leaf (11.6755 {+/-}1.9860 mg/kg dry weight) and root (51.4251 {+/-}5.0320 mg/kg dry weight) of B. juncea. However, the highest amount of Cr was found in the leaf (5.9871{+/-}0.9032 mg/kg dry weight) of B. juncea; and in the root (46.4739{+/-}2.2920 mg/kg dry weight) of O. sativa respectively. Although the highest amount (0.9624{+/-}0.0920 mg/kg dry weight) of Cd was found in the leaf of B. juncea; the amount of Cd in the root was approximately same in all the three plants. This research also found more amounts of heavy metals compared to other studies using Indian mustard, rice and wheat against the three most environmental pollutants viz., Pb, Cr and Cd. Moreover, this study revealed that B. juncea is the highest hyperaccumulator species regarding Pb, Cr and Cd accumulation and can be used to clean up the polluted Buriganga riverbank soil.


title: Genome-wide analysis of bromodomain gene family in Arabidopsis and Rice

abstract: The bromodomain containing proteins (Brd-proteins) comprise a diverse family of  epigenetic mark readers, which are an integral to epigenetic gene regulation, important for diverse cellular processes and environmental stress responses. The Brd-proteins harbour a conserved  bromodomain (BRD) that interacts with the acetylated histones in the chromatin. Presence of several additional domains enhances the structural-functional diversity and the associated functions of the Brd-proteins. Plants contains multiple Brd-proteins, however the extent of diversity and the molecular events responsible are relatively less understood. The present genome-wide analysis of Brd-members in A. thaliana and O. sativa showed extensive diversity in structure of gene and regulatory regions, expression dynamics, and domains/motifs, including bromodomain. More than 40% Brd-genes in both the species were affected by genomic duplication events, while, ~60% A. thaliana and ~40% O. sativa genes showed alternative splicing-mediated changes. These molecular events affected one or more features of the Brd-members (regulatory elements, untranslated regions, and coding regions), which could potentially alter their expression, and associated functions. The phylogenetic analysis divided the A. thaliana and O. sativa Brd-homologs into six major clusters, and displayed consistency with certain domain/motif combination architectures. Homology modelling of most A. thaliana and O. sativa Brd-homologs displayed typical BRD-fold. However, the duplicate/divergent sequences displayed important structural variations, which can potentially alter its interaction with the chromatin, and affect associated function. The results also suggested substantial contribution of various genome duplication events (predominantly block events) in the evolution and expansion of Brd-family among diverse plants, including both monocot and dicot species.


title: SAI: Fast and automated quantification of stomatal parameters on microscope images

abstract: Using microscopy to investigate stomatal behaviour is a common technique in plant physiology research. Manual inspection and measurement of stomatal features is a low throughput process in terms of time and human effort, which relies on expert knowledge to identify and measure stomata accurately. This process represents a significant bottleneck in research pipelines, adding significant researcher time to any project that requires it. To alleviate this, we introduce StomaAI (SAI): a reliable and user-friendly tool that measures stomata of the model plant Arabidopsis (dicot) and the crop plant barley (monocot grass) via the application of deep computer vision. We evaluated the reliability of predicted measurements: SAI is capable of producing measurements consistent with human experts and successfully reproduced conclusions of published datasets. Hence, SAI boosts the number of images that biologists can evaluate in a fraction of the time so is capable of obtaining more accurate and representative results.


title: Biosynthesis and apoplast accumulation of the apocarotenoid pigment azafrin in parasitizing roots of Escobedia grandiflora

abstract:
O_LIThe herbaceous hemiparasite Escobedia grandiflora (Orobanchaceae) is used in traditional medicine in the Andean region. Their roots accumulate an orange pigment with a significant relevance as a cooking dye that exhibits antioxidant and cardioprotective properties.
C_LIO_LIThe present work combined metabolic and cytological analyses with de novo transcriptome assembly, gene expression studies, and phylogenetic analyses to confirm the chemical identity of the pigment and investigate its biosynthesis and function in Escobedia roots.
C_LIO_LIThe pigment was conclusively shown to be azafrin, an apocarotenoid likely derived from the cleavage of {beta}-carotene. Candidate genes for the production of azafrin in Escobedia roots are proposed based on RNA-seq supported by RT-qPCR and phylogeny reconstruction analyses. In particular, our data suggest that azafrin production relies a carotenoid cleavage dioxygenase (CCD) different from CCD7 and similar to CCD4 enzymes. We also show that azafrin is delivered to the root apoplast and that it accumulates in the area where the Escobedia haustorium contacts the hosts root, suggesting a role of azafrin in the parasitization process.
C_LIO_LIAltogether, our work represents an unprecedented step forward in our understanding of the Escobedia parasitization system, but it also provides vital information towards the eventual domestication of this valuable medicinal plant.
C_LI


title: Simple and efficient modification of Golden Gate design standards and parts using oligo stitching

abstract:
The assembly of DNA parts is a critical aspect of contemporary biological research. Gibson assembly and Golden Gate cloning are two popular options. Here, we explore the use of single stranded DNA oligos with Gibson assembly to augment Golden Gate cloning workflows in a process called "oligo stitching". Our results show that oligo stitching can efficiently convert Golden Gate parts between different assembly standards and directly assemble incompatible Golden Gate parts without PCR amplification. Building on previous reports, we show that it can also be used to assemble de novo sequences. As a final application, we show that restriction enzyme recognition sites can be removed from plasmids and utilize the same concept to perform saturation mutagenesis. Given oligo stitchings versatility and high efficiency, we expect that it will be a useful addition to the molecular biologists toolbox.

Abstract Graphic O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

category: synthetic biology


title: A systems biology approach to elucidate the post-translational regulome of coronary artery disease

abstract: Coronary Artery Disease is a major killer in India and world at large but the molecular regulators which modulate clinically relevant pathways are not completely understood. This study was aimed at identifying essential post-translational modifications (PTM) regulome network and its master regulator modulating the CAD associated pathways. 995 CAD associated genes were taken from InCardiome database (www.tri-incardiome.org) were analyzed for all possible PTMs. Two important interdependent molecular processes which define the function of a protein are protein-protein interactions and PTMs of which PTMs play regulatory role. Using PTMCode2 we evaluated the co-evolving amino acid residues for important PTMs and found that serine-serine phosphorylation is highly represented combinatorial regulator in these set of proteins. Furthermore, in the CAD associated pathways we again found that serine phosphorylation was dominant player in all the processes of atherosclerosis. In order to identify the master regulator kinase, we further assessed the kinome network associated with CAD and identified 5 most important kinases namely GSK3B, PRKCA, PRKCD, SRC and PRKACA which might modulate clinically important pathways. GSK3B with the highest network parameters (node degree and betweenness centrality) was identified as master regulator and 1 U/l increase of phsophoGSK3B (on a log scale) increased the odds ratio (OR) by 4.07 fold (AUC 0.620) and 6.27 fold (AUC 0.752) upon addition of conventional risk factors (CRFs).


title: SC-RNA-seq assisted synthesis of a Boolean network to model early haematopoiesis aging

abstract: Hematopoietic stem cell (HSC) aging is a multifactorial event that leads to changes in HSC properties and function. These changes are intrinsically coordinated and affect the early hematopoiesis, involving hematopoietic stem and progenitor cells (HSPCs). The objective of this work is to better understand the mechanisms and factors controlling these changes. We have therefore developed an original strategy to construct a Boolean network of genes explaining the priming and homeostasis of HSCs (graphical abstract). Based on our previous scRNA-seq data, we performed an exhaustive analysis of the transcriptional network and identified active transcription modules or regulons along the differentiation trajectory of selected HSPC states. This global view of transcriptional regulation led us to focus on 15 components, 13 selected TFs (Tal1, Fli1, Gata2, Gata1, Zfpm1, Egr1, Junb, Ikzf1, Myc, Cebpa, Bclaf1, Klf1, Spi1) and 2 complexes regulating the ability of HSC to cycle (CDK4/6 - Cyclin D and CIP/KIP). We then defined the connections controlling the differentiation dynamics of HSC states and constructed an influence graph between the TFs involved in the dynamics by mixing observations from our scRNA-seq data and knowledge from the literature. Then, using answer set programming (ASP) and in silico perturbation analysis, we obtained a Boolean model which is the solution of a Boolean satisfiability problem. Finally, perturbation of the model based on age-related changes revealed important regulations, such as the overactivation of Egr1 and Junb or the loss of Cebpa activation by Gata2, which were found to be relevant for the myeloid bias of aged HSC. Our work shows the efficiency of the combination of manual and systematic methods to elaborate a Boolean model. The developed strategy led to the proposal of new regulatory mechanisms underlying the differentiation bias of aged HSCs, explaining the decreased transcriptional priming of HSCs to all mature cell types except megakaryocytes.

category: systems biology


title: A novel Boolean network inference strategy to model early hematopoiesis aging


title: First genetic data for the Critically Endangered Cuban endemic Zapata Rail Cyanolimnas cerverai, and the taxonomic implications

abstract:
The taxonomic affinity of the near-flightless Zapata Rail Cyanolimnas cerverai, a Critically Endangered and highly localized species endemic to Cuba, has long been debated. Morphological analyses have suggested that this species, which constitutes a monotypic genus, could be related either to the extinct Tahitian Cave Rails (Nesotrochis sp.) or to the South American rail tribe Pardirallini, i.e., the genera Neocrex, Mustelirallus, and Pardirallus. Whilst pronounced phenotypic convergence-and divergence-among rails have repeatedly proven morphology-based phylogenies unreliable, thus far no attempt to sequence DNA from the enigmatic Cyanolimnas has succeeded. In this study, we extracted historic DNA from a museum specimen collected in 1927 and sequenced multiple short fragments that allowed us to assemble a partial sequence of the mitochondrial cytochrome oxidase I gene. Phylogenetic analyses confirm that Cyanolimnas belongs in tribe Pardirallini as sister to genus Neocrex, from which it diverged about six million years ago. Their divergence from Mustelirallus was estimated at about eight million years ago. Based on morphology and our mitochondrial phylogeny, we conclude that it is unjustified to retain the monotypic genus Cyanolimnas and tentatively recommend that C. cerverai and the two Neocrex species are ascribed to genus Mustelirallus.

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

category: zoology


title: Reconciling GenBank names with standardized avian taxonomies to improve linkage between phylogeny and phenotype

abstract:
Biodiversity research has advanced by testing expectations of ecological and evolutionary hypotheses through the linking of large-scale genetic, distributional, and trait datasets. The rise of molecular systematics over the past 30 years has resulted in a wealth of DNA sequence data from around the globe, facilitating biodiversity research. However, advances in molecular systematics also have created taxonomic instability, as new estimates of evolutionary relationships and interpretations of species limits have led to widespread scientific name changes. Taxonomic instability, or "splits, lumps, and shuffles", present logistical challenges to large-scale biodiversity research because species or populations may be listed under different names in different data sources, or because different species or populations may be listed under previous names. Consequently, distributional and trait data are often difficult to link directly to DNA sequence data without extensive and time consuming curation. Here, we present RANT: Reconciliation of Avian NCBI Taxonomy. RANT applies taxonomic reconciliation to standardize all avian names in use in NCBI GenBank, a primary source of genetic data, to a widely-used and regularly-updated avian taxonomy: eBird/Clements. Of 14,341 avian species or subspecies names used by GenBank, 11,031 names directly matched an eBird/Clements name, which were linked to over 6 million nucleotide sequences. For the remaining unique avian names in GenBank, we used Avibases taxonomic concepts, taxonomic descriptions in Cornells Birds of the World, and DNA sequence metadata to identify corresponding eBird/Clements names. Reconciled names were linked to over 600,000 nucleotide sequences, approximately 9% of all avian sequences on GenBank. Nearly 10% of eBird/Clements names had nucleotide sequences listed under two or more GenBank names. Our avian GenBank naming reconciliation is open source and available at GitHub, where it can be updated to correspond with future annual eBird/Clements taxonomic updates.

LAY SUMMARY- 23% of avian names on GenBank do not match eBird/Clements, a widely-used standardized avian taxonomy
- 600,000 nucleotide sequences on GenBank are associated with names that do not match eBird/Clements
- 10% of eBird/Clements names have nucleotide sequences listed under multiple GenBank names
- We provide an open source taxonomic reconciliation to mitigate difficulties associated with non-standardized name use for GenBank data


title: Transient oscillations of neural firing rate associated with routing of evidence in a perceptual decision

abstract: To form a perceptual decision the brain must acquire samples of evidence from the environment and incorporate them in computations that mediate choice behavior. While much is known about the neural circuits that process sensory information and those that form decisions, less is known about the mechanisms that establish the functional linkage between them. We trained monkeys to make difficult decisions about the net direction of visual motion under conditions that required trial-by-trial control of functional connectivity. In one condition, the motion appeared at different locations on different trials. In the other, two motion patches appeared, only one of which was informative. Neurons in the parietal cortex produced brief oscillations in their firing rate at the time routing was established: upon onset of the motion display when its location was unpredictable across trials, and upon onset of an attention cue that indicated in which of two locations an informative patch of dots would appear. The oscillation was absent when the stimulus location was fixed across trials. We interpret the oscillation as a manifestation of the mechanism that establishes the source and destination of flexibly routed information, but not the transmission of the information per se.

category: neuroscience


title: An Optrode Array for Spatiotemporally Precise Large-Scale Optogenetic Stimulation of Deep Cortical Layers in Non-human Primates

abstract: Optogenetics has transformed studies of neural circuit function, but remains challenging to apply in non-human primates (NHPs). A major challenge is delivering intense and spatially precise patterned photostimulation across large volumes in deep tissue. Here, we have developed and tested the Utah Optrode Array (UOA) to meet this critical need. The UOA is a 10x10 glass waveguide array bonded to an electrically-addressable LED array. In vivo electrophysiology and immediate early gene (c-fos) immunohistochemistry demonstrate that the UOA allows for large-scale spatiotemporally precise neuromodulation of deep tissue in macaque primary visual cortex. Specifically, the UOA permits either focal (confined to single layers or columns), or large-scale (across multiple layers or columns) photostimulation of deep cortical layers, simply by varying the number of simultaneously activated LEDs and/or the light irradiance. These results establish the UOA as a powerful tool for studying targeted neural populations within single or across multiple deep layers in complex NHP circuits.


title: Automatic post-processing and merging of multiple spike-sorting analyses with Lussac

abstract: The increasing site counts of multielectrodes used in extracellular recording preclude traditional manual spike-sorting and have led to the development of several new software suites with increased automation. It remains difficult, however, to find parameters enabling optimal processing of signals from all neuronal types simultaneously. Here, we describe a procedure for automatically post-processing and merging the outputs of multiple spike-sorting analyses, enabling the accumulation of results optimised for different activity features. This approach was motivated by and first applied to the analysis of cerebellar activity, in which the complex spikes challenge standard analyses. The core of our modular procedure involves the identification of cluster-fusion candidates and assurance that only fusions that increase a clearly defined cluster-quality metric are performed; important subsidiary steps that have also been automated include spike alignment and duplicate removal. The procedure was validated by recovery of injected spike waveforms. In tests combining analyses from Kilosort and MountainSort, our procedure yielded a 50 % increase in the detected number of Purkinje cell clusters with both simple and complex spikes. A further validation used the synthetic SpikeForest dataset approximating neocortical activity, in which our package also improved performance. Combining multiple analyses therefore offers a general method for improving spike-sorting, and furthermore Lussac reduces manual input and increases the objectivity of analyses.


title: A Robust and Automated Spike Sorting Method based on the Density Peaks

abstract:
ObjectiveSpike sorting is a fundamental step in extracting single-unit activity from neural ensemble recordings, which play an important role in basic neuroscience and neurotechnologies. A few algorithms have been applied in spike sorting. However, when noise level or waveform similarity becomes relatively high, their robustness still faces a big challenge.

ApproachIn this study, we propose a spike sorting method combining Linear Discriminant Analysis (LDA) and Density Peaks (DP) for feature extraction and clustering. Relying on the joint optimization of LDA and DP: DP provides more accurate classification labels for LDA, LDA extracts more discriminative features to cluster for DP, and the algorithm achieves high performance after iteration. We first compared the proposed LDA-DP algorithm with several algorithms on one publicly available simulated dataset and one real rodent neural dataset with different noise levels. We further demonstrated the performance of the LDA-DP method on a real neural dataset from non-human primates with more complex distribution characteristics.

Main resultsThe results show that our LDA-DP algorithm extracts a more discriminative feature subspace and achieves better cluster quality than previously established methods in both simulated and real data. Especially in the neural recordings with high noise levels or waveform similarity, the LDA-DP still yields a robust performance with automatic detection of the number of clusters.

SignificanceThe proposed LDA-DP algorithm achieved high sorting accuracy and robustness to noise, which offers a promising tool for spike sorting and facilitates the following analysis of neural population activity.


title: A Robust Spike Sorting Method based on the Joint Optimization of Linear Discrimination Analysis and Density Peaks


title: Unique neural coding of crucial versus irrelevant plant odors in a hawkmoth

abstract: The sense of smell is pivotal for nocturnal moths to locate feeding and oviposition sites. However, these crucial resources are often rare and their bouquets are intermingled with volatiles emanating from surrounding  background plants. Here we asked if the olfactory system of female hawkmoths, Manduca sexta, could differentiate between crucial and background cues. To answer this question, we collected nocturnal headspaces of numerous plants in a natural habitat of M. sexta. We analyzed the chemical composition of these headspaces, and used them as stimuli in physiological experiments at the antenna and in the brain. The intense odors of floral nectar sources evoked strong responses in virgin and mated female moths, most likely enabling the localization of profitable flowers at a distance. Bouquets of larval host plants and most background plants, in contrast, were subtle, thus potentially complicating host identification. However, despite being subtle, antennal responses and brain activation patterns evoked by the smell of larval host plants were clearly different from those evoked by other plants. Interestingly, this difference was even more pronounced in the antennal lobe of mated females, revealing a status-dependent tuning of their olfactory system towards oviposition sites. Our study suggests that female moths possess unique neural coding strategies to find not only conspicuous floral cues but also inconspicuous bouquets of larval host plants within a complex olfactory landscape.


title: Integrating region- and network-level contributions to episodic recollection using multilevel structural equation modeling

abstract: The brain is composed of networks of interacting brain regions that support higher order cognition. For instance, a posterior medial (PM) network appears to support recollection and other forms of episodic construction. Past research has focused largely on the roles of individual brain regions in recollection or on their mutual engagement as part of an integrated network. Yet the relationship between these region- and network-level contributions remains poorly understood. Here we applied multilevel structural equation modeling (SEM) to examine the functional organization of the PM network and its relationship to episodic memory outcomes. We evaluated two aspects of functional heterogeneity in the PM network: first, the organization of individual regions into subnetworks, and second, the presence of regionally-specific contributions while accounting for network-level effects. The results suggest that the PM network is composed of ventral and dorsal subnetworks, with the ventral subnetwork making a unique contribution to recollection, and that memory-related activity in individual regions is well accounted for by these network-level effects. These findings highlight the importance of considering both region and network levels of analysis when studying brain-behavior relationships.


title: Shoulder muscle recruitment during the delay period encodes a reach movement plan which is sensitive to task context

abstract:
Understanding how the brain transforms motor plans into the appropriate pattern of muscle activity is a central question in motor control. Whereas some neurophysiological studies in non-human primates have shown the presence of early recruitment of motor units, other studies have reported recruitment of motor units to occur just prior to movement initiation. To address this issue in humans, and test whether early recruitment is task dependent, we studied anterior deltoid muscle activity using high density surface EMG during visually guided delayed and immediate movement tasks while subjects used either hand or isometric cursor movements. Whereas spatially specific delay period activity was present for hand movements, it was absent for isometric movements. Nonetheless, muscle activity was correlated with the time it took to initiate movements in both the task conditions. Further, we used an accumulator framework to assess initiation of movements in different task contexts. Interestingly, we found consistent but different parameters of the accumulator model to play an important role in initiating hand and cursor movements: whereas the growth rate of muscle activity was an important determinant of reaction times in delayed and immediate hand movement tasks, the onset of muscle activity recruitment played a consistent and important role in determining the reaction time for initiating cursor movements during the delayed and immediate tasks. Overall, our results reveal an unexpected heterogeneity of early recruitment as seen with high density surface EMG that may relate to the differential extent of planning engendered during actual hand movements versus isometric movements.

Significance statementWe studied the spatial and temporal aspects of response preparation in anterior deltoid muscles using high density surface EMG. Our results showed that a readiness signal could be accessed from muscle activity but were absent during isometric cursor movements. Using an accumulator framework, we show that growth rate and onset of motor unit recruitment systematically explained variability in time to movement onset based on task context.


title: Motor cortical influence relies on task-specific activity covariation

abstract:
During limb movement, spinal circuits facilitate the alternating activation of antagonistic flexor and extensor muscles. Yet antagonist cocontraction is often required to stabilize joints, like when loads are handled. Previous results suggest that these different muscle activation patterns are mediated by separate flexion- and extension-related motor cortical output populations, while others suggest recruitment of task-specific populations. To distinguish between hypotheses, we developed a paradigm in which mice toggle between forelimb tasks requiring antagonist alternation or cocontraction and measured activity in motor cortical layer 5b. Our results conformed to neither hypothesis: consistent flexion- and extension-related activity was not observed across tasks, and no task-specific populations were observed. Instead, activity covariation among motor cortical neurons dramatically changed between tasks, thereby altering the relation between neural and muscle activity. This was also observed specifically for corticospinal neurons. Collectively, our findings indicate that motor cortex drives different muscle activation patterns via task-specific activity covariation.

HIGHLIGHTSMice perform two forelimb tasks involving distinct antagonist muscle activity in a novel paradigm

L5b motor cortical neurons are not organized by task-specific activity

L5b motor cortical neurons do not encode muscle activity consistently across tasks

Task-specific muscle activity is driven by a change in motor cortical activity covariation

eTOC BLURBWarriner et al. simultaneously measured muscle and motor cortical activity in mouse during antagonist forelimb muscle alternation and cocontraction to reveal that these distinct muscle activation patterns are not driven through consistent flexion and extension programs nor through the activity of discrete, task-specific neuronal subsets. Instead, distinct patterns involve task-specific changes in firing pattern covariation among layer 5b neurons, and corticospinal neurons in particular, which change their relationship to muscle activity across tasks.


title: Interaural time difference sensitivity under binaural cochlear implant stimulation even at high pulse rates up to 900 pps

abstract: Spatial hearing remains one of the major challenges for bilateral cochlear implant (biCI) users, and early deaf patients in particular are often completely insensitive to interaural time differences (ITDs) delivered through biCIs. One popular theory held is that this may be due to a lack of early binaural experience. However, previously we have shown that neonatally deafened rats fitted with biCIs in adulthood quickly learn to discriminate ITDs as well as their normal hearing litter mates (Rosskothen-Kuhl et al. 2021), and perform orders of magnitude better than human biCI users. Our unique behaving biCI rat model allows us to investigate other possible limiting factors of prosthetic binaural hearing, such as the effect of stimulus pulse rate and envelope shape. Previous work has indicated that ITD sensitivity may decline substantially at the high pulse rates often used in clinical practice. We therefore measured behavioral ITD thresholds in neonatally deafened, adult implanted biCI rats to pulse trains of 50, 300, 900 and 1800 pulses per second (pps), with either rectangular or Hanning window envelopes. Our rats exhibited very high sensitivity to ITDs at pulse rates up to 900 pps for both envelope shapes, a pulse rate commonly used in clinical practice. However, ITD sensitivity declined to near zero at 1800 pps, for both Hanning and rectangular windowed pulse trains. Current clinical cochlear implant (CI) processors are often set to pulse rates [≥]900 pps, but ITD sensitivity in human CI listeners is often reported to decline sharply above [~]300 pps. Our results suggest that the relatively poor ITD sensitivity seen at >300 pps in human CI users may not reflect the hard upper limit of biCI ITD performance in the mammalian auditory pathway. Perhaps with training or better CI strategies good binaural hearing may be achievable at pulse rates high enough to allow good sampling of speech envelopes while delivering usable ITDs.


title: Network States Classification based on Local Field Potential Recordings in the Awake Mouse Neocortex

abstract:
Recent studies using intracellular recordings in awake behaving mice revealed that cortical network states, defined based on membrane potential features, modulate sensory responses and perceptual outcomes. Single cell intracellular recordings are difficult to achieve and have low yield compared to extracellular recordings of population signals, such as local field potentials (LFPs). However, it is currently unclear how to identify these behaviorally-relevant network states from the LFP. We used simultaneous LFP and intracellular recordings in the somatosensory cortex of awake mice to design and calibrate a model-based analysis method, the Network State Index (NSI), that enables network state classification from the LFP. We used the NSI to analyze the relationship between single-cell (intracellular) and population (LFP) signals over different network states of wakefulness. We found that graded levels of population signal faithfully predicted the levels of single cell depolarization in non-rhythmic regimes whereas, in delta ([2-4 Hz]) oscillatory regimes, the graded levels of rhythmicity in the LFP mapped into a stereotypical oscillatory pattern of membrane potential. Finally, we showed that the variability of network states, beyond the occurrence of slow oscillatory activity, critically shaped the average correlations between single cell and population signals. NSI-based characterization provides a ready-to-use tool to understand from LFP recordings how the modulation of local network dynamics shapes the flexibility of sensory processing during behavior.

Significance statementSensation during behaviour is strongly modulated by the animals internal state. Such context-dependent modulation of sensory processing is believed to largely stem from top-down control of network states in sensory cortices, with different network states being associated with distinct computational properties of the circuit. So far, a detailed characterization of network states in the awake cortex has mostly been achieved through single-cell intracellular recordings, which however cannot be easily recorded. Here, we developed a new method to classify network states from the easily accessible extracellular LFP recordings of population activity. Given the widespread use of LFPs, our work provides a critical methodology to greatly expand our understanding of the mechanisms underlying state-dependent computations in neocortex.


title: Neurobeachin controls the asymmetric subcellular distribution of electrical synapse proteins

abstract: Brain function relies upon the carefully coordinated development of specialized synaptic connections between neurons. The subcellular positioning of synapses and their molecular composition regulate neural computation, forming the fundamental basis of neural circuits. Like chemical synapses, electrical synapses are constructed from an assortment of cell adhesion, scaffolding, and regulatory molecules, as well as channel-forming Connexin proteins, yet little is known about how these molecules localize at specified subcellular compartments of the neuron. Here we investigated the cell biological relationship between the autism- and epilepsy-associated gene Neurobeachin, the gap junction forming neuronal Connexins, and electrical synapse scaffold Zonula Occludens 1 (ZO1). Using the model electrical synapses of the zebrafish Mauthner circuit we found that Neurobeachin localizes to the electrical synapse independent of the ZO1 scaffold and Connexins. By contrast, we show that Neurobeachin functions postsynaptically where it is required for the robust localization of ZO1 and Connexins. We demonstrate that Neurobeachin binds ZO1 but not Connexin proteins. Finally, we find that Neurobeachin is required to restrict postsynaptic electrical synapse proteins to dendritic synapses. Altogether the findings reveal a mechanism for the asymmetric synaptic localization of electrical synapse components providing a basis for the subcellular specialization of neuronal gap junctions.


title: A parvocellular-magnocellular functional gradient in human visual cortex

abstract: The magnocellular and parvocellular systems are major visual recognition pathways, with distinct histological and physiological properties. Despite their critical role, there is limited evidence on the specific contributions these visual pathways make to visual recognition in general and to word reading in particular. Using a multimodal functional MRI approach and individual-subject analyses, here we investigate the involvement of visual cortex regions in the recognition of magnocellular- and parvocellular-biased words and images. Our results reveal a functional gradient in the activation profile of both left and right visual cortex: posterior regions are more strongly recruited for processing parvocellular-biased, while anterior regions are more involved in processing magnocellular-biased stimuli. Furthermore, functional connectivity analyses show clustering in the strength of functional coupling among visual cortex regions as a function of the distance between regions, with greater coupling within and less coupling across posterior and anterior regions. Finally, we found minimal differences in lateralization for word and image recognition in these visual cortical regions. These results were replicated in a retest session with a subset of participants. Our findings underscore a functional division of labor in the visual cortex as a function determined by a parvocellular or magnocellular bias in properties of the stimuli and further reveal that in this context the visual cortex is not particularly biased towards words or images.


title: Single-cell immune repertoire sequencing of B and T cells in murine models of infection and autoimmunity

abstract: Adaptive immune repertoires are composed by the ensemble of B and T cell receptors (BCR, TCR) within an individual and reflect both past and current immune responses. Recent advances in single-cell sequencing enable recovery of the complete adaptive immune receptor sequences in addition to transcriptional information. Such high-dimensional datasets enable the molecular quantification of clonal selection of B and T cells across a wide variety of conditions such as infection and disease. Due to costs, time required for the analysis and current practices of academic publishing, small-scale sequencing studies are often not made publicly available, despite having informative potential to elucidate immunological principles and guide future-studies. Here, we performed single-cell sequencing of B and T cells to profile clonal selection across murine models of viral infection and autoimmune disease. Specifically, we recovered transcriptome and immune repertoire information for polyclonal T follicular helper cells following acute and chronic viral infection, CD8+ T cells with binding specificity restricted to two distinct peptides of lymphocytic choriomeningitis virus, and B and T cells isolated from the nervous system in the context of experimental autoimmune encephalomyelitis. We could relate repertoire features such as clonal expansion, germline gene usage, and clonal convergence to cell phenotypes spanning activation, memory, naive, antibody secretion, T cell inflation, and regulation. Together, this dataset provides a resource for experimental and computational immunologists that can be integrated with future single-cell immune repertoire and transcriptome sequencing datasets.

category: immunology


title: Pancreas resident macrophage-induced fibrosis has divergent roles in pancreas inflammatory injury and PDAC

abstract: Tissue-resident macrophages (TRMs) are long-lived cells that maintain locally and can be phenotypically distinct from monocyte-derived macrophages (MDMs). However, whether TRMs and MDMs have functional distinction under differing pathologies is not understood. Here, we show a significant portion of macrophages that accumulated during pancreatitis and pancreatic cancer were expanded from TRMs. We further established that pancreas TRMs have a distinct extracellular matrix remodeling phenotype that was critical for maintaining tissue homeostasis during inflammation. Loss of TRMs led to exacerbation of severe pancreatitis and animal death, due to impaired acinar cell survival and recovery. In pancreatitis, TRMs elicited protective effects by triggering the accumulation and activation of fibroblasts, which was necessary for initiating fibrosis as a wound healing response. The same TRM-driven fibrosis, however, drove pancreas cancer pathogenesis and progression. Together, these findings indicate that TRMs play divergent roles in the pathogenesis of pancreatitis and cancer through regulation of stromagenesis.


title: Functional, transcriptional, and microbial shifts associated with healthy pulmonary aging: insights from rhesus macaques

abstract: Older individuals are at increased risk of developing severe respiratory infections due to age-related changes in the immunological, microbial, and functional landscape of the lung. However, our understanding of the impact of age on the respiratory tract remains limited as samples from healthy humans are challenging to obtain and confounding variables such as smoking and environmental pollutant exposure make it difficult to assess the true impact of aging. On the other hand, studies in rodent models are biased by their specific pathogen free status. In this study, we utilize a rhesus macaque model of healthy aging to examine the functional, immunological, and microbial consequences of aging in the lung. Pulmonary function testing in this large (n=34 adult, n=49 aged) cross-sectional study established age and sex differences similar to humans supporting the translational accuracy of this model. Additionally, an increased abundance of myeloid cells (alveolar and infiltrating macrophages) and a concomitant decrease in T-cells were also observed in aged animals. Single cell RNA sequencing indicated a transcriptional shift in the pulmonary CD8+ T-cell population from GRZMB expressing cells to IFN expressing cells, while frequency of IL-1B expressing alveolar macrophages was significantly reduced. Interestingly, the lung microbiome of many animals was dominated by a single microbe, Tropheryma spp., the prevalence of which decreased with age. These data provide a comprehensive picture of the functional, microbial and immunological changes of the lung in healthy macaque aging and provide insight into the increased prevalence and severity of respiratory disease in the elderly.


title: Keap1 moderates the transcription of virus induced genes through G9a-GLP and NFκB p50 recruitment, and H3K9me2 deposition

abstract:
Cells must moderate transcription that is induced by virus infection to mitigate deleterious consequences of inflammation. We investigated the mechanisms whereby Keap1 moderates the transcription of genes that are induced by Sendai virus infection in mouse embryo fibroblasts (MEFs). Virus infection induced Keap1 to bind Ifnb1, Tnf and Il6, and reduced Keap1 binding at Cdkn1a and Ccng1. Keap1 was required for G9a and GLP to bind and to deposit H3K9me2 at these genes upon virus infection. Keap1 moderated the transcription of genes that were induced by virus infection in concert with G9a, GLP, and NF[kcy]B p50 recruitment. G9a-GLP lysine methyltransferase activity was required for Keap1 to moderate the transcription of virus induced genes. G9a-GLP inhibitors enhanced the transcription of virus induced genes, and they augmented Keap1 and NF[kcy]B p50 recruitment, in parallel with the inhibition of H3K9me2 deposition. The interdependent effects of Keap1 and G9a-GLP on transcription and on the recruitment of each other constitute a feedback circuit that moderates the transcription of virus induced genes. G9a-GLP inhibitors augmented Keap1 binding to different genes in virus infected and in uninfected MEFs, whereas they inhibited H3K9me2 deposition that was induced by virus infection selectively. G9a-GLP inhibitors stabilized Keap1 retention in permeabilized MEFs and augmented Keap1 binding to specific genes in parallel. Keap1 was required for NF[kcy]B p50 recruitment, and for the augmentation of NF[kcy]B binding by G9a-GLP inhibitors. Keap1 and the electrophile tBHQ attenuated virus induced gene transcription through independent mechanisms, and they regulated the recruitment of different NF[kcy]B subunits.

ImportanceExcess and maladaptive immune responses to virus infections are a major contributing factor to the morbidity and mortality of COVID-19 and other diseases. Conversely, inadequate immune responses to vaccines and pathogens by individuals with suppressed immune function expose them to infections. Currently available drugs that enable therapeutic management of immune responses have low specificity and can blunt beneficial immune functions. The molecular mechanisms that moderate the transcription of genes that are induced by virus infection are incompletely understood. Characterization of the mechanisms whereby Keap1, G9a-GLP and NF[kcy]B p50 moderate virus induced gene transcription in mouse embryo fibroblasts represents the first step toward the identification of new targets for therapeutic agents that can modulate immune responsiveness.


title: A conundrum of r-protein stability: unbalanced stoichiometry of r-proteins during stationary phase in Escherichia coli

abstract:
Bacterial ribosomes are composed of three RNA (rRNA) and over 50 ribosomal protein (r-protein) molecules. R-proteins are essential for ribosome assembly, structural stability, and also participate in almost all ribosome functions. Ribosomal components are present in stoichiometric amounts in the mature 70S ribosomes during the exponential and early stationary growth phases. Ribosomes are degraded in stationary phase, however, the stability and fate of ribosomal proteins during stationary growth phase are not known. In this study, we report a quantitative analysis of ribosomal components during the extended stationary phase in Escherichia coli. We show that: (a) the quantity of ribosomes per cell decreases in the stationary phase. (b) 70S ribosomes contain r-proteins in stoichiometric amounts. (c) 30S subunits are degraded faster than 50S. (d) In total proteome, the quantity of 20 r-proteins decreases during 14 days (short-lived r-proteins) concomitantly with the reduction of cellular RNA. (e) 30 r-proteins are stable and form a pool of free r-proteins (stable r-proteins). Thus r-proteins are present in non-stochiometric amounts in the proteome of E. coli during the extended stationary phase.

IMPORTANCERibosome degradation has been extensively described from the viewpoint of its main component - rRNA. Here we aim to complement our knowledge by quantitatively analyzing r-protein degradation and stability in the ribosomes as well as in the whole-cell proteome during stationary phase in E. coli. R-proteins are considered to be very stable in the proteome. Here we show that a specific set of r-proteins are rapidly degraded after release from the rRNA. The degradation of r-proteins is an intriguing new aspect of r-protein metabolism in bacteria.

category: molecular biology


title: Dissecting aggregation and seeding dynamics of α-Syn polymorphs using the phasor approach to FLIM

abstract: Synucleinopathies are a heterogenous group of neurodegenerative diseases characterized by the progressive accumulation of pathological -synuclein (-Syn). The importance of structural polymorphism of -Syn assemblies for distinct synucleinopathies and their progression is increasingly recognized. However, the underlying mechanisms are poorly understood. Here we use fluorescence lifetime imaging microscopy (FLIM) to investigate seeded aggregation of -Syn in a biosensor cell line. We show that conformationally distinct -Syn polymorphs exhibit characteristic fluorescence lifetimes. FLIM further revealed that -Syn polymorphs were differentially processed by cellular clearance pathways, yielding fibrillar species with increased seeding capacity. Thus, FLIM is not only a powerful tool to distinguish different amyloid structures, but also to monitor the dynamic process of amyloid remodeling by the cellular environment. Our data suggest that the accumulation of highly seeding competent degradation products for particular polymorphs may account for accelerated disease progression in some patients.


title: Ethylene Insensitive 3-Like 2 (EIL2) is a Brassicaceae-specific transcriptional regulator involved in fine-tuning ethylene-dependent hypocotyl elongation, lateral root formation and flowering time in Arabidopsis thaliana

abstract:
Ethylene signaling directs a pleiotropy of developmental processes in plants. In Arabidopsis, ethylene signaling converges at the master transcription factor Ethylene Insensitive 3 (EIN3), which has five homologs, EIN3-like 1-5 (EIL1-5). EIL1 is most characterized and operates similar as EIN3, while EIL3-5 are not involved in ethylene signaling. EIL2 remains less investigated. Our phylogenetic analysis revealed that EIL2 homologs are only retrieved in the Brassicaceae family, suggesting EIL2 diverged to have specific functions in the mustard family. By characterizing eil2 mutants, we found that EIL2 is involved in regulating ethylene-specific developmental processes in Arabidopsis thaliana, albeit in a more subtle way compared to EIN3/EIL1. EIL2 steers ethylene-triggered hypocotyl elongation in light-grown seedlings and it is involved in lateral root formation. Furthermore, EIL2 takes part in regulating flowering time as eil2 mutants flower on average one day earlier and have fewer leaves. A pEIL2:EIL2:GFP translational reporter line revealed that EIL2 protein abundance is restricted to the stele of young developing roots. EIL2 expression, and not EIL2 protein stability, is regulated by ethylene in an EIN3/EIL1-dependent way. Despite EIL2 taking part in several developmental processes, the precise upstream and downstream regulation of this ethylene- and Brassicaceae-specific transcription factor remains to be elucidated.

HighlightEIL2 is an ethylene-dependent transcription factor that evolved exclusively in the mustard family and operates downstream from EIN3/EIL1. EIL2 is involved in fine-tuning hypocotyl elongation, lateral root formation and flowering time in Arabidopsis thaliana.


title: TORC1, BORC, and ARL-8 cycling drive vesiculation of cell corpse phagolysosomes

abstract: The dynamics of phagolysosomes leading to cargo clearance are important to provide cells with metabolites and avoid auto-immune responses, but little is known about how phagolysosomes finally resolve cell corpses and cell debris. We previously discovered that polar body phagolysosomes tubulate into small vesicles to facilitate corpse clearance within 1.5 hours in C. elegans. Here, we show that vesiculation depends on activating TORC1 through amino acid release by the solute transporter SLC-36.1. Downstream of TORC1, BLOC-1-related complex (BORC) recruits the Arf-like GTPase ARL-8 to the phagolysosome for tubulation. Disrupting the regulated GTP-GDP cycle of ARL-8 reduces tubulation, delays corpse clearance, and mislocalizes ARL-8 away from lysosomes. We also demonstrate that mammalian phagocytes use TORC1, BORC, and ARL8B to promote phagolysosomal degradation, confirming the conserved importance of this pathway. Thus, by observing single phagolysosomes over time, we identified the molecular pathway regulating phagolysosome vesiculation that promotes efficient resolution.

category: cell biology


title: The Requirement of Ubiquitin C-Terminal Hydrolase L1 (UCHL1) in Mouse Ovarian Development and Fertility

abstract:
Ubiquitin C-Terminal Hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme enriched in neuronal and gonadal tissues known to regulate the cellular stores of mono-ubiquitin and protein turnover. While its function in maintaining proper motor neuron function is well-established, investigation into its role in the health and function of reproductive processes is only just beginning to be studied. Single-cell-sequencing analysis of all ovarian cells from the murine perinatal period revealed that Uchl1 is very highly expressed in the developing oocyte population, an observation which was corroborated by high levels of oocyte-enriched UCHL1 protein expression in oocytes of all stages throughout the mouse reproductive lifespan. To better understand the role UCHL1 may be playing in oocytes, we utilized a UCHL1-deficient mouse line, finding reduced number of litters, reduced litter sizes, altered folliculogenesis, morphologically abnormal oocytes, disrupted estrous cyclicity and apparent endocrine dysfunction in these animals compared to their wild-type and heterozygous littermates. These data reveal a novel role of UCHL1 in female fertility as well as overall ovarian function, and suggest a potentially essential role for the ubiquitin proteasome pathway in mediating reproductive health.

Summary sentenceUbiquitin C-Terminal Hydrolase L1 (UCHL1) is required for proper ovarian folliculogenesis, estrous cyclicity, and fertility in the female mouse.

category: developmental biology


title: Comparing dormancy in two distantly related tunicates reveals morphological, molecular, and ecological convergences and repeated co-option

abstract: Many asexually-propagating marine invertebrates can survive extreme environmental conditions by developing dormant structures, i.e., morphologically simplified bodies that retain the capacity to completely regenerate a functional adult when conditions return to normal. Here, we examine the environmental, morphological, and molecular characteristics of dormancy in two distantly related clonal tunicate species: Polyandrocarpa zorritensis and Clavelina lepadiformis. In both species, we report that the dormant structures are able to withstand harsher temperature and salinity conditions compared to the adult, and are the dominant forms these species employ to survive the colder winter months. By finely controlling the entry and exit of dormancy in laboratory-reared individuals, we were able to select and characterize the morphology of dormant structures associated with their transcriptome dynamics. In both species, we identified putative stem and nutritive cells in structures that resemble the earliest stages of asexual propagation. By characterizing gene expression during dormancy and regeneration into the adult body plan (i.e., germination), we observed that genes which control dormancy and environmental sensing in other metazoans, notably HIF- and insulin signaling genes, are also expressed in tunicate dormancy. Germination-related genes in these two species, such as the retinoic acid pathway, are also found in other unrelated clonal tunicates during asexual development. These results are suggestive of repeated exaptation of conserved eco-physiological and regeneration programs for the origin of novel dormancy-germination processes across distantly related animal taxa.


title: Early tetrapod diversification under neutral theory

abstract: Estimates of deep-time biodiversity typically rely on statistical methods to mitigate the impacts of sampling biases in the fossil record. However, these methods are limited by the spatial and temporal scale of the underlying data. Here we use a spatially explicit mechanistic model, based on neutral theory, to test hypotheses of early tetrapod diversity change during the late Carboniferous and early Permian, critical intervals for the diversification of vertebrate life on land. Our neutral simulations suggest, in contrast to previous studies, that increases in early tetrapod diversity were not driven by local endemism following the  Carboniferous Rainforest Collapse. We show that apparent changes in face-value diversity can instead be explained by variation in sampling intensity through time. Our results further demonstrate the importance of accounting for sampling biases in analyses of the fossil record and demonstrate the vast potential of mechanistic models, including neutral models, for testing hypotheses in palaeobiology.

category: evolutionary biology


title: Combinatorial effects on gene expression at the Lbx1/Fgf8 locus resolve Split-Hand/Foot Malformation type 3

abstract: Split-Hand/Foot Malformation type 3 (SHFM3) is a congenital limb malformation associated with tandem duplications at the LBX1/FGF8 locus. Yet, the disease patho-mechanism remains unsolved. Here we investigated the functional consequences of SHFM3-associated rearrangements on chromatin conformation and gene expression in vivo in transgenic mice. We show that the Lbx1/Fgf8 locus consists of two separate, but interacting, regulatory domains. Re-engineering of a SHFM3-associated duplication and a newly reported inversion in mice resulted in restructuring of the chromatin architecture. This led to an ectopic activation of the Lbx1 and Btrc genes in the apical ectodermal ridge (AER) in an Fgf8-like pattern. Artificial repositioning of the AER-specific enhancers of Fgf8 was sufficient to induce misexpression of Lbx1 and Btrc. We provide evidence that the SHFM3 phenotype is the result of a combinatorial effect on gene misexpression and dosage in the developing limb. Our results reveal new insights into the molecular mechanism underlying SHFM3 and provide novel conceptual framework for how genomic rearrangements can cause gene misexpression and disease.

category: genetics


title: Genome-wide analyses of PAM-relaxed Cas9 genome editors reveal substantial off-target effects by ABE8e in rice

abstract: PAM-relaxed Cas9 nucleases, cytosine base editors and adenine base editors are promising tools for precise genome editing in plants. However, their genome-wide off-target effects are largely undetermined. Here, we conduct whole-genome sequencing (WGS) analyses of transgenic plants edited by xCas9, Cas9-NGv1, Cas9-NG, SpRY, nCas9-NG-PmCDA1, nSpRY-PmCDA1 and nSpRY-ABE8e in rice. Our results reveal different guide RNA (gRNA)-dependent off-target effects with different editors. De novo generated new gRNAs by SpRY editors lead to additional but not substantial off-target mutations. Strikingly, ABE8e results in ~500 genome-wide A-to-G off-target mutations at TA motif sites per transgenic plant. The preference of the TA motif by ABE8e is also observed at the target sites. Finally, we investigate the timeline and mechanism of somaclonal variation due to tissue culture, which chiefly contributes to the background mutations. This study provides a comprehensive understanding on the scales and mechanisms of off-target and background mutations during PAM-relaxed genome editing in plants.


title: Genomic stock structure of the marine teleost tarakihi (Nemadactylus macropterus) provides evidence of fine-scale adaptation and a temperature-associated cline amid panmixia

abstract: Tarakihi (Nemadactylus macropterus) is an important fishery species with widespread distribution around New Zealand and off the southern coasts of Australia. However, little is known about whether the populations are locally adapted or genetically structured. To address this, we conducted whole-genome resequencing of 175 tarakihi from around New Zealand and Tasmania (Australia) to obtain a dataset of 7.5 million genome-wide and high-quality single nucleotide polymorphisms (SNPs). Variant filtering, FST-outlier analysis, and redundancy analysis (RDA) were used to evaluate population structure, adaptive structure, and locus-environment associations. A weak but significant level of neutral genetic differentiation was found between tarakihi from New Zealand and Tasmania (FST = 0.0054-0.0073, P [≤] 0.05), supporting the existence of at least two separate reproductive stocks. No clustering was detected among the New Zealand populations ([FE]ST < 0.001, P = 0.77). Outlier-based, presumably adaptive variation suggests fine-scale adaptive structure between locations around central New Zealand off the east (Wairarapa, Cape Campbell, and Hawkes Bay) and the west coast (Tasman Bay/Golden Bay and Upper West Coast of South Island). Allele frequencies from 55 loci were associated with at least one of six environmental variables, of which 47 correlated strongly with yearly mean water temperature. Although genes associated with these loci are linked to various functions, the most common functions were integral components of membrane and cilium assembly. Projection of the RDA indicates the existence of a latitudinal temperature cline. Our work provides the first genomic insights supporting panmixia of tarakihi in New Zealand and evidence of a genomic cline that appears to be driven by the temperature gradients, together providing crucial information to inform the stock assessment of this species, and to widen the insights of the ecological drivers of adaptive variation in a marine species.

category: genomics


title: Chromosome-scale genome assembly of the diploid oat Avena longiglumis reveals the landscape of repetitive sequences, genes and chromosome evolution in grasses

abstract:
BackgroundOat (Avena sativa, 2n=6x=42) is an important crop, and with its wild relatives including A. longiglumis (ALO, 2n=6x=14), has advantageous agronomic and nutritional traits. A de-novo chromosome-level ALO genome assembly was made to investigate diversity and structural genome variation between Avena species and other Poaceae in an evolutionary context, and develop genomic resources to identify the pangenome and economic traits within Pooideae.

ResultsThe 3.85 gigabase ALO genome (seven pseudo-chromosomes), contained 40,845 protein-coding genes and 87% repetitive sequences (84.21% transposable elements). An LTR retrotransposon family was abundant at all chromosome centromeres, and genes were distributed without major terminal clusters. Comparisons of synteny with A. eriantha and A. strigosa showed evolutionary translocations of terminal segments including many genes. Comparison with rice (x=12) and the ancestral grass karyotype showed synteny and features of chromosome evolution including fusions, translocations and insertions of syntenic blocks across Pooideae species. With a genome size 10 times larger than rice, ALO showed relatively uniform expansion along the chromosome arms, with few gene-poor regions along arms, and no major duplications nor deletions. Linked gene networks were identified (mixed-linkage glucans and cellulose synthase genes), and CYP450 genes may be related to salt-tolerance.

ConclusionsThe high-continuity genome assembly shows gene, chromosomal structural and copy number variation, providing a reference for the Avena pangenome, defining the full spectrum of diversity. Chromosomal rearrangements and genome expansion demonstrate features of evolution across the genus and grass BOP-clade, contributing to exploitation of gene and genome diversity through precision breeding.