title: Genetic structure in patchy populations of a candidate foundation plant: a case study of Leymus chinensis (Poaceae) using genetic and clonal diversity

abstract:
PREMISEThe distribution of genetic diversity on the landscape has critical ecological and evolutionary implications. This may be especially the case on a local scale for foundation plant species since they create and define ecological communities, contributing disproportionately to ecosystem function.

METHODSWe examined the distribution of genetic diversity and clones, which we defined first as unique multi-locus genotypes (MLG), and then by grouping similar MLGs into multi-locus lineages (MLL). We used 186 markers from inter-simple sequence repeats (ISSR) across 358 ramets from 13 patches of the foundation grass Leymus chinensis. We examined the relationship between genetic and clonal diversities, their variation with patch-size, and the effect of the number of markers used to evaluate genetic diversity and structure in this species.

RESULTSEvery ramet had a unique MLG. Almost all patches consisted of individuals belonging to a single MLL. We confirmed this with a clustering algorithm to group related genotypes. The predominance of a single lineage within each patch could be the result of the accumulation of somatic mutations, limited dispersal, some sexual reproduction with partners mainly restricted to the same patch, or a combination of all three.

CONCLUSIONSWe found strong genetic structure among patches of L. chinensis. Consistent with previous work on the species, the clustering of similar genotypes within patches suggests that clonal reproduction combined with somatic mutation, limited dispersal, and some degree of sexual reproduction among neighbors causes individuals within a patch to be more closely related than among patches.


title: Increased apoptotic priming of glioblastoma enables therapeutic targeting by BH3-mimetics

abstract: IDH wild-type glioblastoma (GBM) is the most prevalent malignant primary brain tumour in adults. GBM typically has a poor prognosis, mainly due to a lack of effective treatment options leading to tumour persistence or recurrence. Tackling this, we investigated the therapeutic potential of targeting anti-apoptotic BCL-2 proteins in GBM. Levels of anti- apoptotic BCL-xL and MCL-1 were consistently increased in GBM compared with non- malignant cells and tissue. Moreover, we found that relative to their differentiated counterparts, patient-derived GBM stem-like cells also displayed higher expression of anti- apoptotic BCL-2 family members. Surprisingly, high anti-apoptotic BCL-xL and MCL-1 expression correlated with heightened susceptibility of GBM to BCL-2 family protein- targeting BH3-mimetics. This is indicative of increased apoptotic priming. Indeed, GBM displayed an obligate requirement for MCL-1 expression in both tumour development and maintenance. Investigating this apoptotic sensitivity, we found that sequential inhibition of BCL-xL and MCL-1 led to robust anti-tumour responses in vivo, in the absence of overt toxicity. These data demonstrate that BCL-xL and MCL-1 pro-survival function is a fundamental prerequisite for GBM survival that can be therapeutically exploited by BH3- mimetics.

category: cancer biology


title: Schizophrenia polygenic risk during typical development reflects multiscale cortical organization

abstract: Schizophrenia is widely recognized as a neurodevelopmental disorder. Abnormal cortical development may by revealed using polygenic risk scoring for schizophrenia (PRS-SCZ). We assessed PRS-SCZ and cortical morphometry in typically developing children (3-21 years) using whole genome genotyping and T1-weighted MRI (n=390) from the Pediatric Imaging, Neurocognition and Genetics (PING) cohort. We contextualise the findings using (i) age-matched transcriptomics, (ii) histologically-defined cytoarchitectural types and functionally-defined networks, (iii) case-control differences of schizophrenia and other major psychiatric disorders. Higher PRS-SCZ was associated with greater cortical thickness, which was most prominent in areas with heightened gene expression of dendrites and synapses. PRS-SCZ related increases in vertex-wise cortical thickness were especially focused in the ventral attention network, while koniocortical type cortex (i.e. primary sensory areas) was relatively conserved from PRS-SCZ related differences. The large-scale pattern of cortical thickness increases related to PRS-SCZ mirrored the pattern of cortical thinning in schizophrenia and mood-related psychiatric disorders. Age group models illustrate a possible trajectory from PRS-SCZ associated cortical thickness increases in early childhood towards thinning in late adolescence, which resembles the adult brain phenotype of schizophrenia. Collectively, combining imaging-genetics with multi-scale mapping, our work provides novel insight into how genetic risk for schizophrenia impacts the cortex early in life.

category: neuroscience


title: Nanopore sequencing of RNA and cDNA molecules expands the transcriptomic toolbox in prokaryotes

abstract: High-throughput sequencing dramatically changed our view of transcriptome architectures and allowed for ground-breaking discoveries in RNA biology. Recently, sequencing of full-length transcripts based on the single-molecule sequencing platform from Oxford Nanopore Technologies (ONT) was introduced and is widely employed to sequence eukaryotic and viral RNAs. However, experimental approaches implementing this technique for prokaryotic transcriptomes remain scarce. Here, we present an experimental and bioinformatic workflow for ONT RNA-seq in the bacterial model organism Escherichia coli, which can be applied to any microorganism. Our study highlights critical steps of library preparation and computational analysis and compares the results to gold standards in the field. Furthermore, we comprehensively evaluate the applicability and advantages of different ONT-based RNA sequencing protocols, including direct RNA, direct cDNA, and PCR-cDNA. We find that cDNA-seq offers improved yield and accuracy without bias in quantification compared to direct RNA sequencing. Notably, cDNA-seq can be readily used for simultaneous transcript quantification, accurate detection of transcript 5' and 3' boundaries, analysis of transcriptional units and transcriptional heterogeneity. In summary, we establish Nanopore RNA-seq to be a ready-to-use tool allowing rapid, cost-effective, and accurate annotation of multiple transcriptomic features thereby advancing it to become a standard method for RNA analysis in prokaryotes.

category: microbiology


title: Plasmodium falciparum protein Pfs16 is a target for transmission-blocking antimalarial drug development

abstract: Phenotypic cell-based screens are critical to the discovery of new antimalarial lead compounds. However, identification and validation of cellular targets of lead compounds is required following discovery in a phenotypic screen. We recently discovered a Plasmodium transmission-blocking N-((4-hydroxychroman-4-yl)methyl)-sulfonamide (N-4HCS) compound, DDD01035881, in a phenotypic screen. DDD01035881 and its potent derivatives have been shown to block Plasmodium male gamete formation (microgametogenesis) with nanomolar activity. Here, we synthesised a photoactivatable N-4HCS derivative, probe 2, to identify the N-4HCS cellular target. Using probe 2 in photo-affinity labelling coupled with mass spectrometry, we identified the 16 kDa Plasmodium falciparum parasitophorous vacuole membrane protein Pfs16 as the likely cellular target of the N-4HCS series. Further validating Pfs16 as the cellular target of the N-4HCS series, the Cellular Thermal Shift Assay (CETSA) confirmed DDD01035881 stabilised Pfs16 in lysate from activated mature gametocytes. Additionally, photo-affinity labelling combined with in-gel fluorescence and immunoblot analysis confirmed the N-4HCS series interacted with Pfs16. High-resolution, widefield fluorescence and electron microscopy of N-4HCS-inhibited parasites was found to result in a cell morphology entirely consistent with targeted gene disruption of Pfs16. Taken together, these data strongly implicate Pfs16 as the target of DDD01035881 and establish the N-4HCS scaffold family as a powerful starting point from which future transmission-blocking antimalarials can be developed.


title: VirClust, a tool for hierarchical clustering, core gene detection and annotation of (prokaryotic) viruses

abstract: Recent years have seen major changes in the classification criteria and taxonomy of viruses. The current classification scheme, also called "megataxonomy of viruses", recognizes five different viral realms, defined based on the presence of viral hallmark genes. Within the realms, viruses are classified into hierarchical taxons, ideally defined by their shared genes. Therefore, there is currently a need for virus classification tools based on such shared genes / proteins. Here, VirClust is presented - a novel tool capable of performing i) hierarchical clustering of viruses based on intergenomic distances calculated from their protein cluster content, ii) identification of core proteins and iii) annotation of viral proteins. VirClust groups proteins into clusters both based on BLASTP sequence similarity, which identifies more related proteins, and also based on hidden markow models (HMM), which identifies more distantly related proteins. Furthermore, VirClust provides an integrated visualization of the hierarchical clustering tree and of the distribution of the protein content, which allows the identification of the genomic features responsible for the respective clustering. By using different intergenomic distances, the hierarchical trees produced by VirClust can be split into viral genome clusters of different taxonomic ranks. VirClust is freely available, as web-service (virclust.icbm.de) and stand-alone tool.


title: Effectiveness of antifungal treatments during chytridiomycosis epizootics in populations of an endangered frog

abstract: The recently-emerged amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has had an unprecedented impact on global amphibian populations, and highlights the urgent need to develop effective mitigation strategies against this pathogen. We conducted field antifungal treatment experiments in populations of the endangered mountain yellow-legged frog during or immediately after Bd-caused mass die-off events. The objective of the treatments was to reduce Bd infection intensity ("load") and in doing so alter frog-Bd dynamics and increase the probability of frog population persistence despite ongoing Bd infection. Experiments included treatment of early life stages (tadpoles and subadults) with the antifungal drug itraconazole, treatment of adults with itraconazole, and augmentation of the skin microbiome of subadults with Janthinobacterium lividum, a commensal bacterium with antifungal properties. All itraconazole treatments caused immediate reductions in Bd load, and produced longer-term effects that differed between life stages. In experiments focused on early life stages, Bd load was reduced in the two months immediately following treatment and was associated with increased survival of subadults. However, Bd load and frog survival returned to pre-treatment levels in less than one year, and treatment had no effect on population persistence. In adults, treatment reduced Bd load and increased frog survival over the three-year post-treatment period, consistent with frogs having developed an effective adaptive immune response against Bd. Despite this protracted period of reduced impacts of Bd on adults, recruitment of new individuals into the adult population was limited and the population eventually declined to near-extirpation. In the microbiome augmentation experiment, bathing frogs in a J. lividum solution after Bd load reduction with itraconazole increased concentrations of this bacterium on frogs, but concentrations declined to baseline levels within one month and did not have a protective effect against Bd infection. Collectively, these results suggest that Bd mitigation efforts focused on frog populations that have recently declined due to Bd emergence are ineffective in causing long-term changes in frog-Bd dynamics and increasing population persistence, due largely to the inability of early life stages to mount an effective immune response against Bd and resulting high susceptibility. This results in repeated recruitment failure and a low probability of population persistence in the face of ongoing Bd infection.

category: ecology


title: Monitoring fish spawning sites in freshwater ecosystems using low-cost UAV data: A case study of salmonids in lakes in Iceland

abstract: Low-cost unmanned aerial vehicles (UAV), widely known as drones, have become ubiquitous and improved considerably in their technical capabilities and data quality. This opens new opportunities for their utilisation in scientific research that can help to reduce equipment and data collection costs. Remote sensing methods in ecological fieldwork can be a suitable approach to complementing, augmenting or even replacing certain aspects of fieldwork. In this study we tested the suitability of UAV for the detection of salmonid spawning grounds in two lakes in Iceland, Thingvallavatn and Ellidavatn. Salmonids are very susceptible to environmental changes, especially during embryonic development when highly oxygenated water flow and low temperatures are required. Monitoring the changes of the redd density over time will help understand the population dynamics of salmonid species and create strategies for species conservation. As part of this pilot study, we conducted aerial surveys during the spawning seasons in both locations in 2018 recording standard photographs in the visible spectrum (red, green, blue) to fully cover the respective areas of interest. Different flight altitudes were recorded to test the effects of image resolution on the final analyses. The images were then processed by applying standard remote sensing analyses in the software ENVI. The maximum likelihood classification combined with post-classification improvement methods resulted in satisfactory accuracies that are valuable for further monitoring efforts. From these findings we established a workflow that allows the implementation of UAV in ecological fieldwork for regular long-term observations. We discuss our experiences with regards to their utility, their limitations and identify future directions of research for implementing the potential that low-cost approaches have in supporting ecological studies of freshwater ecosystems.


title: Single-nucleus transcriptomes reveal functional and evolutionary properties of cell types in the Drosophila accessory gland

abstract:
Many traits responsible for male reproduction evolve quickly, including gene expression phenotypes in germline and somatic male reproductive tissues. Rapid male evolution in polyandrous species is thought to be driven by competition among males for fertilizations and conflicts between male and female fitness interests that manifest in post-copulatory phenotypes. In Drosophila, seminal fluid proteins secreted by three major cell types of the male accessory gland and ejaculatory duct are required for female sperm storage and use, and influence female post-copulatory traits. Recent work has shown that these cell types have overlapping but distinct effects on female post-copulatory biology, yet relatively little is known about their evolutionary properties. Here we use single-nucleus RNA-Seq of the accessory gland and ejaculatory duct from Drosophila melanogaster and two closely related species to comprehensively describe the cell diversity of these tissues and their transcriptome evolution for the first time. We find that seminal fluid transcripts are strongly partitioned across the major cell types, and expression of many other genes additionally define each cell type. We also report previously undocumented diversity in main cells. Transcriptome divergence was found to be heterogeneous across cell types and lineages, revealing a complex evolutionary process. Furthermore, protein adaptation varied across cell types, with potential consequences for our understanding of selection on male post-copulatory traits.

SIGNIFICANCE STATEMENTRapid evolution of male traits may result from competition among males or antagonistic interactions between the sexes over control of reproduction. In animals with internal fertilization, interactions may occur in the female reproductive tract. Drosophila seminal fluid proteins, which are secreted by three major cell types of the male accessory gland and ejaculatory duct, are required for female sperm storage and use, and influence female behavior and physiology. These cell types have distinct effects on females, yet relatively little is known about their evolutionary properties. Here we characterize diversity and transcriptome evolution of seminal fluid-producing tissues at the cell level. These data reveal new functional properties of these cells and complex evolutionary patterns that vary across cell types and lineages.

category: evolutionary biology


title: In silico study for decoding the correlated role of MCM7 gene in progression of breast cancer and Alzheimers disorder

abstract: Breast cancer and Alzheimers disease (AD) are two of the progressive and detrimental disorders affecting large population around the globe. While the chemotherapy of breast cancer is well established and enriched, the AD still lacks it due to unrecognized peripheral biomarkers for detection and targeted therapy. This study aimed to identify common molecular signature markers in breast cancer (grade 1, 2, and 3) and AD for the diagnosis and prognosis. We used two microarray datasets (GSE42568, GSE33000) respectively for both disorders that led to identification of two common differentially expressed genes (DEGs), namely MCM7 and CD209, as common players in both these two conditions. While the pattern of expression of CD209 gene running upregulated in both disorders, the MCM7 showed unusual contrary in its pattern of expression. The expression of MCM7 is downregulated in breast cancer but upregulated in AD. Gene set and protein overrepresentation analysis, protein-protein interaction (PPI), and protein subcellular localizations analyses of this underrated MCM7 gene was performed with further prediction and validation of its structure. The findings may pave the way in designing therapeutic approaches to ameliorate AD.

category: bioinformatics


title: Pharmacologic and genetic inhibition of cholesterol esterification reduces tumour burden: a pan-cancer systematic review and meta-analysis of preclinical models

abstract: Cholesterol esterification proteins Sterol-O acyltransferases (SOAT) 1 and 2 are emerging prognostic markers in many cancers. These enzymes utilise fatty acids conjugated to coenzyme A to esterify cholesterol. Cholesterol esterification is tightly regulated and enables formation of lipid droplets that act as storage organelles for lipid soluble vitamins and minerals, and as cholesterol reservoirs. In cancer, this provides rapid access to cholesterol to maintain continual synthesis of the plasma membrane. In this systematic review and meta-analysis, we summarise the current depth of understanding of the role of this metabolic pathway in pan-cancer development. A systematic search of PubMed, Scopus, and Web of Science for preclinical studies identified eight studies where cholesteryl ester concentrations were compared between tumour and adjacent-normal tissue, and 24 studies where cholesterol esterification was blocked by pharmacological or genetic approaches. Tumour tissue had a significantly greater concentration of cholesteryl esters than non-tumour tissue (p<0.0001). Pharmacological or genetic inhibition of SOAT was associated with significantly smaller tumours of all types (p[≤]0.002). SOAT inhibition increased tumour apoptosis (p=0.007), CD8+ lymphocyte infiltration and cytotoxicity (p[≤]0.05), and reduced proliferation (p=0.0003) and metastasis (p<0.0001). Significant risk of publication bias was found and may have contributed to a 32% overestimation of the meta-analysed effect size was overestimated. Avasimibe, the most frequently used SOAT inhibitor, was effective at doses equivalent to those previously reported to be safe and tolerable in humans. This work indicates that SOAT inhibition should be explored in clinical trials as an adjunct to existing anti-neoplastic agents.


title: Transcriptome features of striated muscle aging and predictability of protein level changes

abstract: RNA and protein levels correlate only partially and some transcripts are better correlated with their protein counterparts than others. This suggests that in aging and disease studies, some transcriptomics markers may carry more information in predicting protein-level changes. Here we applied a computational data analysis workflow to predict which transcriptomic changes are more likely relevant to protein-level regulation in striated muscle aging. The protein predictability of each transcript is estimated from existing large proteogenomics data sets, then transferred to new total RNA sequencing data comparing skeletal muscle and cardiac muscle in young adult (~4 months) mice vs. early aging (~20 months) mice. Aging cardiac and skeletal muscles both invoke transcriptomic changes in innate immune system and mitochondria pathways but diverge in extracellular matrix processes. On an individual gene level, we identified 611 age-associated signatures in skeletal and cardiac muscles at 10% FDR, including a number of myokine and cardiokine encoding genes. We estimate that about 48% of the aging-associated transcripts may predict protein levels well (r [≥] 0.5). In parallel, a comparison of the identified aging-regulated genes with public human transcriptomics data showed that only 35-45% of the identified genes show an age-dependent expression in corresponding human tissues. Finally, integrating both RNA-protein correlation and human conservation across data sources, we nominate 134 prioritized aging striated muscle signature genes that are predicted to correlate strongly with protein levels and that show age-dependent expression in humans. These prioritized signatures may hold promise to understanding heart and skeletal muscle physiology in human and mouse aging.

category: physiology


title: Specific and dynamic lignification at the cell-type level controls plant physiology and adaptability

abstract: The biopolymer lignin, deposited in the cell walls of vascular cells, is essential for long-distance water conduction and structural support of plants. Independently of the species, each different vascular cell type contains a conserved lignin chemistry with specific aromatic and aliphatic substitutions. Yet, the biological role of this conserved and specific lignin chemistry for each cell type remained unclear. Herein, we investigate the role of specific lignin chemistries for cellular function by producing single cell analyses on vascular cell morphotypes, all enabling sap conduction but differing in morphology. We found that specific lignin chemistries accumulate in each morphotype. Moreover, lignin accumulates dynamically, increasing in quantity and changing composition, to alter the cell wall biomechanics of each morphotype during their maturation. For similar aromatic substitution, residues with alcohol aliphatic functions increased stiffness whereas aldehydes increased flexibility. Modifying this specific lignin chemistry impairs the cell wall biomechanics of each morphotype and consequently reduces their capacity to optimally conduct water in normal conditions, and to recover from drought. Altogether, lignin chemistry is differently controlled for each sap conducting cell types during their maturation to dynamically adjust their biomechanics and hydraulic properties to adapt to developmental and environmental constraints.

category: plant biology


title: Plant biomechanics and resilience to environmental changes are controlled by specific lignin chemistries in each vascular cell type and morphotype


title: Mathematical modeling of the effects of Wnt-10b on bone metabolism

abstract: Bone health is determined by many factors including bone metabolism or remodeling. Wnt-10b has been shown to alter osteoblastogenesis through pre-osteoblast proliferation and differentiation as well as the osteoblast apoptosis rate, which collectively lead to the increase of bone density. To model this change, we adapted a previously published model of bone remodeling. The resulting model is a single compartment system that includes ordinary differential equations for active osteoclasts, pre-osteoblasts, osteoblasts, and osteocytes and a differential equation that tracks the amount of bone present at the remodeling site. Our alterations to the original model consist of extending it past a single remodeling cycle and implementing a direct relationship to Wnt-10b. Four new parameters were estimated and validated using normalized data from mice. The model connects Wnt-10b to bone metabolism and predicts the change in trabecular bone volume caused by a change in Wnt-10b. We find that this model predicts the expected increase in pre-osteoblasts and osteoblasts while also pointing to a decrease in osteoclasts when Wnt-10b is increased. Keywords: Bone remodeling, Wnt signaling, Mathematical modeling, Bone cells

category: systems biology


title: Tangential high-density electrode insertions allow to simultaneously measure neuronal activity across an extended region of the visual field in mouse superior colliculus

abstract: The superior colliculus (SC) is a midbrain structure that plays a central role in visual processing. Although we have learned a considerable amount about the function of single SC neurons, the way in which sensory information is represented and processed on the population level in awake behaving animals and across a large region of the retinotopic map is still largely unknown. Partially because the SC is anatomically located below the cortical sheet and the transverse sinus, it is technically difficult to measure neuronal activity from a large population of neurons in SC. To address this, we propose a tangential recording configuration using high-density electrode probes (Neuropixels) in mouse SC in vivo that permits a large number of recording sites (~200) accessibility inside the SC circuitry. This approach thereby provides a unique opportunity to measure the activity of SC neuronal populations composing up to ~2 mm of SC tissue and characterized by receptive fields covering an extended region in the visual field. Here we describe how to perform tangential recordings along the anterior-posterior and the medio-lateral axis of the mouse SC in vivo and how to combine this approach with optogenetic tools for cell-type identification on the population level.


title: Coordinated multivoxel coding beyond univariate effects is not likely to be observable in fMRI data

abstract: Simultaneous recording of activity across brain regions can contain additional information compared to regional recordings done in isolation. In particular, multivariate pattern analysis (MVPA) across voxels has been interpreted as evidence for distributed coding of cognitive or sensorimotor processes beyond what can be gleaned from a collection of univariate responses (UVR) using functional magnetic resonance imaging (fMRI). Here, we argue that regardless of patterns revealed, conventional MVPA is merely a decoding tool with increased sensitivity arising from considering a large number of  weak classifiers (i.e. single voxels) in higher dimensions. We propose instead that  real multivoxel coding should result in changes in higher-order statistics across voxels between conditions such as second-order multivariate responses (sMVR). Surprisingly, analysis of conditions with robust multivariate responses (MVR) revealed by MVPA failed to show significant sMVR in two species (humans and macaques). Further analysis showed that while both MVR and sMVR can be readily observed in the spiking activity of neuronal populations, the slow and nonlinear hemodynamic coupling and low spatial resolution of fMRI activations make the observation of higher-order statistics between voxels highly unlikely. These results reveal inherent limitations of fMRI signals for studying coordinated coding across voxels. Together, these findings suggest that care should be taken in interpreting significant MVPA results as representing anything beyond a collection of univariate effects.


title: Age-related increases in reaction time result from slower preparation, not delayed initiation

abstract: Recent work indicates that healthy younger adults can prepare accurate responses faster than their voluntary reaction times indicate, leaving a seemingly unnecessary delay of 80-100ms before responding. Here we examined how the preparation of movements, initiation of movements, and the delay between them are affected by ageing. Participants made planar reaching movements in two conditions. The  Free Reaction Time condition assessed the voluntary reaction times with which participants responded to the appearance of a stimulus. The  Forced Reaction Time condition assessed the minimum time actually needed to prepare accurate movements by controlling the time allowed for movement preparation. The time taken to both initiate movements in the Free Reaction Time and to prepare movements in the Forced Response condition increased with age. Notably, the time required to prepare accurate movements was significantly shorter than participants self-selected initiation times; however, the delay between movement preparation and initiation remained consistent across the lifespan (~90ms). These results indicate that the slower reaction times of healthy older adults are not due to an increased hesitancy to respond, but can instead be attributed to changes in their ability to process stimuli and prepare movements accordingly, consistent with age-related changes in brain structure and function.


title: Brain network dynamics codify heterogeneity in seizure propagation

abstract: Dynamic functional brain connectivity facilitates adaptive cognition and behavior. Abnormal alterations within such connectivity could result in disrupted functions observed across various neurological conditions. As one of the most common neurological disorders, epilepsy is defined by the seemingly random occurrence of spontaneous seizures. A central but unresolved question concerns the mechanisms by which extraordinarily diverse dynamics of seizures emerge. Here, we apply a graph-theoretical approach to assess dynamic reconfigurations in the functional brain connectivity before, during, and after seizures that display heterogeneous propagation patterns despite sharing similar origins. We demonstrate unique reconfigurations in globally-defined network properties preceding seizure onset that predict propagation patterns of impending seizures, and in locally-defined network properties that differentiate post-onset dynamics. These results characterize quantitative network features underlying the heterogeneity of seizure dynamics and the accompanying clinical manifestations. Decoding these network properties could improve personalized preventative treatment strategies for epilepsy as well as other neurological disorders.


title: Masked features of task states found in individual brain networks

abstract:
Completing complex tasks requires flexible integration of functions across brain regions. While studies have shown that functional networks are altered across tasks, recent work highlights that brain networks exhibit substantial individual differences. Here we asked whether individual differences are important for predicting brain network interactions across cognitive states. We trained classifiers to decode state using data from single person "precision" fMRI datasets across 5 diverse cognitive states. Classifiers were then tested on either independent sessions from the same person or new individuals. Classifiers were able to decode task states in both the same and new participants above chance. However, classification performance was significantly higher within a person, a pattern consistent across model types, datasets, tasks, and feature subsets. This suggests that individualized approaches can uncover robust features of brain states, including features obscured in cross-subject analyses. Individualized approaches have potential to deepen our understanding of brain interactions during complex cognition.

Citation Diversity StatementRecently, the field of neuroscience has reported a bias in citation practices such that papers from minority groups are more often under-cited relative to the number of papers in the field1. The authors of this paper were proactive in consideration of selecting references that reflect diversity of the field in thought, contribution, and gender. Utilizing previously derived databases 1,2 we obtained the predicted gender of authors referenced in this manuscript. By this measure (and excluding self-citations to the first and last authors of our current paper), our references contain 13.87% woman(first)/woman(last), 23.3% man/woman, 23.3% woman/man, and 39.53% man/man. This method is limited in that a) names, pronouns, and social media profiles used to construct the databases may not, in every case, be indicative of gender identity and b) it cannot account for intersex, non-binary, or transgender people. Second, we obtained the predicted racial/ethnic category of the first and last author of each reference by databases that store the probability of a first and last name being carried by an author of color3. By this measure (and excluding self-citations), our references contain 10.83% author of color (first)/author of color(last), 10.64% white author/author of color, 23.55% author of color/white author, and 54.98% white author/white author. This method is limited in that a) names and Florida Voter Data to make the predictions may not be indicative of racial/ethnic identity, and b) it cannot account for Indigenous and mixed-race authors, or those who may face differential biases due to the ambiguous racialization or ethnicization of their names. We look forward to future work that could help us to better understand how to support equitable practices in science.


title: CCPE: Cell Cycle Pseudotime Estimation for Single Cell RNA-seq Data

abstract: The rapid development of single-cell RNA-sequencing (scRNA-seq) technologies makes it possible to characterize cellular heterogeneity by detecting and quantifying transcriptional changes at the single-cell level. Pseudotime analysis enables to characterize the continuous progression of various biological processes, such as cell cycle. Cell cycle plays an important regulatory role in cell fate decisions and differentiation and is also often regarded as a confounder in scRNA-seq data analysis when analyzing the role of other factors on transcriptional regulation. Therefore, accurate prediction of cell cycle pseudotime and identify cell stages are important steps for characterizing the development-related biological processes, identifying important regulatory molecules and promoting the analysis of transcriptional heterogeneity. Here, we develop CCPE, a novel cell cycle pseudotime estimation method to characterize cell cycle timing and determine cell cycle phases from single-cell RNA-seq data. CCPE uses a discriminative helix to characterize the circular process and estimates pseudotime in the cell cycle. We evaluated the model performance based on a variety of simulated and real scRNA-seq datasets. Our results indicate that CCPE is an effective method for cell cycle estimation and competitive in various downstream analyses compared with other existing methods. CCPE successfully identified cell cycle marker genes and is robust to dropout events in scRNA-seq data. CCPE also has excellent performance on small datasets with fewer genes or cells. Accurate prediction of the cell cycle in CCPE effectively contributes to cell cycle effect removal across cell types or conditions.


title: Deep learning models for identification of splice junctions across species

abstract:
Deep learning models like convolutional neural networks (CNN) and recurrent neural networks (RNN) have been frequently used to identify splice sites from genome sequences. Most of the deep learning applications identify splice sites from a single species. Furthermore, the models generally identify and interpret only the canonical splice sites. However, a model capable of identifying both canonical and non-canonical splice sites from multiple species with comparable accuracy is more generalizable and robust. We choose some state-of-the-art CNN and RNN models and compare their performances in identifying novel canonical and non-canonical splice sites in homo sapiens, mus musculus, and drosophila melanogaster.

The RNN-based model named SpliceViNCI outperforms its counterparts in identifying splice sites from multiple species as well as on unseen species. SpliceViNCI maintains its performance when trained with imbalanced data making it more robust. We observe that all the models perform better when trained with more than one species. SpliceViNCI outperforms the counterparts when trained with such an augmented dataset. We further extract and compare the features learned by SpliceViNCI when trained with single and multiple species. We validate the extracted features with knowledge from the literature.


title: Counterdiabatic control of biophysical processes

abstract: The biochemical reaction networks that regulate living systems are all stochastic to varying degrees. The resulting randomness affects biological outcomes at multiple scales, from the functional states of single proteins in a cell to the evolutionary trajectory of whole populations. Controlling how the distribution of these outcomes changes over time--via external interventions like time-varying concentrations of chemical species--is a complex challenge. In this work, we show how counterdiabatic (CD) driving, first developed to control quantum systems, provides a versatile tool for steering biological processes. We develop a practical graph-theoretic framework for CD driving in discrete-state continuous-time Markov networks. Though CD driving is limited to target trajectories that are instantaneous stationary states, we show how to generalize the approach to allow for non-stationary targets and local control--where only a subset of system states are targeted. The latter is particularly useful for biological implementations where there may be only a small number of available external control knobs, insufficient for global control. We derive simple graphical criteria for when local versus global control is possible. Finally, we illustrate the formalism with global control of a genetic regulatory switch and local control in chaperone-assisted protein folding. The derived control protocols in the chaperone system closely resemble natural control strategies seen in experimental measurements of heat shock response in yeast and E. coli.

category: biophysics


title: Shortcuts in stochastic systems and control of biophysical processes


title: Emerging Hantaviruses in Central Argentina: first case of Hantavirus Pulmonary Syndrome caused by Alto Paraguay Virus and a novel orthohantavirus in Scapteromys aquaticus rodent.

abstract:
Orthohantaviruses are emerging rodent-borne pathogens that cause Hantavirus Pulmonary Syndrome in humans. They have a wide range of rodent reservoir hosts and are transmitted to humans through aerosolized viral particles generated by the excretions of infected individuals. Since the first description of HPS in Argentina, new hantaviruses have been reported throughout the country, most of which are pathogenic to humans.

We present here the first HPS case infected with Alto Paraguay virus reported in Argentina. Until now, Alto Paraguay virus was considered a non-pathogenic orthohantavirus since it was identified in a rodent, Hollochilus chacarius. In addition to this, with the goal of identifying potential hantavirus host species in the province of Santa Fe, we finally describe a novel orthohantavirus found in the native rodent Scapteromys aquaticus, which differed from other hantaviruses described in the country so far.

Our findings implicate an epidemiological warning regarding these new orthohantaviruses circulating in Central Argentina as well as new rodent species that must be considered as hosts from now on.

category: pathology


title: Rapid Characterization of Human Serum Albumin Binding for Per- And Polyfluoroalkyl Substances Using Differential Scanning Fluorimetry

abstract: Per- and polyfluoroalkyl substances (PFAS) are a diverse class of synthetic chemicals that accumulate in the environment. Many proteins, including the primary human serum transport protein albumin (HSA), bind PFAS. The predictive power of physiologically based pharmacokinetic modeling approaches are currently limited by a lack of experimental data defining albumin binding properties for most PFAS. A novel thermal denaturation assay was optimized to evaluate changes in thermal stability of HSA in the presence of increasing concentrations of known ligands and a structurally diverse set of PFAS. Assay performance was initially evaluated for fatty acids and HSA binding drugs ibuprofen and warfarin. Concentration response relationships were determined and dissociation constants (Kd) for each compound were calculated using regression analysis of the dose-dependent changes in HSA melting temperature. Estimated Kd values for HSA binding of octanoic acid, decanoic acid, hexadecenoic acid, ibuprofen and warfarin agreed with established values. The binding affinities for 24 PFAS that included perfluoroalkyl carboxylic acids (C4-C12), perfluoroalkyl sulfonic acids (C4-C8), mono- and polyether perfluoroalkyl ether acids, and polyfluoroalkyl fluorotelomer substances were determined. These results demonstrate the utility of this differential scanning fluorimetry assay as a rapid high through-put approach for determining the relative protein binding properties and identification of chemical structures involved in binding for large numbers of structurally diverse PFAS.

category: pharmacology and toxicology


title: Mechanosensory trichome cells evoke a mechanical stimuli-induced immune response in plants

abstract: Perception of pathogen-derived ligands by corresponding host receptors is a pivotal strategy in eukaryotic innate immunity. In plants, this is complemented by circadian anticipation of infection timing, promoting basal resistance even in the absence of pathogen threat. Here, we report that trichomes, hair-like structures on the epidermis, directly sense external mechanical forces caused by raindrops to anticipate waterborne infections in Arabidopsis thaliana. Exposure of leaf surfaces to mechanical stimuli initiates the concentric propagation of intercellular calcium waves away from trichomes to induce defence-related genes. Propagating calcium waves enable effective immunity against pathogenic microbes through the calmodulin-binding transcription activator 3 (CAMTA3) and mitogen-activated protein kinases. We propose a novel layer of plant immunity in which trichomes function as mechanosensory cells to detect potential risks.


title: The Genetic Architecture of Strawberry Yield and Fruit Quality Traits

abstract:
Over the last two centuries breeders have drastically modified the fruit quality of strawberries through artificial selection. However, there remains significant variation in quality across germplasm with scope for further improvements to be made. We report extensive phenotyping of fruit quality and yield traits in a multi-parental strawberry population to allow genomic prediction and QTL identification, thereby enabling the description of genetic architecture to inform the efficacy of implementing advanced breeding strategies.

A trade-off was observed between two essential traits: sugar content and class one yield. This result highlights an established dilemma for strawberry breeders and a need to uncouple the relationship, particularly under June-bearing, protected production systems comparable to this study. A large effect QTL was associated with perceived acidity and pH whereas multiple loci were associated with firmness, we therefore recommend the implementation of both MAS and genomic prediction to capture the observed variation respectively.

Ultimately, our results suggest that the best method to improve strawberry yield is through selecting parental lines based upon the number of marketable fruit produced per plant. Strawberry number metrics were less influenced by environmental fluctuations and had a larger additive genetic component when compared to mass traits. As such, selecting using "number" traits should lead to faster genetic gain. Finally, we identify a large effect locus associated with an increase in class one fruit.


title: Developmental plasticity in male courtship in Bicyclus anynana butterflies is driven by hormone regulation of the yellow gene

abstract:
The organizational role for hormones in the regulation of sexual behavior is currently poorly explored. Previous work showed that seasonal variation in levels of the steroid hormone 20-hydroxyecdysone (20E) during pupal development regulates plasticity in male courtship behavior in Bicyclus anynana butterflies. Wet season (WS) males, reared at high temperature, have high levels of 20-hydroxyecdysone (20E) during pupation and become active courters. Dry season (DS) males, reared at low temperatures, have lower levels of 20E and lower courtship rates. Rescue of WS courtship rates can be achieved via injection of 20E into DS male pupae, but it is still unknown whether 20E alters gene expression in the pupal brain, and if so, the identity of those targets. Using transcriptomics, qPCR, and behavioral assays with a transgenic knockout, we show that higher expression levels of the yellow gene in DS male pupal brains, relative to WS brains, represses courtship in DS males. Furthermore, injecting DS males with 20E downregulates yellow to WS levels 4 hours post-injection, revealing a hormone sensitive window that determines courtship behavior. These findings are in striking contrast to Drosophila, where yellow is required for active male courtship behavior. We conclude that 20E plays an organizational role during pupal brain development by regulating the expression of yellow, which is a repressor of the neural circuity for male courtship behavior in B. anynana. This work shows that similar to vertebrates, hormones can also play an organizational role in insect brains, leading to permanent changes in adult sexual behavior.

Significance StatementBehavioral plasticity in adult insects is known to be regulated by hormones, which activate neural circuits in response to environmental cues. Here, we show that hormones can also regulate adult behavioral plasticity by altering gene expression during brain development, adjusting the insects behavior to predictable seasonal environmental variation. We show that seasonal changes in the hormone 20E alters expression of the yellow gene in the developing pupal brain of Bicyclus anynana butterflies, which leads to differences in male courtship behavior between the dry and wet seasonal forms. This work provides one of the first examples of the organizational role of hormones in altering gene expression and adult sexual behavior in the developing insect brain.


title: Eye-hand coordination during online reach corrections is task-dependent

abstract:
To produce accurate movements, the human motor system needs to deal with errors that can occur due to inherent noise, changes in the body, or disturbances in the environment. Here, we investigated the temporal coordination of rapid corrections of the eye and hand in response to a change in visual target location during the movement. In addition to a  classic double-step task in which the target stepped to a new position, participants performed a set of modified double-step tasks in which the change in movement goal was indicated by the appearance of an additional target, or by a spatial or symbolic cue. We found that both the absolute correction latencies of the eye and hand and the relative eye-hand correction latencies were dependent on the visual characteristics of the target change, with increasingly longer latencies in tasks that required more visual and cognitive processing. Typically, the hand started correcting slightly earlier than the eye, especially when the target change was indicated by a symbolic cue, and in conditions where visual feedback of the hand position was provided during the reach. Our results indicate that the oculomotor and limb-motor system can be differentially influenced by processing requirements of the task and emphasize that temporal eye-hand coordination is flexible rather than rigid.

New & NoteworthyEye movements support hand movements in many situations. Here we used variations of a double-step task to investigate temporal coupling of corrective hand and eye movements in response to target displacements. Correction latency coupling depended on the visual and cognitive processing demands of the task. The hand started correcting before the eye, especially when the task required decoding a symbolic cue. These findings highlight the flexibility and task-dependency of eye-hand coordination.


title: miR-544-3p mediates arthritis pain through regulation of FcγRI

abstract: Chronic or episodic joint pain is a major symptom in rheumatoid arthritis (RA) and its adequate treatment represents an unmet medical need. However, the cellular and molecular mechanisms underlying RA pain remain elusive. Non-coding microRNAs (miRNAs) have been implicated in the pathogenesis of RA as negative regulators of the stability or translation of specific target mRNAs. Yet, their significance in RA pain is still not well defined. We and other groups recently identified neuronally expressed Fc{gamma}RI as a key driver of arthritis pain in mouse RA models. Thus, we tested the hypothesis that miRNAs that target and regulate neuronal Fc{gamma}RI attenuate RA pain. Here, we show that miR-544-3p was robustly downregulated whereas Fc{gamma}RI was significantly upregulated in the dorsal root ganglion (DRG) in mouse RA models. Intrathecal injection of miR-544-3p mimic attenuated established mechanical and heat hyperalgesia in a mouse model of collagen II-induced arthritis (CIA). Moreover, this effect was likely mediated, at least in part, by Fc{gamma}RI since miR-544-3p mimic downregulated Fc{gamma}RI in the DRG during arthritis and genetic deletion of Fc{gamma}RI produced similar antihyperalgesic effects in the CIA model. This notion was further supported by a dual luciferase assay showing that miR-544-3p targeted Fc{gamma}RI by directly binding to its 3UTR. In addition, Fc{gamma}RI expression in DRG neurons in vitro was downregulated by miR-544-3p mimic and upregulated by miR-544-3p inhibitor. In naive mice, miR-544-3p mimic alleviated acute joint pain hypersensitivity induced by IgG immune complex (IgG-IC), whereas miR-544-3p inhibitor potentiated the pro-nociceptive behavioral effect of IgG-IC. These findings suggest that miR-544-3p causally participates in the maintenance of arthritis pain by targeting neuronal Fc{gamma}RI, and thus define miR-544-3p as a new potential therapeutic target for treating RA pain.


title: Optogenetic stimulation of glutamatergic neurons in the cuneiform nucleus controls locomotor movements in a mouse model of Parkinson's disease

abstract:
In Parkinsons disease (PD), the loss of midbrain dopaminergic cells results in severe locomotor deficits such a gait freezing and akinesia. Growing evidence indicates that these deficits can be attributed to decreased activity in the Mesencephalic Locomotor Region (MLR), a brainstem region controlling locomotion. Clinicians are exploring deep brain stimulation of the MLR as a treatment option to improve locomotor function. The results are variable, from modest to promising. However, within the MLR, clinicians have targeted the pedunculopontine nucleus exclusively, while leaving the cuneiform nucleus unexplored. To our knowledge, the effects of cuneiform nucleus stimulation have never been determined in parkinsonian conditions in any animal model. Here, we addressed this issue in a mouse model of Parkinsons disease based on bilateral striatal injection of 6-hydroxydopamine (6-OHDA), which damaged the nigrostriatal pathway and decreased locomotor activity. We show that selective optogenetic stimulation of glutamatergic neurons in the cuneiform nucleus in mice expressing channelrhodopsin in a Cre-dependent manner in Vglut2-positive neurons (Vglut2-ChR2-EYFP mice) increased the number of locomotor initiations, increased the time spent in locomotion, and controlled locomotor speed. Using deep learning-based movement analysis, we found that limb kinematics of optogenetic-evoked locomotion in pathological conditions were largely similar to those recorded in freely moving animals. Our work identifies the glutamatergic neurons of the cuneiform nucleus as a potentially clinically relevant target to improve locomotor activity in parkinsonian conditions. Our study should open new avenues to develop targeted stimulation of these neurons using deep brain stimulation, pharmacotherapy or optogenetics.

SIGNIFICANCE STATEMENTIn Parkinsons disease, alleviating locomotor deficits is a challenge. Clinicians are exploring deep brain stimulation of the Mesencephalic Locomotor Region, a brainstem region controlling locomotion, but results are mixed. However, the best target in this region in Parkinsons disease remains unknown. Indeed, this region which comprises the pedunculopontine and cuneiform nuclei, contains different cell types with opposing effects on locomotor output. Here, using a mouse model where midbrain dopaminergic cells were damaged by a neurotoxin, we demonstrate that optogenetic activation of glutamatergic neurons in the cuneiform nucleus increases locomotion, controls speed, and evokes limb movements similar to those observed during spontaneous locomotion in intact animals. Our study identifies a potentially clinically relevant target to improve locomotor function in Parkinsons disease.


title: Plastic cell morphology changes during dispersal

abstract:
Dispersal is the movement of organisms from one habitat to another that potentially results in gene flow. It is often found to be plastic, allowing organisms to adjust dispersal movements depending on environmental conditions. A fundamental aim in ecology is to understand the determinants underlying dispersal and its plasticity. We utilized 22 strains of the ciliate Tetrahymena thermophila to determine if different phenotypic dispersal strategies co-exist within a species and which mechanisms underlie this variability. We quantified the cell morphologies impacting cell motility and dispersal. Distinct differences in innate cellular morphology and dispersal rates were detected, but no universally utilized combinations of morphological parameters correlate with dispersal. Rather, multiple distinct and plastic morphological changes impact cilia-dependent motility during dispersal, especially in proficient dispersing strains facing challenging environmental conditions. Combining ecology and cell biology experiments, we show that dispersal can be promoted through a panel of plastic motility-associated changes to cell morphology and motile cilia.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/448130v1_ufig1.gif" ALT="Figure 1">
View larger version (45K):
org.highwire.dtl.DTLVardef@1aad069org.highwire.dtl.DTLVardef@158adbdorg.highwire.dtl.DTLVardef@f6570forg.highwire.dtl.DTLVardef@cdc249_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LITetrahymena thermophila exhibits intra-specific diversity in morphology and dispersal.
C_LIO_LICell motility behavior during dispersal changes with cilia length and cell shape.
C_LIO_LICells from proficient dispersing strains transiently change basal body and cilia position.
C_LIO_LIStarvation-induced dispersal triggers increased basal body and cilia density and caudal cilium formation in rapid-swimming cells.
C_LI

category: cell biology


title: TDAExplore: quantitative image analysis through topology-based machine learning

abstract: Machine learning has greatly expanded the ability to classify images. However, many machine learning classifiers require thousands of images for training and lack quantitative descriptors of how images were grouped. We overcome these limitations with a machine learning approach based on topological data analysis, where a data set of 20-30 images is sufficient to accurately train the classifier. Our method quantifies differences between groups and identifies subcellular regions with the largest dissimilarities.


title: A comprehensive series of temporal transcription factors in the fly visual system

abstract: The brain consists of thousands of different neuronal types that are generated through multiple divisions of neuronal stem cells. These stem cells have the capacity to generate different neuronal types at different stages of their development. In Drosophila, this temporal patterning is driven by the successive expression of temporal transcription factors (tTFs). While a number of tTFs are known in different animals and across various parts of the nervous system, these have been mostly identified by informed guesses and antibody availability. We used single-cell mRNA sequencing to identify the complete series of tTFs that specify most Drosophila medulla neurons in the optic lobe. We tested the genetic interactions among these tTFs. While we verify the general principle that tTFs regulate the progression of the series by activating the next tTFs in the series and repressing the previous ones, we also identify more complex regulations. Two of the tTFs, Eyeless and Dichaete, act as hubs integrating the input of several upstream tTFs before allowing the series to progress and in turn regulating the expression of several downstream tTFs. Moreover, we show that tTFs not only specify neuronal identity by controlling the expression of cell type-specific genes. Finally, we describe the very first steps of neuronal differentiation and find that terminal differentiation genes, such as neurotransmitter-related genes, are present as transcripts, but not as proteins, in immature larval neurons days before they are being used in functioning neurons; we show that these mechanisms are conserved in humans. Our results offer a comprehensive description of a temporal series of tTFs in a neuronal system, offering mechanistic insights into the regulation of the progression of the series and the regulation of neuronal diversity. This represents a proof-of-principle for the use of single-cell mRNA sequencing for the comparison of temporal patterning across phyla that can lead to an understanding of how the human brain develops and how it has evolved.

category: developmental biology


title: Quantifying concordant genetic effects of de novo mutations on multiple disorders

abstract: Exome sequencing on tens of thousands of parent-proband trios has identified numerous deleterious de novo mutations (DNMs) and implicated risk genes for many disorders. Recent studies have suggested shared genes and pathways are enriched for DNMs across multiple disorders. However, existing analytic strategies only focus on genes that reach statistical significance for multiple disorders and require large trio samples in each study. As a result, these methods are not able to characterize the full landscape of genetic sharing due to polygenicity and incomplete penetrance. In this work, we introduce EncoreDNM, a novel statistical framework to quantify shared genetic effects between two disorders characterized by concordant enrichment of DNMs in the exome. EncoreDNM makes use of exome-wide, summary-level DNM data, including genes that do not reach statistical significance in single-disorder analysis, to evaluate the overall and annotation-partitioned genetic sharing between two disorders. Applying EncoreDNM to DNM data of nine disorders, we identified abundant pairwise enrichment correlations, especially in genes intolerant to pathogenic mutations and genes highly expressed in fetal tissues. These results suggest that EncoreDNM improves current analytic approaches and may have broad applications in DNM studies.

category: genetics


title: Horizontal transfer and subsequent explosive expansion of a DNA transposon in sea kraits (Laticauda)

abstract: Transposable elements (TEs) are self replicating genetic sequences and are often described as important "drivers of evolution". This driving force is because TEs promote genomic novelty by enabling rearrangement, and through exaptation as coding and regulatory elements. However, most TE insertions will be neutral or harmful, therefore host genomes have evolved machinery to supress TE expansion. Through horizontal transposon transfer (HTT) TEs can colonise new genomes, and since new hosts may not be able to shut them down, these TEs may proliferate rapidly. Here we describe HTT of the Harbinger-Snek DNA transposon into sea kraits (Laticauda), and its subsequent explosive expansion within Laticauda genomes. This HTT occurred following the divergence of Laticauda from terrestrial Australian elapids ~15-25 Mya. This has resulted in numerous insertions into introns and regulatory regions, with some insertions into exons which appear to have altered UTRs or added sequence to coding exons. Harbinger-Snek has rapidly expanded to make up 8-12% of Laticauda spp. genomes; this is the fastest known expansion of TEs in amniotes following HTT. Genomic changes caused by this rapid expansion may have contributed to adaptation to the amphibious-marine habitat.

category: genomics


title: Measuring the repertoire of age-related behavioral changes in Drosophila melanogaster

abstract: Aging affects almost all aspects of an organism - its morphology, its physiology, its behavior. Isolating which biological mechanisms are regulating these changes, however, has proven difficult, potentially due to our inability to characterize the full repertoire of an animals behavior across the lifespan. Using data from fruit flies (D. melanogaster) we measure the full repertoire of behaviors as a function of age. We observe a sexually dimorphic pattern of changes in the behavioral repertoire during aging. Although the stereotypy of the behaviors and the complexity of the repertoire overall remains relatively unchanged, we find evidence that the observed alterations in behavior can be explained by changing the flys overall energy budget, suggesting potential connections between metabolism, aging, and behavior.

category: animal behavior and cognition


title: Single-Cell Classification Using Graph Convolutional Networks

abstract:
BackgroundAnalyzing single-cell RNA sequencing (scRNAseq) data plays an important role in understanding the intrinsic and extrinsic cellular processes in biological and biomedical research. One significant effort in this area is the identification of cell types. With the availability of a huge amount of single cell sequencing data and discovering more and more cell types, classifying cells into known cell types has become a priority nowadays. Several methods have been introduced to classify cells utilizing gene expression data. However, incorporating biological gene interaction networks has been proved valuable in cell classification procedures.

ResultsIn this study, we propose a multimodal end-to-end deep learning model, named sigGCN, for cell classification that combines a graph convolutional network (GCN) and a neural network to exploit gene interaction networks. We used standard classification metrics to evaluate the performance of the proposed method on the within-dataset classification and the cross-dataset classification. We compared the performance of the proposed method with those of the existing cell classification tools and traditional machine learning classification methods.

ConclusionsResults indicate that the proposed method outperforms other commonly used methods in terms of classification accuracy and F1 scores. This study shows that the integration of prior knowledge about gene interactions with gene expressions using GCN methodologies can extract effective features improving the performance of cell classification.


title: STRipy: a graphical application for enhanced genotyping of pathogenic short tandem repeats in sequencing data

abstract:
Short tandem repeats (STRs) are highly polymorphic with high mutation rates and expansions of STRs have been implicated as the causal variant in diseases. The application of genome sequencing in patients has recently allowed many new discoveries with over 50 disease causing loci known to date. There are several tools which allow genotyping of STRs from high-throughput sequencing (HTS) data. However, running these tools out of the box only allow around half of the known disease-causing loci to be genotyped, with lengths often limited to either read or fragment length which is less than the pathogenic cut-off for some diseases. While analysis tools can be customised to genotype extra loci, this requires proficiency in bioinformatics to set up, use, and analyse the resulting data, limiting their widespread usage by other researchers and clinicians.

To address these issues, we have created a new software called STRipy that has an intuitive graphical interface and requires no specific skills for usage, thus significantly simplifying detection of STRs expansions from human HTS data. STRipy is able to target all known disease-causing STRs with genotyping performed with an established tool, ExpansionHunter, that is incorporated into the software. We have created additional functionality into STRipy to work with long alleles exceeding the fragment length.

STRipy was validated using over 60 thousand simulated samples and was shown to work on whole genome sequencing of biological samples with pathogenic variants. Finally, we have used STRipy to acquire genotypes of pathogenic loci for thousands of samples from various populations which are provided to the user along with the data from the literature to assist with results interpretation. We believe the simplicity and breadth of STRipy will increase the testing of STR diseases in current datasets resulting in further diagnoses of rare diseases caused by STRs expansions.


title: Quantitative imaging of intracellular density with ratiometric stimulated Raman scattering microscopy

abstract: Cell size and density impact a wide range of physiological functions, including tissue homeostasis, growth regulation, and osmoregulation. Both are tightly regulated in mammalian cells. In comparison, density variation of a given cell type is much smaller than cell size, indicating that maintenance of cell type-specific density is important for cell function. Despite this importance, little is known about how cell density affects cell function and how it is controlled. Current tools for intracellular cell density measurements are limited either to suspended cells or cells growing on 2D substrates, neither of which recapitulate the physiology of single cells in intact tissue. While optical measurements have the potential to measure cell density in situ and noninvasively, light scattering in multicellular systems prevents direct quantification. Here, we introduce an intracellular density imaging technique based on ratiometric stimulated Raman scattering microscopy (rSRS). It quantifies intracellular drymass density through vibrational imaging of macromolecules. Moreover, water is used as an internal standard to correct for aberration and light scattering. We demonstrate real-time measurement of intracellular density quantification and show that density is tightly regulated across different cell types and can be used to differentiate cell types as well as cell states. We further demonstrate dynamic imaging of density change in response to osmotic challenge as well as intracellular density imaging of a 3D tumor spheroid. Our technique has the potential for imaging intracellular density in intact tissue and understanding density regulation and its role in tissue homeostasis.


title: Scaffold Protein RACK1 inhibitor compounds prevent the Focal Adhesion Kinase mediated breast cancer cell migration and invasion potential

abstract: Scaffold protein RACK1 mediates cancer cell migration mostly through regulation of focal adhesion (FA) assembly by promoting a focal adhesion kinase (FAK) activation downstream of the integrin clustering and adhesion at the extracellular matrix (ECM). Here we demonstrated the efficacy of our recently developed RACK1 Y246 phosphorylation inhibitor compounds (SD29 and SD29-14) to inhibit the migration and invasion of MCF7 and MDA-MB-231 breast cancer cell lines. Using multiple assays, our results confirmed that inhibitor compounds effectively prevent the filopodia/lamellipodia development and inhibits the migration of breast cancer cells. A mechanistic model of the inhibitor compounds has been developed. Migration and invasion capabilities of the cancer cells define the metastasis of cancer. Thus, our results suggest a potential therapeutic mechanism of the inhibitors to prevent metastasis in diverse cancers.

category: cancer biology


title: The U1 snRNP component RBP45d regulates temperature-responsive flowering in Arabidopsis thaliana

abstract: Pre-mRNA splicing is a crucial step of gene expression whereby the spliceosome produces constitutively and alternatively spliced transcripts that not only diversify the transcriptome but also play essential functions during plant development and responses to environmental changes. Numerous evidences indicate that regulation at the pre-mRNA splicing step is important for flowering time control, however the components and detailed mechanism underlying this process remain largely unknown. Here, we identified a previously unknown splicing factor in Arabidopsis thaliana, RNA BINDING PROTEIN 45d (RBP45d), a member of the RBP45/47 family. Using sequence comparison and biochemical analysis, we determined that RBP45d is a component of the U1 small nuclear ribonucleoprotein (U1 snRNP) with functions distinct from other family members. RBP45d associates with the U1 snRNP by interacting with pre-mRNA-processing factor 39a (PRP39a) and directly regulates alternative splicing (AS) for a specific set of genes. Plants with loss of RBP45d function exhibit defects in temperature-induced flowering potentially due to the mis-regulation of temperature-sensitive AS by RBP45d and PRP39a of the key flowering gene FLOWERING LOCUS M. Taken together, we report that RBP45d is a novel U1 snRNP component in plants that functions together with PRP39a in temperature-mediated flowering.


title: Reservoir water in Singapore contains ESBL-producing and carbapenem-resistant bacteria with conjugatable conserved gene cluster transfer between different species.

abstract: As the role of the aquatic environment in the "One Health" approach has called increasing attention, the studies of Antimicrobial resistance (AMR) spreading in the water bodies have been reported worldwide. However, there are still limited studies on the AMR carrier in the reservoir water in Singapore. Since 2018, our group has collect water samples from six reservoirs in Singapore and isolated the beta-lactam resistant bacteria from them. We then characterized the isolates with Whole-genome sequencing (WGS) and successfully identified ESBL-producing bacteria from three sampling reservoirs, and confirmed their resistance with both phenotypic and sequencing methods. To better understand the AMR spreading locally, we compared our isolates with isolates from other WGS studies in Singapore covered humans, food, and the enviroment. From there, we noticed the same sequence type (ST) as ST10, ST23, and ST38 has been shared among the environment, food, and humans, as well as the same beta-lactamase genes, are widely distributed among multiple sources. Further genetic environment comparison of beta-lactamase has suggested their spreading as conserved gene clusters among different species and sources. And this hypothesis has been supported by the successful conjugation of blaCTX-M-15 from Klebsiella pneumonia to Escherichia coli (E .coli). We also applied the shotgun metagenomic sequencing to understand the community of bacteria in reservoir water and detect the AMR genes. The composition of bacteria has shown different diversity among different samples. Besides, different beta-lactamase genes have been identified compared to culture depended methods. Here, we suggest that sequencing analysis has great potential in understanding AMR spreading in the "One-Health" approach. A genetic-based AMR risk assessment is in urgent need in Singapore.


title: Proliferation maintains the undifferentiated status of stem cells: the role of the planarian cell cycle regulator Cdh1

abstract:
The coincidence of cell cycle arrest and differentiation has been described in a wide variety of stem cells and organisms for decades, but the causal relationship is still unclear due to the complicated regulation of the cell cycle. Here, we used the planarian Dugesia japonica since it possesses a quite simple cell cycle regulation in which cdh1 is the only factor that arrests the cell cycle. When cdh1 was functionally inhibited, the planarians could not maintain their tissue homeostasis and could not regenerate their missing body parts. While the ablation of cdh1 caused pronounced propagation of the stem cells, the progenitor and differentiated cells were decreased. Further analysis indicated that the stem cells without cdh1 expression did not undergo differentiation even though they received ERK signaling activation as an induction signal. These results suggested that stem cells could not acquire differentiation competence without cell cycle arrest. Thus, we propose that cell cycle regulation determines the differentiation competence and that cell cycle exit to G0 enables stem cells to undergo differentiation.

Summary statementBy using planarians, which have quite simple cell cycle regulation, we revealed that stem cells with cell cycle progression could not undergo differentiation even though an induction signal was activated.


title: Bottom-up but not Top-down Attention Dominates the Value Representation in the Orbitofrontal Cortex

abstract: The orbitofrontal cortex (OFC) is essential for value-based learning and decision making. Understanding the attentional modulation of the representation of value in the OFC provides us key information on its functional roles and links the OFC to other cognitive processes. We examined how top-down and bottom-up attention modulates the value encoding in the OFC. Two macaque monkeys were trained to detect a luminance change at a cued location between a pair of visual stimuli, which were over-trained pictures associated with different amount of juice rewards and, thus, different salience. While the monkeys behavior and the DLPFC neuronal activities indicated that the monkeys actively directed their attention toward the cued location during the task, the OFC neurons value encoding, however, was dominated by the bottom-up attention based on stimulus salience and only reflected the top-down attention weakly. The disassociation between the top-down and bottom-up attention signals in the OFC indicates that the OFC occupies an early stage of value information processing in the brain.

category: neuroscience


title: Early functional connectivity in the developing sensorimotor network that is independent of sensory experience

abstract: Neonatal sensory experience shapes development of neural pathways carrying sensory information to the cortex. These pathways link to wider functional networks that coordinate activity of separate cortical regions, but it remains unknown when these broader networks emerge or how their maturation is influenced by sensory experience. By imaging activity across the cortex in neonatal mice, we have found unexpectedly early emergence of coordinated activity within a sensorimotor network that includes whisker-related somatosensory cortex and motor cortex. This network is spontaneously active but is not engaged by sensory stimulation, even though whisker deflection reliably drives cortical activity within barrel cortex. Acute silencing of the sensory periphery ablated spontaneous activity that was restricted to barrel cortex but spared this early sensorimotor network coactivity, suggesting that it is driven from elsewhere. Furthermore, perturbing sensory experience by whisker trimming did not impact emergence or early maturation of spontaneous activity in the sensorimotor network. As such, functional sensorimotor cortical networks develop early, in parallel with development of ascending sensory pathways, and their initial maturation is independent of sensory experience.


title: Bacteriophage T4 Vaccine Platform for Next-generation Influenza Vaccine Development

abstract: Developing influenza vaccines that protect against a broad range of viruses is a public health priority, and several conserved viral proteins or domains have been identified as promising targets for such vaccine development. However, none of the targets is immunogenic, and vaccine platforms that can incorporate multiple antigens with enhanced immunogenicity are desperately needed. In this study, we provided proof-of-concept for the development of next-generation influenza vaccine using T4 phage virus-like particle (VLP) platform. With extracellular domain of influenza matrix protein 2 (M2e) as a readout, we showed that more than 1,280 M2e molecules can be assembled on a 120x90 nanometer phage capsid to form T4-M2e VLPs, which are highly immunogenic and induced complete protection against influenza virus challenge without any addition adjuvant. Potentially, additional conserved antigens or molecular adjuvants could be incorporated into the T4-M2e VLPs to customize influenza vaccines to address different issues. All the components of T4 VLP vaccines can be mass-produced in E. coli in a short time, therefore, providing a rapid approach to deal with the potential influenza pandemic.

category: microbiology


title: Development Of Novel Multiplex PCR For Rapid Diagnosis Of Coinfected Hemo-Parasites In Cattle

abstract: A novel, rapid and specific multiplex polymerase chain reaction has been developed for the diagnosis of hemo-parasitic infection in bovine blood by three of the most common hemo-parasites. The reported method relied on the detection of the three different bovine hemoparasites isolated from red blood cells (RBCs) of cattle by conventional Giemsa stained blood smear (GSBS) and confirmed by multiplex PCR. The designed multiplex primer sets can amplify 205, 313 and 422 bp fragments of apocytochrome b, sporozoite and macroschizont 2 (spm2) and 16S rRNA gene for Babesia bigemina, Theileria annulata and Anaplasma marginale, respectively. This multiplex PCR was sensitive with the ability to detect the presence of 150 ng of genomic DNA. The primers used in this multiplex PCR also showed highly specific amplification of specific gene fragments of each respective parasite DNA without the presence of non-specific and non-target PCR products. This multiplex PCR system was used to diagnose GSBS confirmed blood samples (N=12) found infected or co-infected with hemoparasites. A comparison of the two detection methods revealed that 58.33% of specimens showed concordant diagnoses with both techniques. The specificity, positive predictive value and kappa coefficient of agreement was highest for diagnosis of B. bigemina and lowest for A. marginale. The overall Kappa coefficient for diagnosis based on GSBS for multiple pathogen compared to multiplex PCR was 0.56 slightly behind the threshold of 0.6 of agreement. Therefore, confirmation should always be made based on PCR to rule out false positive due to differences in subjective observations, stain particles and false negative due to low level of parasitaemia. The simplicity and rapidity of this specific multiplex PCR method make it suitable for large-scale epidemiological studies and for follow-up of drug treatments.

category: molecular biology


title: Interphase microtubule disassembly is a signaling cue that drives cell rounding at mitotic entry.

abstract: Reorganization of the cortical actin cytoskeleton at mitotic entry is essential to increase membrane tension for cell rounding1,2. This spherical shape is necessary for the biogenesis and organization of the mitotic spindle2-6. Proteins of the Ezrin, Radixin, Moesin (ERM) family play essential roles in mitotic morphogenesis by linking actomyosin forces to the plasma membrane2,3,7-10. While ERMs drive metaphase cell rounding, the cell-cycle signals that prompt their conformational activation in mitosis are unknown11. We screened a library of small molecules using novel ERM biosensors12 and we unexpectedly found that drugs that disassemble microtubules promote ERM activation. Remarkably, cells disassemble their interphase microtubules while entering mitosis13. We further discovered that this disassembly of microtubules acts as a cell-cycle signal that directs ERM activation and metaphase cell rounding. We show that GEF-H1, a Rho-GEF inhibited by microtubule binding, acts downstream of microtubule disassembly to activate ERMs via RhoA and its kinase effector SLK. In addition, we demonstrate that GEF-H1 and Ect2, another Rho-GEF responsible for the generation of mitotic actomyosin forces6,14, act together to drive metaphase ERM activation and cell rounding. In summary, we report microtubule disassembly as a cell cycle signal that triggers a signaling network ensuring that actomyosin forces are efficiently integrated at the plasma membrane to promote cell rounding at mitotic entry.


title: Multiple recent sex chromosome fusions in Drosophila virilis associated with elevated satellite DNA abundance

abstract: Repetitive satellite DNA is highly variable both within and between species, and is often located near centromeres. However, the abundance or array length of satellite DNA may be constrained or have maximum limits. Drosophila virilis contains among the highest relative satellite abundances, with almost half its genome composed of three related 7 bp satellites. We discovered a strain of D. virilis that has 15% more pericentromeric satellite DNA compared to other strains, and also underwent two independent centromere-to-centromere sex chromosome fusion events. These fusions are presumably caused by DNA breakage near the pericentromeric satellites followed by repair using similar repetitive regions of nonhomologous chromosomes. We hypothesized that excess satellite DNA might increase the risk of DNA breaks and genome instability when stressed, which would be consistent with the apparent high rate of fusions we found in this strain. To directly quantify DNA breakage levels between strains with different satellite DNA abundances, we performed the comet assay after feeding flies gemcitabine and administering low-dose gamma radiation. We found a positive correlation between the rate of DNA breakage and satellite DNA abundance. This was further supported by a significant decrease in DNA breakage in an otherwise genetically identical substrain that lost the chromosome fusion and several megabases of satellite DNA. We find that the centromere-to-centromere fusions resulted in up to a 21% nondisjunction rate between the X and Y chromosomes in males, adding a fitness cost. Finally, we propose a model consistent with our data that implicates genome instability as a critical evolutionary constraint to satellite abundance.

category: evolutionary biology


title: Evolution of the open-access CIViC knowledgebase is driven by the needsof the cancer variant interpretation community

abstract: CIViC (Clinical Interpretation of Variants in Cancer; civicdb.org) is a crowd-sourced, public domain knowledgebase composed of literature-derived evidence characterizing the clinical utility of cancer variants. As clinical sequencing becomes more prevalent in cancer management, the need for cancer variant interpretation has grown beyond the capability of any single institution. With nearly 300 contributors, CIViC contains peer-reviewed, published literature curated and expert-moderated into structured data units (Evidence Items) that can be accessed globally and in real time, reducing barriers to clinical variant knowledge sharing. We have extended CIViCs functionality to support emergent variant interpretation guidelines, increase interoperability with other variant resources, and promote widespread dissemination of structured curated data. To support the full breadth of variant interpretation from basic to translational, including integration of somatic and germline variant knowledge and inference of drug response, we have enabled curation of three new evidence types (predisposing, oncogenic and functional). The growing CIViC knowledgebase distributes clinically-relevant cancer variant data currently representing >2500 variants in >400 genes from >2800 publications.


title: Unexpected gene activation following CRISPR-Cas9-mediated genome editing

abstract: The discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) and its development as a genome editing tool has revolutionized the field of molecular biology. In the DNA damage field, CRISPR has brought an alternative to induce endogenous double-strand breaks (DSB) at desired genomic locations and study the DNA damage response and its consequences. Many systems for sgRNA delivery have been reported in order to efficiency generate this DSB, including lentiviral vectors. However, some of the consequences of these systems are yet not well understood. Here we report that lentiviral-based sgRNA vectors can integrate into the endogenous genomic target location, leading to undesired activation of the target gene. By generating a DSB in the regulatory region of the ABCB1 gene using a lentiviral sgRNA vector, we can induce the formation of taxol-resistant colonies. We show that these colonies upregulated ABCB1 via integration of the EEF1A1 and the U6 promoters from the sgRNA vector. We believe that this is an unreported CRISPR/Cas9 artefact that researchers need to be aware of when using lentiviral vectors for genome editing.


title: Epigenetic features improve TALE target prediction

abstract:
The yield of many crop plants can be substantially reduced by plant-pathogenic Xanthomonas bacteria. The infection strategy of many Xanthomonas strains is based on transcription activator-like effectors (TALEs), which are secreted into the host cells and act as transcriptional activators of plant genes that are beneficial for the bacteria.

The modular DNA binding domain of TALEs contains tandem repeats, each comprising two hyper-variable amino acids. These repeat-variable diresidues (RVDs) bind to a continuous DNA stretch (a target box) and determine the specificity of a TALE. All available tools for the prediction of TALE targets within the host plant suffer from many false positives. In this paper we propose a strategy to improve prediction accuracy by considering the epigenetic state of the host plant genome in the region of the target box. To this end, we extend our previously published tool PrediTALE by two epigenetic features: (i) We allow for filtering target boxes according to chromatin accessibility and (ii) we allow for considering the methylation state of cytosines within the target box during prediction, since DNA methylation may affect the binding specificity of RVDs. Here, we determine the epigenetic features from publicly available DNase-seq, ATAC-seq, and WGBS-seq data in rice.

We benchmark the utility of both epigenetic features separately and in combination, deriving ground-truth from RNA-seq infections studies in rice. We find an improvement for each individual epigenetic feature, but especially the combination of both. Having established an advantage in TALE target predicting considering epigenetic features, we use these data for promoterome and genome-wide scans by our new tool EpiTALE, leading to several novel putative virulence targets.

Our results suggest that it would be worthwhile to collect condition-specific chromatin accessibility data and methylation information when studying putative virulence targets of Xan-thomonas TALEs.

category: bioinformatics


title: Oncogenic RAS sensitizes cells to drug-induced replication stress via transcriptional silencing of P53

abstract: Cancer cells often experience high basal levels of DNA replication stress (RS), for example due to hyperactivation of oncoproteins like MYC or RAS. Therefore, cancer cells are considered to be sensitive to drugs that exacerbate the level of RS or block the intra S-phase checkpoint. Consequently, RS-inducing drugs including ATR and CHK1 inhibitors are used or evaluated as anti-cancer therapies. However, drug resistance and lack of biomarkers predicting therapeutic efficacy limit efficient use. This raises the question what determines sensitivity of individual cancer cells to RS. Here, we report that oncogenic RAS does not only enhance the sensitivity to ATR/CHK1 inhibitors by directly causing RS. Instead, we observed that HRASG12V dampens the activation of the P53-dependent transcriptional response to drug-induced RS, which in turn confers sensitivity to RS. We demonstrate that inducible expression of HRASG12V sensitized retina pigment epithelial (RPE-hTERT) as well as osteosarcoma (U2OS) cells to ATR and CHK1 inhibitors. Using RNA-sequencing of FACS-sorted cells we discovered that P53 signaling is the sole transcriptional response to RS. However, oncogenic RAS attenuates the transcription of P53 and its target genes. Accordingly, live cell imaging showed that HRASG12V exacerbates RS in S/G2-phase, which could be rescued by stabilization of P53. Thus, our results demonstrate that transcriptional control of P53 is a prime determinant in the response to ATR/CHK1 inhibitors and show that hyperactivation of the MAPK pathway impedes this response. Our findings suggest that the level of oncogenic MAPK signaling could predict sensitivity to intra-S-phase inhibition in cancers with intact P53.


title: Neuroblastoma signalling models unveil combination therapies targeting feedback-mediated resistance

abstract: Very high risk neuroblastoma is characterised by increased MAPK signalling, and targeting MAPK signalling is a promising therapeutic strategy. We used a deeply characterised panel of neuroblastoma cell lines and found that the sensitivity to MEK inhibitors varied drastically between these cell lines. By generating quantitative perturbation data and mathematical modelling, we determined potential resistance mechanisms. We found that negative feedbacks within MAPK signalling and to the IGF receptor mediate re-activation of MAPK signalling upon treatment in resistant cell lines. By using cell-line specific models, we predict that combinations of MEK inhibitors with RAF or IGFR inhibitors can overcome resistance, and tested these predictions experimentally. In addition, phospo-proteomics profiles confirm the cell-specific feedback effects and synergy of MEK and IGFR targeted treatements. Our study shows that a quantitative understanding of signalling and feedback mechanisms facilitated by models can help to develop and optimise therapeutic strategies, and our findings should be considered for the planning of future clinical trials introducing MEKi in the treatment of neuroblastoma.


title: Nash equilibria in human sensorimotor interactions explained by Q-Learning

abstract: The Nash equilibrium concept has previously been shown to be an important tool to understand human sensorimotor interactions, where different actors vie for minimizing their respective effort while engaging in a multi-agent motor task. However, it is not clear how such equilibria are reached. Here, we compare different reinforcement learning models based on haptic feedback to human behavior in sensorimotor versions of three classic games, including the Prisoners Dilemma, and the symmetric and asymmetric matching pennies games. We find that a discrete analysis that reduces the continuous sensorimotor interaction to binary choices as in classical matrix games does not allow to distinguish between the different learning algorithms, but that a more detailed continuous analysis with continuous formulations of the learning algorithms and the game-theoretic solutions affords different predictions. In particular, we find that Q-learning with intrinsic costs that disfavor deviations from average behavior explains the observed data best, even though all learning algorithms equally converge to admissible Nash equilibrium solutions. We therefore conclude that it is important to study different learning algorithms for understanding sensorimotor interactions, as such behavior cannot be inferred from a game-theoretic analysis alone, that simply focuses on the Nash equilibrium concept, as different learning algorithms impose preferences on the set of possible equilibrium solutions due to the inherent learning dynamics.


title: EEG-based visual deviance detection in freely behaving mice

abstract: The mouse is widely used as an experimental model to study visual processing. To probe how the visual system detects changes in the environment, functional paradigms in freely behaving mice are strongly needed. We developed and validated the first EEG-based method to investigate visual deviance detection in freely behaving mice. Mice with EEG implants were exposed to a visual deviant detection paradigm that involved changes in light intensity as standard and deviant stimuli. By subtracting the standard from the deviant evoked waveform, deviant detection was evident as bi-phasic negativity (starting around 70 ms) in the difference waveform. Additionally, deviance-associated evoked (beta/gamma) and induced (gamma) oscillatory responses were found. We showed that the results were stimulus-independent by applying a "flip-flop" design and the results showed good repeatability in an independent measurement. Together, we put forward a validated, easy-to-use paradigm to measure visual deviance processing in freely behaving mice.


title: Serotonergic neurons mediate operant conditioning in Drosophila larvae

abstract: Observed across species, operant conditioning facilitates learned associations between behaviours and outcomes, biasing future action selection to maximise reward and avoid punishment. To elucidate the underlying neural mechanisms, we built a high-throughput tracker for Drosophila melanogaster larvae, combining real-time behaviour detection with closed-loop optogenetic and thermogenetic stimulation capabilities. We demonstrate operant conditioning in Drosophila larvae by inducing a bend direction preference through optogenetic activation of reward-encoding serotonergic neurons. Specifically, we establish that the ventral nerve cord is necessary for this memory formation. Our results extend the role of serotonergic neurons for learning in insects as well as the existence of learning circuits outside the mushroom body. This work supports future studies on the function of serotonin and the mechanisms underlying operant conditioning at both circuit and cellular levels.


title: A high-throughput multiplexing and selection strategy to complete bacterial genomes

abstract:
BackgroundBacterial whole-genome sequencing based on short-read sequencing data often results in a draft assembly formed by contiguous sequences. The introduction of long-read sequencing technologies permits to unambiguously bridge those contiguous sequences into complete genomes. However, the elevated costs associated with long-read sequencing frequently limit the number of bacterial isolates that can be long-read sequenced.

Here we evaluated the recently released 96 barcoding kit from Oxford Nanopore Technologies (ONT) to generate complete genomes on a high-throughput basis. In addition, we propose a long-read isolate selection strategy that optimizes a representative selection of isolates from large-scale bacterial collections.

ResultsDespite an uneven distribution of long-reads per barcode, near-complete chromosomal sequences (assembly contiguity = 0.89) were generated for 96 Escherichia coli isolates with associated short-read sequencing data. The assembly contiguity of the plasmid replicons was even higher (0.98) which indicated the suitability of the multiplexing strategy for studies focused on resolving plasmid sequences. We benchmarked hybrid and ONT-only assemblies and showed that the combination of ONT sequencing data with short-read sequencing data is still highly desirable: (i) to perform an unbiased selection of isolates for long-read sequencing, (ii) to achieve an optimal genome accuracy and completeness, and (iii) to include small plasmids underrepresented in the ONT library.

ConclusionsThe proposed long-read isolate selection ensures completing bacterial genomes of isolates that span the genome diversity inherent in large collections of bacterial isolates. We show the potential of using this multiplexing approach to close bacterial genomes on a high-throughput basis.