title: mobileOG-db: a manually curated database of protein families mediating the life cycle of bacterial mobile genetic elements

abstract: Currently available databases of bacterial mobile genetic elements (MGEs) contain both "core" and accessory MGE functional modules, the latter of which are often only transiently associated with the element. The presence of these accessory genes, which are often close homologs to primarily immobile genes, limits the usability of these databases for MGE annotation. To overcome this limitation, we analysed 10,776,212 protein sequences derived from seven MGE databases to compile a comprehensive database of 6,140 manually curated protein families that are linked to the "life cycle" (integration, excision, replication/recombination/repair, transfer, and stability/defense) of all major classes of bacterial MGEs. We overlay experimental information where available to create a tiered annotation scheme of high-quality annotations and annotations inferred exclusively through bioinformatic evidence. We additionally provide an MGE-class label for each entry (e.g., plasmid, integrative element) derived from the source database, and assign a list of keywords to each entry to delineate different MGE functional modules and to facilitate annotation. The resulting database, mobileOG-db (for mobile orthologous groups), provides a simple and readily interpretable foundation for an array of MGE-centred analyses. mobileOG-db can be accessed at mobileogdb.flsi.cloud.vt.edu/, where users can browse and design, refine, and analyse custom subsets of the dynamic mobilome.


title: Successive remodeling of IgG glycans using a solid-phase enzymatic platform

abstract:
The success of glycoprotein-based drugs in various disease treatments has become widespread. Frequently, therapeutic glycoproteins exhibit a heterogeneous array of glycans that are intended to mimic human glycopatterns. While immunogenic responses to biologic drugs are uncommon, enabling exquisite control of glycosylation with minimized microheterogeneity would improve their safety, efficacy and bioavailability. Therefore, close attention has been drawn to the development of glycoengineering strategies to control the glycan structures. With the accumulation of knowledge about the glycan biosynthesis enzymes, enzymatic glycan remodeling provides a potential strategy to construct highly ordered glycans with improved efficiency and biocompatibility. In this study, we quantitatively evaluate more than 30 enzymes for glycoengineering immobilized immunoglobulin G, an impactful glycoprotein class in the pharmaceutical field. We demonstrate successive glycan remodeling in a solid-phase platform, which enabled IgG glycan harmonization into a series of complex-type N-glycoforms with high yield and efficiency while retaining native IgG binding affinity.

SignificanceGlycosylation plays critical functional and structural roles in protein biology. However, our understanding of how discrete glycan structures affect protein behaviors remains extremely limited due to the naturally occurring microheterogeneity. Through the use of characterized glycoengineering enzyme combination, we report a solid-phase glycan remodeling (SPGR) platform that enables efficient IgG glycan harmonization into several glycoforms of interest with high biocompatibility to the substrates. It provides an efficient strategy to screen the biological behavior of distinct glycoforms, building a fundamental understanding of glycosylation.

category: biochemistry


title: Goal-Dependent Hippocampal Representations Facilitate Self-Control

abstract: Mnemonic influences on decision-making processes are important for linking past experiences and simulations of the future with current goals. The ways in which mnemonic information is represented may be especially critical in situations where one needs to overcome past rewarding experiences and exert self-control. We propose that self-control success or failure may depend on how information is retrieved from memory and how effectively this memory retrieval process can be modified to achieve a specific goal. We test our hypothesis using representational similarity analyses of human neuroimaging data during a dietary self-control task in which individuals must overcome taste temptations to choose healthy foods. We find that self-control is indeed associated with the way individuals represent taste information in the brain and how taste representations adapt to align with different goals. These results provide new insights into the processes leading to self-control success and indicate the need to update the classical view of self-control to continue to advance our understanding of its behavioral and neural underpinnings.

category: neuroscience


title: A Large-Scale Investigation of White Matter Microstructural Associations with Reading Ability

abstract: Reading involves the functioning of a widely distributed brain network, and white matter tracts are responsible for transmitting information between constituent network nodes. Several studies have analyzed fiber bundle microstructural properties to shed insights into the neural basis of reading abilities and disabilities. Findings have been inconsistent, potentially due to small sample sizes and varying methodology. To address this, we analyzed a large data set of 686 children ages 5-18 using state-of-the-art neuroimaging acquisitions and processing techniques. We searched for associations between fractional anisotropy (FA) and single-word and single-nonword reading skills in children with diverse reading abilities across multiple tracts previously thought to contribute to reading. We also looked for group differences in tract FA between typically reading children and children with reading disabilities. FA of the white matter increased with age across all participants. There were no significant correlations between overall reading abilities and tract FAs across all children, and no significant group differences in tract FA between children with and without reading disabilities. There were associations between FA and nonword reading ability in older children (ages 9 and above). Higher FA in the right superior longitudinal fasciculus (SLF) and left inferior cerebellar peduncle (ICP) correlated with better nonword reading skills. These results suggest that letter-sound correspondence skills, as measured by nonword reading, are associated with greater white matter coherence among older children in these two tracts, as indexed by higher FA.


title: NKG2D signaling regulates IL-17A-producing γδT cells to promote cancer progression

abstract: {gamma}{delta}T cells are unconventional T cells particularly abundant in mucosal tissues that play an important role in tissue surveillance and homeostasis. {gamma}{delta}T cell activation is mediated by the T cell receptor composed of {gamma} and {delta} chains, as well as activating receptors for stress-induced ligands, such as NKG2D. Contrary to the well-established anti-tumor function of {gamma}{delta}T cells, recent studies have shown that {gamma}{delta}T cells can promote tumor development in certain contexts. However, the mechanisms leading to this diseasepromoting role remain poorly understood. Here, we show that mice lacking {gamma}{delta}T cells survive longer in a mouse model of intestinal cancer, further supporting their pro-tumoral role. In a surprising conceptual twist, we found that these pro-tumor {gamma}{delta}T cells are regulated by NKG2D signaling, a receptor normally associated with cancer cell killing. Germline deletion of Klrk1, the gene encoding NKG2D, reduced the frequency of {gamma}{delta}T cells in the tumor microenvironment and delayed tumor progression. We further show that blocking NKG2D reduces the capability of {gamma}{delta}T cells to produce IL-17A in the pre-metastatic lung and that co-culture of lung T cells with NKG2D ligand-expressing tumor cells specifically increases the frequency of {gamma}{delta}T cells. Together, these data support the hypothesis that in a tumor microenvironment where NKG2D ligands are constitutively expressed, {gamma}{delta}T cells accumulate in an NKG2D-dependent manner and drive tumor progression by secreting pro-inflammatory cytokines, such as IL-17A.

category: immunology


title: Wear and Tear of the Intestinal Visceral Musculature by Intrinsic and Extrinsic Factors

abstract: The gut visceral musculature plays essential roles in not only moving substances through the lumen but also maintaining the function and physiology of the gut. Although the development of the visceral musculature has been studied in multiple model organisms, how it degenerates is poorly understood. Here, we employ the Drosophila midgut as a model to demonstrate that the visceral musculature is disrupted by intrinsic and extrinsic factors, such as aging, feeding, chemical-induced tissue damage, and oncogenic transformation in the epithelium. Notably, we define four prominent visceral musculature disruption phenotypes, which we refer as  sprout,  discontinuity,  furcation, and  crossover of the longitudinal muscle. Given that the occurrence of these phenotypes is increased during aging and under various stresses, we propose that these phenotypes can be used as quantitative readouts of deterioration of the visceral musculature. Intriguingly, administration of a tissue-damaging chemical dextran sulfate sodium (DSS) induced similar visceral musculature disruption phenotypes in zebrafish larvae, indicating that ingestion of a tissue-damaging chemical can disrupt the visceral musculature in a vertebrate as well. Our study provides insights into the deterioration of the gut visceral musculature and lays a groundwork for investigating the underlying mechanisms in Drosophila as well as other animals.

category: developmental biology


title: Best templates outperform homology models in predicting the impact of mutations on protein stability

abstract:
MotivationPrediction of protein stability change upon mutation ({Delta}{Delta}G) is crucial for facilitating protein engineering and understanding of protein folding principles. Robust prediction of protein folding free energy change requires the knowledge of protein three-dimensional (3D) structure. Unfortunately, protein 3D structure is not always available. In this case, one can still predict the protein stability change by constructing a homology model of the protein; however, the accuracy of homology model-based {Delta}{Delta}G predictions is unknown. The perspectives of using 3D structures of the best templates are also unclear.

ResultsTo investigate these questions, we used the most popular and accurate publicly available tools: FoldX for stability change prediction and I-Tasser for homology modeling. We found that both homology models and best templates worsen the {Delta}{Delta}G prediction, with best templates performing 1.5 times better than homology models. For AlphaFold models, we also found that the best templates seem to outperform protein models. Our findings imply using the 3D structures of the best templates for {Delta}{Delta}G prediction if the 3D protein structure is unavailable.

Contactd.ivankov@skoltech.ru

category: bioinformatics


title: Dissociating the Neural Correlates of Planning and Executing Hierarchical Task Sets

abstract: Task processing (e.g., the preparation and execution of responses) and task representation (e.g., the activation and maintenance of stimulus-response and context information) are two facets of cognitive control supported by lateral frontal cortex (LFC). However, the mechanistic overlap (or distinction) between these two facets is unknown. We explored this by combining a complex task mapping with a pre-cueing procedure. Participants made match/non-match judgments on pairs of stimuli during fMRI recording. Pre-cues on each trial gave variable amounts of information to the participant in anticipation of the stimulus. Our results demonstrated that regions throughout LFC were more active at the stimulus (when responses could be executed) than at the cue (when they could only be prepared), indicating that they supported execution of the task agnostic to the specific task representation. A subset of regions in left caudal LFC showed increased activity with more cue information at the cue and the reverse at the stimulus, suggesting their involvement in reducing uncertainty within the task representation. These results suggest that one component of task processing is preparing and executing the task according to the relevant representation, confined to left caudal LFC, while non-representational functions that occur primarily during execution are supported by different regions throughout the rest of LFC. We further conducted an exploratory investigation of connectivity between the two groups of regions in this study and their potential relationship to the fronto-parietal and cingulo-opercular networks. Regions with both patterns of activity appear to be part of the fronto-parietal network.


title: Dissociating the Neural Correlates of Planning and Executing Tasks with Nested Task Sets


title: The brain tunes to unfamiliar voices during sleeping

abstract:
The brain continues to respond selectively to environmental stimuli even during sleep. However, the functional role of such responses, and whether they reflect information processing or rather sensory inhibition is not fully understood.

Here, we presented 17 human sleepers (14 females) with their own name and two unfamiliar first names, spoken by either a familiar voice (FV) or an unfamiliar voice (UFV), while recording polysomnography during a full nights sleep. We detected K-complexes, sleep spindles, and micro-arousals, and then assessed event-related potentials, oscillatory power as well as intertrial phase synchronization in response to the different stimuli presented during non-rapid eye movement (NREM) sleep.

We show that UFVs evoke more K-complexes and micro-arousals than FVs. When both stimuli evoke a K-complex, we observed larger evoked potentials, higher oscillatory power in the high beta (>16Hz) frequency range, and stronger time-locking in the delta band (1-4 Hz) in response to UFVs relative to FVs. Crucially, these differences in brain responses disappear when no K-complexes are evoked by the auditory stimuli.

Our findings highlight discrepancies in brain responses to auditory stimuli based on their relevance to the sleeper and propose a key role for K-complexes in the modulation of sensory processing during sleep. We argue that such content-specific, dynamic reactivity to external sensory information enables the brain to enter a  sentinel processing mode in which it engages in the many important processes that are ongoing during sleep while still maintaining the ability to process vital information in the surrounding.

Significance statementPrevious research has shown that sensory processing continues during sleep. Here, we studied the capacity of the sleeping brain to extract and process relevant sensory information. We presented sleepers with their own names and unfamiliar names spoken by either a familiar (FV) or an unfamiliar voice (UFV). During non-rapid eye movement (NREM) sleep, UFVs elicited more K-complexes and micro-arousals than FVs. By contrasting stimuli which evoked K-complexes, we demonstrate that UFVs triggered larger evoked potentials, stronger time-locking in the delta (1-4Hz) band, and higher oscillatory power (>16Hz) relative to FVs. These differences in brain responses disappeared when no K-complexes were evoked. Our results suggest a pivotal role for K-complexes in the selective processing of relevant information during NREM sleep.


title: A functional network target for tic reduction during thalamic stimulation for Tourette Syndrome

abstract:
BackgroundDeep brain stimulation (DBS) of the medial thalamus is an evolving therapy for severe, treatment-refractory Tourette syndrome (TS). It remains unanswered which functionally connected networks need to be modulated to obtain optimal treatment results.

MethodsWe assessed treatment response of 15 patients with TS untergoing thalamic DBS six and twelve months postoperatively using the Yale Global Tic Severity Scale (YGTSS) tic score. For each time point, functional connectivity maps seeding from stimulation sites were calculated based on a normative functional connectome derived from 1000 healthy subjects. Resulting maps were analyzed in a voxel-wise mixed model for repeated measurements to identify patterns of connectivity associated with tic reduction.

ResultsConnectivity of stimulation to the medial frontal cortex, bilateral insulae and sensorimotor cortex was associated with tic reduction. Connectivity with the temporal lobe, cerebellum, ventral striatum and orbitofrontal cortex was negatively associated. The overall connectivity pattern was robust to leave-one-out cross-validation, explaining 25 % of outcome variance (R = 0.500; p = 0.005).

ConclusionsWe delineated a functional connectivity profile seeding from stimulation sites associated with TS-DBS outcome. This pattern comprised areas linked to the processing of premonitory urges and tic execution, thereby extending our current understanding of effective neuromodulation for TS.


title: Microenvironmental correlates of immune checkpoint inhibitor response in human melanoma brain metastases revealed by T cell receptor and single-cell RNA sequencing

abstract: Melanoma-derived brain metastases (MBM) represent an unmet clinical need due to central nervous system (CNS) progression as a frequent, end-stage site of disease. Immune checkpoint inhibition (ICI) represents a clinical opportunity against MBM; however, the MBM tumor microenvironment (TME) has not been fully elucidated in the context of ICI. To dissect unique MBM-TME elements and correlates of MBM-ICI response, we collected 32 fresh MBM and performed single cell RNA sequencing of the MBM-TME and T cell receptor clonotyping on T cells from MBM and matched blood and extracranial lesions. We observed myeloid phenotypic heterogeneity, most notably multiple distinct neutrophil states including an IL-8 expressing population that correlated with malignant cell epithelial-to-mesenchymal transition. Additionally, we observe significant relationships between intracranial T cell phenotypes and the distribution of T cell clonotypes intracranially and peripherally. We found that the phenotype, clonotype, and overall number of MBM-infiltrating T cells were associated with response to ICI, suggesting that ICI-responsive MBMs interact with peripheral blood in a manner similar to extracranial lesions. These data demonstrate unique features of the MBM-TME, which may represent potential targets to improve clinical outcomes for patients with MBM.

category: cancer biology


title: Using Continuous Directed Evolution to Improve Enzymes for Plant Applications

abstract:
Continuous directed evolution of enzymes and other proteins in microbial hosts is capable of outperforming classical directed evolution by executing hypermutation and selection concurrently in vivo, at scale, with minimal manual input. Provided that a target enzymes activity can be coupled to growth of the host cells, the activity can be improved simply by selecting for growth. Like all directed evolution, the continuous version requires no prior mechanistic knowledge of the target. Continuous directed evolution is thus a powerful new way to modify plant or non-plant enzymes for use in plant metabolic research and engineering. Here, we first describe the basic features of the Saccharomyces cerevisiae OrthoRep system for continuous directed evolution and compare it briefly with other systems. We then give a step-by-step account of three ways in which OrthoRep can be deployed to evolve primary metabolic enzymes, using a THI4 thiazole synthase as an example and illustrating the mutational outcomes obtained. We close by outlining applications of OrthoRep that serve growing demands (i) to change the characteristics of plant enzymes destined for return to plants, and (ii) to adapt ( plantize) enzymes from prokaryotes - especially exotic prokaryotes - to function well in mild, plant-like conditions.

One-sentence summaryContinuous directed evolution using the yeast OrthoRep system is a powerful new way to improve enzymes for use in plant engineering as illustrated by  plantizing a bacterial thiamin synthesis enzyme..

category: synthetic biology


title: Zika virus induces mitotic catastrophe in human neural progenitors by triggering unscheduled mitotic entry in the presence of DNA damage while functionally depleting nuclear PNKP

abstract:
Vertical transmission of Zika virus (ZIKV) leads with high frequency to congenital ZIKV syndrome (CZS), whose worst outcome is microcephaly. However, the mechanisms of congenital ZIKV neurodevelopmental pathologies, including direct cytotoxicity to neural progenitor cells (NPC), placental insufficiency, and immune responses, remain incompletely understood. At the cellular level, microcephaly typically results from death or insufficient proliferation of NPC or cortical neurons. NPCs replicate fast, requiring efficient DNA damage responses to ensure genome stability. Like congenital ZIKV infection, mutations in the polynucleotide 5-kinase 3-phosphatase (PNKP) gene, which encodes a critical DNA damage repair enzyme, results in recessive syndromes often characterized by congenital microcephaly with seizures (MCSZ). We thus tested whether there were any links between ZIKV and PNKP.

Here we show that two PNKP phosphatase inhibitors or PNKP knockout inhibited ZIKV replication. PNKP relocalized from the nucleus to the cytoplasm in infected cells, co-localizing with the marker of ZIKV replication factories (RF) NS1 and resulting in functional nuclear PNKP depletion. Although infected NPC accumulated DNA damage, they failed to activate the DNA damage checkpoint kinases Chk1 and Chk2. ZIKV also induced activation of cytoplasmic CycA/CDK1 complexes, which trigger unscheduled mitotic entry. Inhibition of CDK1 activity inhibited ZIKV replication and the formation of RF, supporting a role of cytoplasmic CycA/CDK1 in RF morphogenesis. In brief, ZIKV infection induces mitotic catastrophe resulting from unscheduled mitotic entry in the presence of DNA damage. PNKP and CycA/CDK1 are thus host factors participating in ZIKV replication in NPC, and pathogenesis to neural progenitor cells.

SignificanceThe 2015-2017 Zika virus (ZIKV) outbreak in Brazil and subsequent international epidemic revealed the strong association between ZIKV infection and congenital malformations, mostly neurodevelopmental defects up to microcephaly. The scale and global expansion of the epidemic, the new ZIKV outbreaks (Kerala state, India, 2021), and the potential burden of future ones pose a serious ongoing risk. However, the cellular and molecular mechanisms resulting in microcephaly remain incompletely understood. Here we show that ZIKV infection of neuronal progenitor cells results in cytoplasmic sequestration of an essential DNA repair protein itself associated with microcephaly, with the consequent accumulation of DNA damage, together with an unscheduled activation of cytoplasmic CDK1/Cyclin A complexes in the presence of DNA damage. These alterations result in mitotic catastrophe of neuronal progenitors, which would lead to a depletion of cortical neurons during development.

category: microbiology


title: Accuracy of mutual predictions of plant and microbe communities varies along a successional gradient in an alpine glacier forefield

abstract:
O_LIDue to climate warming, recently deglaciated glacier forefields create virtually uninhabited substrates waiting for initial colonization of bacteria, fungi and plants and serve as an ideal ecosystem for studying transformations in community composition and diversity over time and the interactions between taxonomic groups.
C_LIO_LIIn this study, we investigated the composition and diversity of bacteria, and fungi, plants and environmental factors (pH, temperature, plot age and soil nutrients) along a 1.5km glacier forefield. We used random forest analysis to detect how well the composition and diversity of taxonomic groups and environmental factors can be mutually predicted.
C_LIO_LICommunity composition and diversity of taxonomic groups predicted each other more accurately than environmental factors predicted the taxonomic groups; within the taxonomic groups bacteria and fungi predicted each other best and the taxas composition was better predicted than diversity indices. Additionally, accuracy of prediction among taxonomic groups and environmental factors considerably varied along the successional gradient.
C_LIO_LIAlthough our results are no direct indication of interactions between the taxa investigated and the environmental conditions, the accurate predictions among bacteria, fungi, and plants do provide insights into the concerted community assembly of different taxa in response to changing environments along a successional gradient.
C_LI

category: ecology


title: An integrated model for termination of RNA polymerase III transcription

abstract: RNA polymerase III (RNAPIII) synthesizes essential and abundant non-coding RNAs such as tRNAs. Controlling RNAPIII span of activity by accurate and efficient termination is a challenging necessity to ensure robust gene expression and to prevent conflicts with other DNA-associated machineries. The mechanism of RNAPIII termination is believed to be simpler than that of other eukaryotic RNA polymerases, solely relying on the recognition of a T-tract in the non-template strand. Here we combine high-resolution genome-wide analyses and in vitro transcription termination assays to revisit the mechanism of RNAPIII transcription termination in budding yeast. We show that T-tracts are necessary but not always sufficient for termination and that secondary structures of the nascent RNAs are important auxiliary cis-acting elements. Moreover, we show that the helicase Sen1 plays a key role in a fail-safe termination pathway. Our results provide a comprehensive model illustrating how multiple mechanisms cooperate to ensure efficient RNAPIII transcription termination.

category: genomics


title: Combining genetic resources and elite material populations to improve the accuracy of genomic prediction in apple

abstract: Genomic selection is an attractive strategy for apple breeding that could reduce the length of breeding cycles. A possible limitation to the practical implementation of this approach lies in the creation of a training set large and diverse enough to ensure accurate predictions. In this study, we investigated the potential of combining two available populations, i.e. genetic resources and elite material, in order to obtain a large training set with a high genetic diversity. We compared the predictive ability of genomic predictions within-population, across-population or when combining both populations, and tested a model accounting for population-specific marker effects in this last case. The obtained predictive abilities were moderate to high according to the studied trait and were always highest when the two populations were combined into a unique training set. We also investigated the potential of such a training set to predict hybrids resulting from crosses between the two populations, with a focus on the method to design the training set and the best proportion of each population to optimize predictions. The measured predictive abilities were very similar for all the proportions, except for the extreme cases where only one of the two populations was used in the training set, in which case predictive abilities could be lower than when using both populations. Using an optimization algorithm to choose the genotypes in the training set also led to higher predictive abilities than when the genotypes were chosen at random. Our results provide guidelines to initiate breeding programs that use genomic selection when the implementation of the training set is a limitation.


title: Light Potentials of Photosynthetic Energy Storage in the Field: What limits the ability to use or dissipate rapidly increased light energy?

abstract: The responses of plant photosynthesis to rapid fluctuations in environmental conditions are thought to be critical for efficient capture of light energy. Such responses are not well represented under laboratory conditions, but have also been difficult to probe in complex field environments. We demonstrate an open science approach to this problem that combines multifaceted measurements of photosynthesis and environmental conditions, and an unsupervised statistical clustering approach. In a selected set of data on mint (Mentha sp.), we show that the "light potential" for increasing linear electron flow (LEF) and nonphotochemical quenching (NPQ) upon rapid light increases are strongly suppressed in leaves previously exposed to low ambient PAR or low leaf temperatures, factors that can act both independently and cooperatively. Further analyses allowed us to test specific mechanisms. With decreasing leaf temperature or PAR, limitations to photosynthesis during high light fluctuations shifted from rapidly-induced NPQ to photosynthetic control (PCON) of electron flow at the cytochrome b6f complex. At low temperatures, high light induced lumen acidification, but did not induce NPQ, leading to accumulation of reduced electron transfer intermediates, a situation likely to induce photodamage, and represents a potential target for improving the efficiency and robustness of photosynthesis. Finally, we discuss the implications of the approach for open science efforts to understand and improve crop productivity.

category: plant biology


title: A promiscuity locus confers Lotus burttii nodulation with rhizobia from five different genera

abstract: Legumes acquire access to atmospheric nitrogen through nitrogen fixation by rhizobia in root nodules. Rhizobia are soil dwelling organisms and there is a tremendous diversity of rhizobial species in different habitats. From the legume perspective, host range is a compromise between the ability to colonize new habitats, where the preferred symbiotic partner may be absent, and guarding against infection by suboptimal nitrogen fixers. Here, we investigate natural variation in rhizobial host range across Lotus species. We find that Lotus burttii is considerably more promiscuous than Lotus japonicus, represented by the Gifu accession, in its interactions with rhizobia. This promiscuity allows Lotus burttii to form nodules with Mesorhizobium, Rhizobium, Sinorhizobium, Bradyrhizobium, and Allorhizobium species that represent five distinct genera. Using recombinant inbred lines, we have mapped the Gifu/burttii promiscuity QTL to the same genetic locus regardless of rhizobial genus, suggesting a general genetic mechanism for host-range expansion. The Gifu/burttii QTL now provides an opportunity for genetic and mechanistic understanding of promiscuous legume-rhizobia interactions.


title: Genome survey sequencing of wild cotton (Gossypium robinsonii) reveals insights into proteomic responses of pollen to extreme heat

abstract: Heat stress specifically affects fertility by impairing pollen viability but cotton wild relatives successfully reproduce in hot savannas where they evolved. An Australian heat-tolerant cotton (Gossypium robinsonii) was exposed to heat events during pollen development, then mature pollen was subjected to deep proteomic analysis using 57,023 predicted genes from a genomic database we assembled for the same species. Three stages of pollen development, including tetrads, uninucleate and binucleate microspores were exposed to 36{degrees}C or 40{degrees}C for 5 d and the resulting mature pollen was collected at anthesis (p-TE, p-UN and p-BN, respectively). Using SWATH-MS proteomic analysis, 2,704 proteins were identified and quantified across all pollen samples analyzed. Proteins predominantly decreased in abundance at all stages in response to heat, particularly after exposure of tetrads to 40{degrees}C. Functional enrichment analyses demonstrated that extreme heat increased the abundance of proteins that contributed to increased mRNA splicing via spliceosome, initiation of cytoplasmic translation and protein refolding in p-TE40. However, other functional categories that contributed to intercellular transport were inhibited in p-TE40, linked potentially to Rab proteins. We ascribe the resilience of reproductive processes in G. robinsonii at temperatures up to 40{degrees}C, relative to commercial cotton, to a targeted reduction in protein transport.


title: Multiplexed and reproducible high content screening of live and fixed cells using the Dye Drop method

abstract: High throughput measurement of cell perturbation, by libraries of small molecules or gene knockouts, is a key step in functional genomics and pre-clinical drug development. However, it is difficult to perform viable, single-cell assays in 384-well plates, limiting many studies to simple well-average measurements (e.g. CellTiter-Glo(R)). Here we describe a public domain "Dye Drop" method in which sequential density displacement is used to perform multi-step assays for cell viability and EdU incorporation followed by immunofluorescence imaging. The method is rapid, reproducible, can be readily customized, and is compatible with either manual or automated laboratory equipment. We demonstrate Dye Drop in the collection of dose-response data for 67 drugs in 58 breast cancer cell lines and separate cytostatic and cytotoxic responses, thereby providing new insight into the effects of specific drugs on cell cycle progression and cell viability. Dye Drop substantially improves the tradeoff between data content and cost, enabling collection of large information-rich datasets.

category: systems biology


title: Comparative analysis of mandible morphology in four ant species with different foraging and nesting habits

abstract: Mandibles of ants can be considered as one of the most vital tools for the survival and success of their colonies since these are extensively utilised for defence, nest maintenance and foraging activities. We hypothesised that mandibular design is strongly dependent on the respective ecological niche and foraging habit of an ant species. In the present study, we compared the external morphology and zinc content in the mandibles of four species of ants by using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). The ant mandible morphology varied significantly in accordance with their species-specific foraging and nesting strategies. The sickle-shaped mandibles of the strongly predaceous, Oecophylla smaragdina worker ants were characterised by a large number of pointed teeth which would be of immense utility for subduing the prey, while the shovel-shaped, highly sclerotized mandibles of Cataglyphus longipedem ants appear to be adaptations for the solitary scavenging habit and nest maintenance in arid habitats. The large-sized mandibles of Camponotus compressus ants and the stout mandibles of the predatory, Tetraponera. rufonigra forager ants, have apparently evolved for collection of sugary secretions by the former and for the solitary foraging and arboreal nesting habit of the latter. The mandibular zinc content was highest in T. rufonigra ants and the lowest in the mandibles of the sugar-loving Cm. compressus ants. The diversity in the arrangement of bristles and the type of mandibular concavities, have also evolved accordingly. Thus, this study might prove to be instrumental in evaluating the various physical mechanisms involved in the evolution of insect mandibles for their defined function.

category: zoology


title: Recurrent de novo single point mutation on the gene encoding Na+ /K+ pump results in epilepsy

abstract: The etiology of epilepsy remains undefined in two-thirds of patients. Here, we identified a de novo mutation of ATP1A2 (c.2426 T>G, p.Leu809Arg), which encodes the 2 subunit of Na+/K+-ATPase, from a family with idiopathic epilepsy. This mutation caused seizures in the study patients. We generated the point mutation mouse model Atp1a2L809R, which recapitulated the epilepsy observed in the study patients. In Atp1a2L809R/WT mice, convulsions were observed and cognitive and memory function was impaired. This mutation affected the potassium binding function of the protein, disabling its ion transport ability, thereby increasing the frequency of nerve impulses. Our work revealed that ATP1A2L809R mutations cause a predisposition to epilepsy. Moreover, we first provide a point mutation mouse model for epilepsy research and drug screening.


title: Extensive incorporation, polarisation and improved maturation of transplanted human cones in a murine cone degeneration model

abstract:
Once human photoreceptors die, they do not regenerate, thus photoreceptor transplantation has emerged as a potential treatment approach for blinding diseases. Improvements in transplant organization, donor cell maturation and synaptic connectivity to the host will be critical in advancing this technology to clinical practice. Unlike the unstructured grafts of prior cell suspension transplantations into end-stage degeneration models, we describe extensive incorporation of iPSC retinal organoid-derived human photoreceptors into mice with cone dysfunction. This incorporative phenotype was validated in both cone-only as well as pan-photoreceptor transplantations. Rather than forming a glial barrier, Muller cells extend throughout the graft, even forming a common outer limiting membrane. Donor-host interaction appears to promote polarisation as well as development of morphological features critical for light detection, namely formation of inner and well stacked outer segments oriented towards the RPE. Putative synapse formation and graft function is evident both at a structural and electrophysiological level. Overall, these results show that human photoreceptors interact readily with a partially degenerated retina. Moreover, incorporation into the host retina appears to be beneficial to graft maturation, polarisation and function.

HighlightsO_LIGeneration of the first human iPSC cone reporter line
C_LIO_LIHuman cones extensively incorporate into the retina of mice with cone degeneration
C_LIO_LIDonor cone age and time in vivo are important factors for transplant incorporation
C_LIO_LIIncorporation into the host retina correlates with graft polarisation
C_LIO_LIImproved photoreceptor maturation after transplantation in vivo vs. in vitro
C_LIO_LIRe-establishment of cone-mediated light-responses in the cone deficient mouse
C_LI


title: Towards Decoding Selective Attention Through Cochlear Implant Electrodes as Sensors in Subjects with Contralateral Acoustic Hearing

abstract:
ObjectivesFocusing attention on one speaker in a situation with multiple background speakers or noise is referred to as auditory selective attention. Decoding selective attention is an interesting line of research with respect to future brain-guided hearing aids or cochlear implants (CIs) that are designed to adaptively adjust sound processing through cortical feedback loops. This study investigates the feasibility of using the electrodes and backward telemetry of a CI to record electroencephalography (EEG).

ApproachThe study population included 6 normal-hearing (NH) listeners and 5 CI users with contralateral acoustic hearing. Cortical auditory evoked potentials (CAEP) and selective attention were recorded using a state-of-the-art high-density scalp EEG and, in the case of CI users, also using two CI electrodes as sensors in combination with the backward telemetry system of these devices (iEEG).

Main resultsThe peak amplitudes of the CAEPs recorded with iEEG were lower and the latencies were higher than those recorded with scalp EEG. In the selective attention paradigm with multi-channel scalp EEG the mean decoding accuracy across subjects was 92.0 and 92.5% for NH listeners and CI users, respectively. With single-channel scalp EEG the accuracy decreased to 65.6 and to 75.8% for NH listeners and CI users, respectively, and was above chance level in 9 out of 11 subjects. With the single-channel iEEG, the accuracy for CI users decreased to 70% and was above chance level in 3 out of 5 subjects.

SignificanceThis study shows that single-channel EEG is suitable for auditory selective attention decoding, even though it reduces the decoding quality compared to a multi-channel approach. CI-based iEEG can be used for the purpose of recording CAEPs and decoding selective attention. However, the study also points out the need for further technical development for the CI backward telemetry regarding long-term recordings and the optimal sensor positions.


title: Gene signature of circulating platelet-bound neutrophils is associated with poor prognosis in cancer patients

abstract:
Beyond their critical role in hemostasis, platelets physically interact with neutrophils to form neutrophil-platelet aggregates (NPAs), enhancing neutrophil effector functions during inflammation. NPAs may also promote disease worsening in various inflammatory diseases. However, characterization of NPAs in cancer remains totally unexplored. Using ImageStream(R)X (ISX) imaging flow cytometer, we were not only allowed able to detect CD15+ CD14- CD36+ ITGA2B+ NPAs in both healthy donors (HDs) and cancer patients bloods, but we also showed that NPAs result from the binding of platelets preferentially to low-density neutrophils (LDNs) as opposed to normal-density neutrophils (NDNs). By re-analyzing two independent public scRNAseq data of whole blood leukocytes from cancer patients and HDs, we could identify a subset of neutrophils with high platelet gene expression that may correspond to NPAs. Moreover, we showed that cancer patients derived NPAs possessed a distinct molecular signature compared with the other neutrophil subsets, independently of platelet genes. Gene ontology (GO) term enrichment analysis of this NPAs-associated neutrophil transcriptomic signature revealed a significant enrichment of neutrophil degranulation, chemotaxis and trans-endothelial migration GO terms. Lastly, using The Cancer Genome Atlas (TCGA), we could show by multivariate Cox analysis that the NPAs-associated neutrophil transcriptomic signature was associated with a worse patient prognosis in several cancer types. These results suggest that neutrophils from NPAs are systemically primed by platelets empowering them with cancer progression capacities once at tumor site. NPAs may therefore hold clinical utility as novel non-invasive blood prognostic biomarker in cancer patients with solid tumors.

Novelty and ImpactPlatelets physically interact with peripheral blood neutrophils to form neutrophil-platelet aggregates (NPAs), known to promote disease worsening in various inflammatory diseases. However, characterization of NPAs in cancer remains totally unexplored. We showed that NPAs-associated neutrophils were a yet-unreported unique subset of circulating neutrophils associated with a worse patient prognosis in several cancer types. NPAs may hold clinical utility as novel non-invasive blood prognostic biomarker in cancer patients with solid tumors.


title: Multiplex immunohistochemistry differences between Q fever and atherosclerotic abdominal aortic aneurysms indicate immune suppression

abstract:
BackgroundChronic Q fever is a zoonosis caused by the bacterium Coxiella burnetii which can manifest as infection of an abdominal aortic aneurysm (AAA). Antibiotic therapy often fails, resulting in severe morbidity and high mortality. Whereas previous studies have focused on inflammatory processes in blood, the aim of this study was to investigate local inflammation in aortic tissue.

MethodsMultiplex immunohistochemistry was used to investigate local inflammation in Q fever AAAs compared to atherosclerotic AAAs in aorta tissue specimen. Two six-plex panels were used to study both the innate and adaptive immune system.

ResultsQ fever AAAs and atherosclerotic AAAs contained similar numbers of CD68+ macrophages and CD3+ T cells. However, in Q fever AAAs the number of CD68+CD206+ M2 macrophages was increased, while expression of GM-CSF was decreased compared to atherosclerotic AAAs. Furthermore, Q fever AAAs showed an increase in both the number of CD8+ cytotoxic T cells and CD3+FoxP3+ regulatory T cells. Lastly, Q fever AAAs did not contain any well-defined granulomas.

ConclusionsThese findings demonstrate that despite the presence of pro-inflammatory effector cells, there is an immune suppressive micro environment in Q fever AAA resulting in persistent local infection with C. burnetii.


title: The thymic microenvironment gradually modulates the phenotype of thymus-homing peripheral conventional dendritic cells

abstract: Thymic conventional dendritic cells (t-DCs) are crucial for the development of T cells. A substantial fraction of t-DCs originates extrathymically and migrates to the thymus. Here, these cells contribute to key processes of central tolerance like the clonal deletion of self-reactive thymocytes and the generation of regulatory T (Treg) cells. So far, it is only incompletely understood which impact the thymic microenvironment has on thymus-homing conventional DCs (cDCs), which phenotypic changes occur after the entry of peripheral cDCs into the thymus and which functional properties these modulated cells acquire. In the present study, we mimicked the thymus-homing of peripheral cDCs by introducing ex vivo isolated splenic cDCs (sp-DCs) into re-aggregated thymic organ cultures (RTOCs). Already after two days of culture, the transcriptomic profile of sp-DCs was modulated and had acquired certain key signatures of t-DCs. The regulated genes included immunomodulatory cytokines and chemokines as well as co-stimulatory molecules. After four days of culture, sp-DCs appeared to have at least partially acquired the peculiar Treg cell-inducing capacity characteristic of t-DCs. Taken together, our findings indicate that peripheral cDCs possess a high degree of plasticity enabling them to quickly adapt to the thymus-specific microenvironment. We further provide indirect evidence that thymus-specific properties such as the efficient induction of Treg cells under homeostatic conditions can be partially transferred to thymus-homing peripheral cDC subsets.


title: Host lung environment limits Aspergillus fumigatus germination through a SskA-dependent signaling response

abstract:
Aspergillus fumigatus isolates display significant heterogeneity in growth, virulence, pathology, and inflammatory potential in multiple murine models of invasive aspergillosis. Previous studies have linked the initial germination of a fungal isolate in the airways to the inflammatory and pathological potential; but the mechanism(s) regulating A. fumigatus germination in the airways are unresolved. To explore the genetic basis for divergent germination phenotypes, we utilized a serial passaging strategy in which we cultured a slow germinating strain (AF293) in a murine lung based medium for multiple generations. Through this serial passaging approach, a strain emerged with an increased germination rate that induces more inflammation than the parental strain (herein named Lung Homogenate Evolved (LH-EVOL)). We identified a potential loss of function allele of Afu5g08390 (sskA) in the LH-EVOL strain. The LH-EVOL strain had a decreased ability to induce the SakA-dependent stress pathway, similar to AF293 {Delta}sskA and CEA10. In support of the whole genome variant analyses, sskA, sakA, or mpkC loss of function strains in the AF293 parental strain increased germination both in vitro and in vivo. Since the airway surface liquid of the lungs contains low glucose levels, the relationship of low glucose concentration on germination of these mutant AF293 strains was examined; interestingly, in low glucose conditions the sakA pathway mutants exhibited an enhanced germination rate. In conclusion, A. fumigatus germination in the airways is regulated by SskA through the SakA MAPK pathway and drives enhanced disease initiation and inflammation in the lungs.

IMPORTANCEAspergillus fumigatus is an important human fungal pathogen particularly in immunocompromised individuals. Initiation of growth by A. fumigatus in the lung is important for its pathogenicity in murine models. However, our understanding of what regulates fungal germination in the lung environment is lacking. Through a serial passage experiment using lung-based medium, we identified a new strain of A. fumigatus which has increased germination potential and inflammation in the lungs. Using this serially passaged strain we found it had a decreased ability to mediate signaling through the osmotic stress response pathway. This finding was confirmed using genetic null mutants demonstrating that the osmotic stress response pathway is critical for regulating growth in the murine lungs. Our results contribute to the understanding of A. fumigatus adaptation and growth in the host lung environment.


title: Enhancement of the immunogenicity of a Mycobacterium tuberculosis fusion protein using ISCOMATRIX and PLUSCOM nano-adjuvants after nasal administration in mice

abstract:
BackgroundTuberculosis (TB), a contagious disease caused by Mycobacterium tuberculosis (M. tuberculosis), remains a health problem worldwide and this infection has the highest mortality rate among bacterial infections. Current studies suggest that intranasal administration of new tuberculosis vaccines could enhance the immunogenicity of M. tuberculosis antigens. Hence, we aim to evaluate the protective efficacy and immunogenicity of HspX/EsxS fusion protein of M. tuberculosis along with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA through the intranasal administration in mice model.

MethodsIn present study, the recombinant fusion protein was expressed in Escherichia coli and purified and used to prepare different nanoparticle formulations in combination with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA. Mice were intranasally vaccinated with each formulation three times at an interval of 2 weeks. Finally, IFN-{gamma}, IL-4. IL-17 and TGF-{beta} concentration in supernatant of cultured splenocytes of vaccinated mice as well as serum titers of IgG1 and IgG2a and sIgA titers in nasal lavage were determined.

ResultsAccording to obtained results, intranasally vaccinated mice with formulations containing ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA could effectively induced IFN-{gamma} and sIgA responses. Moreover, both HspX/EsxS/ISCOMATRIX/MPLA and HspX/EsxS/PLUSCOM/MPLA and their BCG booster formulation could strongly stimulate the immune system and enhance the immunogenicity of M. tuberculosis antigens.

ConclusionThe results demonstrate the potential of HspX/EsxS-fused protein in combination with ISCOMATRIX, PLUSCOM and MPLA after nasal administration in enhancing immune response against of M. tuberculosis antigens. So, nasal immunization with these formulations, could induce immune responses and considered as new TB vaccine or as BCG booster.


title: Evolutionary analyses of visual opsin genes in anurans reveals diversity and positive selection suggestive of functional adaptation to distinct light environments

abstract: Among major vertebrate groups, anurans (frogs and toads) are understudied with regards to their visual systems and little is known about variation among species that differ in ecology. We sampled North American anurans representing diverse evolutionary and life histories that likely possess visual systems adapted to meet different ecological needs. Using standard molecular techniques, visual opsin genes, which encode the protein component of visual pigments, were obtained from anuran retinas. Additionally, we extracted the visual opsins from publicly available genome and transcriptome assemblies, further increasing the phylogenetic and ecological diversity of our dataset. We found that anurans consistently express four visual opsin genes (RH1, LWS, SWS1, and SWS2, but not RH2) even though reported photoreceptor complements vary widely among species. We found the first evidence of visual opsin duplication in an amphibian with the duplication of the LWS gene in the African bullfrog, which had distinct LWS copies on the sex chromosomes. The proteins encoded by these genes showed considerable sequence variation among species, including at sites known to shift the spectral sensitivity of visual pigments in other vertebrates and thus mediate dim-light and color vision. Using molecular evolutionary analyses of selection (dN/dS) we found significant evidence for positive selection at a subset of sites in the dim-light rod opsin gene RH1 and the long wavelength sensitive cone opsin gene LWS. The function of sites inferred to be under positive selection are largely unknown, but a few are likely to affect spectral sensitivity and other visual pigment functions based on proximity to previously identified sites in other vertebrates. The observed variation cannot fully be explained by evolutionary relationships among species alone. Taken together, our results suggest that other ecological factors, such as habitat and life history, as well as behaviour, may be driving changes to anuran visual systems.

category: evolutionary biology


title: What do we gain when tolerating loss? The information bottleneck, lossy compression, and detecting horizontal gene transfer

abstract: Most microbes have the capacity to acquire genetic material from their environment. Recombination of foreign DNA yields genomes that are, at least in part, incongruent with the vertical history of their species. Dominant approaches for detecting such horizontal gene transfer (HGT) and recombination are phylogenetic, requiring a painstaking series of analyses including sequence-based clustering, alignment, and phylogenetic tree reconstruction. Given the breakneck pace of genome sequencing, these traditional pan-genomic methods do not scale. Here we propose an alignment-free and tree-free technique based on the sequential information bottleneck (SIB), an optimization procedure designed to extract some portion of relevant information from one random variable conditioned on another. In our case, this joint probability distribution tabulates occurrence counts of k-mers with respect to their genomes of origin (the relevance information) with the expectation that HGT and recombination will create a strong signal that distinguishes certain sets of co-occuring k-mers. The technique is conceptualized as a rate-distortion problem. We measure distortion in the relevance information as k-mers are compressed into clusters based on their co-occurrence in the source genomes. This approach is similar to topic mining in the Natural Language Processing (NLP) literature. The result is model-free, unsupervised compression of k-mers into genomic topics that trace tracts of shared genome sequence whether vertically or horizontally acquired. We examine the performance of SIB on simulated data and on the known large-scale recombination event that formed the Staphylococcus aureus ST239 clade. We use this technique to detect recombined regions and recover the vertically inherited core genome with a fraction of the computing power required of current phylogenetic methods.


title: Data-driven, participatory characterization of traditional farmer varieties discloses teff (Eragrostis tef) adaptive and breeding potential under current and future climates

abstract: In smallholder farming systems, traditional farmer varieties of neglected and underutilized species (NUS) support the livelihoods of millions of growers and consumers. NUS combine cultural and agronomic value with local adaptation and transdisciplinary methods are needed to fully evaluate their breeding potential. Here, we assembled and characterized the genetic diversity of a representative collection of 366 Ethiopian teff (Eragrostis tef) farmer varieties and breeding materials, describing their phylogenetic relation and local adaptation on the Ethiopian landscape. We phenotyped the collection for its agronomic performance, involving local farmers expert teff growers in a participatory variety evaluation. Our analyses revealed environmental patterns of teff genetic diversity and allowed us to identify ten genetic clusters associated with climate variation and with uneven spatial distribution. A genome wide association study (GWAS) was used to identify loci and candidate genes related to phenology, yield, local adaptation, and farmers appreciation. The estimated teff genomic offset under climate change scenarios highlighted an area around lake Tana where teff cropping may be most vulnerable to climate change. Our results show that transdisciplinary approaches may efficiently propel untapped NUS farmer varieties into modern breeding to foster more resilient and sustainable cropping systems.

category: genetics


title: PySeq2500: An open source toolkit for repurposing HiSeq 2500 sequencing systems as versatile fluidics and imaging platforms.

abstract: Fluorescence microscopy is a key method in the life sciences. State of the art -omics methods combine fluorescence microscopy with complex protocols to visualize tens to thousands of features in each of millions of pixels across samples. These -omics methods require precise control of temperature, reagent application, and image acquisition parameters during iterative chemistry and imaging cycles conducted over the course of days or weeks. Automated execution of such methods enables robust and reproducible data generation. However, few commercial solutions exist for temperature controlled, fluidics coupled fluorescence imaging, and implementation of bespoke instrumentation requires specialized engineering expertise. Here we present PySeq2500, an open source Python code base and flow cell design that converts the Illumina HiSeq 2500 instrument into an open platform for programmable applications. Customizable PySeq2500 protocols enable experimental designs involving simultaneous 4-channel image acquisition, temperature control, reagent exchange, stable positioning, and sample integrity over extended experiments. To demonstrate accessible automation of complex, multi-day workflows, we use the PySeq2500 system for unattended execution of iterative indirect immunofluorescence imaging (4i). Our automated 4i method uses off-the-shelf antibodies over multiple cycles of staining, imaging, and antibody elution to build highly multiplexed maps of cell types and pathological features in mouse and postmortem human spinal cord sections. As demonstrated here, PySeq2500 enables non-specialists to develop and implement state of the art fluidics coupled imaging methods in a widely available benchtop system.


title: Allele expression biases in mixed-ploid sugarcane accessions

abstract: Allele-specific expression (ASE) represents differences in the magnitude of expression between alleles of the same gene. This is not straightforward for polyploids, especially autopolyploids, as knowledge about the dose of each allele is required for accurate estimation of ASE. This is the case for the genomically complex Saccharum species, characterized by high levels of ploidy and aneuploidy. We used a Beta-Binomial model to test for allelic imbalance in Saccharum, with adaptations for mixed-ploid organisms. The hierarchical Beta-Binomial model was used to test if allele expression followed the expectation based on genomic allele dosage. The highest frequencies of ASE occurred in sugarcane hybrids, suggesting a possible influence of interspecific hybridization in these genotypes. For all accessions, ASEGs were less frequent than those with balanced allelic expression. These genes were related to a broad range of processes, mostly associated with general metabolism, organelles, responses to stress and responses to stimuli. In addition, the frequency of ASEGs in high-level functional terms was similar among the genotypes, with a few genes associated with more specific biological processes. We hypothesize that ASE in Saccharum is largely a genotype-specific phenomenon, as a large number of ASEGs were exclusive to individual accessions.


title: Hi-C-LSTM: Learning representations of chromatin contacts using a recurrent neural network identifies genomic drivers of conformation

abstract: Despite the availability of chromatin conformation capture experiments, understanding the relationship between regulatory elements and conformation remains a challenge. We propose Hi-C-LSTM, a method that produces low-dimensional latent representations that summarize intra-chromosomal Hi-C contacts via a recurrent long short-term memory (LSTM) neural network model. We find that these representations contain all the information needed to recreate the original Hi-C matrix with high accuracy, outperforming existing methods. These representations enable the identification of a variety of conformation-defining genomic elements, including nuclear compartments and conformation-related transcription factors. They furthermore enable in-silico perturbation experiments that measure the influence of cis-regulatory elements on conformation.


title: The genome sequence of Sorbus pohuashanensis provides insights into population evolution and leaf sunburn response

abstract: Sorbus pohuashanensis is a potential horticulture and medicinal plant, but its genomic and genetic background remains unknown. Here, we de novo sequenced and assembled the S. pohuashanensis (Hance) Hedl. reference genome using PacBio long reads. Based on the new reference genome, we resequenced a core collection of 22 Sorbus spp. samples, which were divided into two groups (G1 and G2) based on phylogenetic and PCA analysis. These phylogenetic clusters were highly in accordance with the classification based on leaf shape. Natural hybridization between the G1 and G2 groups was evidenced by a sample (R21) with a highly heterozygous genotype. Nucleotide diversity ({pi}) analysis showed that G1 has a higher diversity than G2, and that G2 originated from G1. During the evolution process, the gene families involved in photosynthesis pathways expanded and gene families involved in energy consumption contracted. Comparative genome analysis showed that S. pohuashanensis has a high level of chromosomal synteny with Malus domestica and Pyrus communis. RNA-seq data suggested that flavonol biosynthesis and heat-shock protein (HSP)-heat-shock factor (HSF) pathways play important roles in protection against sunburn. This research provides new insight into the evolution of Sorbus spp. genomes. In addition, the genomic resources and the identified genetic variations, especially those genes related to stress resistance, will help future efforts to introduce and breed Sorbus spp.


title: Mapping of single-cell landscape of acral melanoma and analysis of molecular regulatory network of tumor microenvironment

abstract: Melanoma is a type of skin malignant tumor with high invasiveness, high metastasis, and poor prognosis. The incidence of melanoma continues to increase. Among them, the subtype of acral melanoma (AM) is more common in Asian populations. AM has higher degree, low immunotherapy response rate. With the help of single-cell sequencing technology provides new technical means for tumor microenvironment research, so that we can more easily explore specific tumor types suitable immunotherapy targets. However, no complete single-cell level differentiation map exists for the AM tumor microenvironment (TME). In this study, we used AM related sample and used the 10x Genomics single-cell transcriptome platform to draw a specific single-cell map of AM, understand the cell composition of AM, and analyze the interaction and molecular regulation of AM TME. Nine cell types were identified, of which malignant cells accounted for the largest proportion, followed by fibroblasts. And the cell interaction network shows that malignant cells, macrophages, B, T and fibroblasts play an important role in AM TME. Our research provides systematic theoretical guidance for the diagnosis and treatment of acral melanoma.


title: Long-Read Sequencing of the Zebrafish Genome Reorganizes Genomic Architecture

abstract: Nanopore sequencing technology has revolutionized the field of genome biology with its ability to generate extra-long reads that can resolve regions of the genome that were previously inaccessible to short-read sequencing platforms. Although long-read sequencing has been used to resolve several vertebrate genomes, a nanopore-based zebrafish assembly has not yet been released. Over 50% of the zebrafish genome consists of difficult to map, highly repetitive, low complexity elements that pose inherent problems for short-read sequencers and assemblers. We used nanopore sequencing to improve upon and resolve the issues plaguing the current zebrafish reference assembly (GRCz11). Our long-read assembly improved the current resolution of the reference genome by identifying 1,697 novel insertions and deletions over 1Kb in length and placing 106 previously unlocalized scaffolds. We also discovered additional sites of retrotransposon integration previously unreported in GRCz11 and observed their expression in adult zebrafish under physiologic conditions, implying they have active mobility in the zebrafish genome and contribute to the ever-changing genomic landscape.


title: Methylation at CpG sites in genomes of aphid Acyrtosiphon pisum and its endosymbiont Buchnera

abstract: Pea aphid Acyrtosiphon pisum, a sap-feeding insect, has established a mutualistic relationship with an endosymbiotic bacteria (Buchnera aphidicola) that constitutes an evolutionary successful symbiosis to synthetize complex chemical compounds from a nutrient deprived diet. In this study, led by the presence of DNMT1 and a putative DNMT3 methylase in the aphid genome, we investigated the distribution of the methyl groups on 5cytosine in CpG motifs on the whole genomes of host and endosymbiont, and looked into their correlation with gene expression. The DNA methylation turned to be present at low level in aphid (around 3% of total genomic cytosine) compared to mammals and plants, but increased to [~]9% in genes. Interestingly, the reduced genome of the endosymbiont Buchnera also shows global low level of methyl cytosine despite the fact that its genome does not shelter any DNA methylase. This finding argues for the import of DNA methylase from the host to the endosymbiont. The observed differences in methylation patterns between two clonal variants (host plus endosymbiont) are reported along with the differences in their transcriptome profiles. Our data allowed to decipher a dynamic combinatorial DNA methylation and epigenetic cross talk between host and symbiont in a clonality context that might count for the aphid adaptation to environment.


title: Multi-omic brain and behavioral correlates of cell-free fetal DNA methylation in macaque maternal obesity models

abstract: Maternal obesity during pregnancy is associated with neurodevelopmental disorder (NDD) risk. We utilized integrative multi-omics to examine maternal obesity effects on offspring neurodevelopment in rhesus macaques by comparison to lean controls and two interventions. Differentially methylated regions (DMRs) from longitudinal maternal blood-derived cell-free fetal DNA (cffDNA) significantly overlapped with DMRs from infant brain. The DMRs were enriched for neurodevelopmental functions, methylation-sensitive developmental transcription factor motifs, and human NDD DMRs identified from brain and placenta. Brain and cffDNA methylation levels from a large region overlapping mir-663 correlated with maternal obesity, metabolic and immune markers, and infant behavior. A DUX4 hippocampal co-methylation network correlated with maternal obesity, infant behavior, infant hippocampal lipidomic and metabolomic profiles, and maternal blood measurements of DUX4 cffDNA methylation, cytokines, and metabolites. Ultimately, maternal obesity altered infant brain and behavior, and these differences were detectable in pregnancy through integrative analyses of cffDNA methylation with immune and metabolic biomarkers.


title: Combined use of Oxford Nanopore and Illumina sequencing yields insights into soybean structural variation biology

abstract:
BackgroundStructural variant (SV) discovery based on short reads is challenging due to their complex signatures and tendency to occur in repeated regions. The increasing availability of long-read technologies has greatly facilitated SV discovery, however these technologies remain too costly to apply routinely to population-level studies. Here, we combined short-read and long-read sequencing technologies to provide a comprehensive population-scale assessment of structural variation in a panel of Canadian soybean cultivars.

ResultsWe used Oxford Nanopore sequencing data ([~]12X mean coverage) for 17 samples to both benchmark SV calls made from the Illumina data and predict SVs that were subsequently genotyped in a population of 102 samples using Illumina data. Benchmarking results show that variants discovered using Oxford Nanopore can be accurately genotyped from the Illumina data. We first use the genotyped SVs for population structure analysis and show that results are comparable to those based on single-nucleotide variants. We observe that the population frequency and distribution within the genome of SVs are constrained by the location of genes. Gene Ontology and PFAM domain enrichment analyses also confirm previous reports that genes harboring high-frequency SVs are enriched for functions in defense response. Finally, we discover polymorphic transposable elements from the SVs and report evidence of the recent activity of a Stowaway MITE.

ConclusionsOur results demonstrate that long-read and short-read sequencing technologies can be efficiently combined to enhance SV analysis in large populations, providing a reusable framework for their study in a wider range of samples and non-model species.


title: Dietary methionine restriction impairs anti-tumor immunity through gut microbiota

abstract: Dietary methionine restriction has been reported to repress cancer growth and improve therapeutic responses in several pre-clinical settings. However, how this dietary intervention impacts cancer progression in the context of the intact immune system is unknown. Here we report that methionine restriction exacerbates cancer growth and influences the outcomes of anti-tumor immunotherapy through gut microbiota and immune suppression in immunocompetent settings. Methionine restriction reduces T cell activation, increases tumor growth, and impairs response to anti-tumor immunotherapy. Mechanistically, methionine restriction alters composition of gut microbiota and reduces microbial production of hydrogen sulfide. Fecal transplantation from methionine-restricted tumor-free animals is sufficient to repress T cell activation and enhance tumor growth in tumor-bearing recipient mice. Conversely, dietary supplementation of a hydrogen sulfide donor or methionine stimulates anti-tumor immunity and suppresses tumor progression. Our findings reveal a vital role of gut microbiota in mediating methionine restriction-induced suppression of anti-tumor immunity and suggest that any possible anti-cancer benefits of methionine restriction require careful considerations of both the microbiota and the immune system.


title: Fluorescence lifetime imaging as an in situ and label-free readout for the chemical composition of lignin

abstract: Important structures and functions within living organisms rely on naturally fluorescent polymeric molecules such as collagen, keratin, elastin, resilin, or lignin. Theoretical physics predict that fluorescence lifetime of these polymers is related to their chemical composition. We verified this prediction for lignin, a major structural element in plant cell walls and one of the most abundant components of wood. Lignin is composed of different types of phenylpropanoid units, and its composition affects its properties, biological functions, and the utilization of wood biomass. We carried out fluorescence lifetime imaging microscopy (FLIM) measurements of wood cell wall lignin in a population of 90 hybrid aspen trees genetically engineered to display differences in cell wall chemistry and structure. We also measured wood cell wall composition by classical analytical methods in the wood cell walls of these trees. Using statistical modelling and machine learning algorithms, we identified parameters of fluorescence lifetime that predict the content of S-type and G-type lignin units, the two main types of units in the lignin of angiosperm plants. Finally, we show how quantitative measurements of lignin chemical composition by FLIM can reveal the dynamics of lignin biosynthesis in two different biological contexts, including in vivo while lignin is being synthesized in the walls of living cells.


title: Improving Sensitivity to Functional Responses without a Loss of Spatiotemporal Precision in Human Brain Imaging

abstract:
As the neuroimaging field moves towards detecting smaller effects at higher spatial resolutions, and faster sampling rates, there is increased attention given to the deleterious contribution of unstructured, thermal noise. Here, we critically evaluate the performance of a recently developed reconstruction method, termed NORDIC, for suppressing thermal noise using datasets acquired with various field strengths, voxel sizes, sampling rates, and task designs.

Following minimal preprocessing, statistical activation (t-values) of NORDIC processed data was compared to the results obtained with alternative denoising methods. Additionally, the consistency of unbiased estimations of task responses at the single-voxel, single run level, using a finite impulse response model were evaluated. To examine the potential impact on effective image resolution, the overall smoothness of the data processed with different methods was examined. Finally, to determine if NORDIC alters or removes important temporal information, an exhaustive K-fold cross validation approach was employed, using unbiased task responses to predict held out timeseries, quantified using R2.

After NORDIC, the t-values are increased, an improvement comparable to what could be achieved by 1.5 voxels smoothing, and task events are clearly visible and have less cross-run error. These advantages are achieved without significant compromises in spatial and temporal resolution. Cross-validated R2s based on the unbiased models show that NORDIC is not distorting the temporal structure of the data and is the best predictor of non-denoised time courses. The results demonstrate that 1 run of NORDIC data is equivalent to using 2 to 3 original runs and performs equally well across a diverse array of functional imaging protocols.

Significance StatementFor functional neuroimaging, the increasing availability of higher field strengths and ever higher spatiotemporal resolutions has led to concomitant increase in concerns about the deleterious effects of thermal noise. Historically this noise source was suppressed using methods that reduce spatial precision such as image blurring or averaging over a large number of trials or sessions, which necessitates large data collection efforts. Here, we critically evaluate the performance of a recently developed reconstruction method, termed NORDIC. Across datasets varying in field strength, voxel sizes, sampling rates, and task designs, NORDIC produces substantial gains in data quality. Both conventional t-statistics derived from general linear models and coefficients of determination for predicting unseen data are improved, without notably increasing image smoothness.


title: Autophagy in DG engrams mediates Rac1-dependent forgetting during aging via microglia-mediated synapse elimination

abstract: Engrams are considered to be substrates for memory storage, and the functional dysregulation of the engrams leads to cognition impairment. However, the pathological changes of the engrams leading to forgetting, which typically involves a failure in memory retrieval, remains unclear. Here we found that the expression of autophagy protein 7 (Atg7) in dentate gyrus (DG) engrams was dramatically increased in aged mice, leading to the activation of surrounding microglia and impair retrieval of conditioned fear memory. Using transcriptomic and fluorescence in situ hybridization analyses, we demonstrated Toll-like receptor (TLR) pathway were upregulated in DG microglia by overexpressing ATG7 in DG engrams. TLR2/4 in the microglia mediates the excessive synapse elimination and impaired retrieval of fear memory induced by ATG7-depedent autophagy in DG engrams. The expression of Rac1, a Rho-GTPases which mediates active forgetting, was upregulated in aged engrams. Optogenetic activation of Rac1 in DG engrams promoted the expression of ATG7 and autophagy in the engrams, the activation of microglia, and thus impaired the retrieval of fear memory. Interference of the Atg7 expression in the engram and microglia activation prevented the impairment of fear memory retrieval induced by activation of Rac1 in DG engrams. Together, our findings revealed autophagy-dependent remodeling of DG engrams by microglia as a novel interference mechanism of memory retrieval.


title: A novel analytical decoder of BOLD signals for dissociating latent neurobehavioral processes

abstract: Brain, as a complex cognitive system, often processes multiple dimension information synchronously and integrate them to adapt dynamic environments and make effective decisions.1-3 How to retrieve latent neurobehavioral processes from complex human neurobiological signals is an important yet previously unresolved challenge.4,5 For instance, the previous literature has proposed two fundamental yet mutually confounded processes during the decision making and affective processing, i.e. valance and arousal.6,7 Here, we develop a novel analytical approach, orthogonal-Decoding multi-Cognitive Processes (DeCoP), with which we dissociate neural responses in processing valence and arousal information during tests of motivational and emotional function. During reward/punishment anticipation, we decode brain-wide responses into spatially overlapping, yet functionally independent, evaluation and readiness networks, i.e., motivational valence and arousal processing, which are modulated differentially by the meso-limbic vs nigro-striatal dopamine systems. Similarly, during emotional reactivity, we decompose amygdala response into independent emotional valence and facial arousal processing features. We demonstrate that DeCoP can resolve paradoxically unexpected brain  inactivation, and be applied more generally to decode multiple latent neurobehavioral processes. Furthermore, we anticipate our approach to advance both the design and hypothesis testing of cognitive experimental task paradigms.


title: Timing and magnitude of climate driven range shifts in transboundary fish stocks challenge their management

abstract: Climate change is shifting the distribution of shared fish stocks between neighboring countries Exclusive Economic Zones (EEZ) and the high seas. The timescale of these transboundary shifts determines how climate change will affect international fisheries governance. Coupling a large ensemble simulation of an Earth system model to a species distribution model, we show that by 2030, 23% of transboundary stocks would have shifted and 78% of the world EEZs will experience at least one shifting stock under a high emission climate change scenario. By the end of this century, 81% of EEZ waters will see at least one shifting stock with a total of 45% of stocks shifting globally, under a high emissions scenario. Importantly, many countries that are highly dependent on fisheries for income, food and nutrition security, as well as livelihoods emerge as hotspots for transboundary shifts showing early, and sometimes past shifts. Existing fisheries agreements need to be assessed for their capacity in addressing transboundary shifts, and strengthened where necessary to limit conflict over these fish stocks while new agreements are urged to considere this problematic in order to be resilient to global change.


title: Transposable element diversity remains high in gigantic genomes

abstract: Transposable elements (TEs) are repetitive sequences of DNA that replicate and proliferate throughout genomes. Taken together, all the TEs in a genome form a diverse community of sequences, which can be studied to draw conclusions about genome evolution. TE diversity can be measured using models for ecological community diversity that consider species richness and evenness. Several models predict TE diversity decreasing as genomes expand because of selection against ectopic recombination and/or competition among TEs to garner host replicative machinery and evade host silencing mechanisms. Salamanders have some of the largest vertebrate genomes and highest TE loads. Salamanders of the genus Plethodon, in particular, have genomes that range in size from 20 to 70 Gb. Here, we use Oxford Nanopore sequencing to generate low-coverage genomic sequences for four species of Plethodon that encompass two independent genome expansion events, one in the eastern clade (P. cinereus, 29.3 Gb vs. P. glutinosus, 38.9 Gb) and one in the western clade (P. vehiculum, 46.4 Gb vs P. idahoensis, 67.0 Gb). We classified the TEs in these genomes and found ~52 TE superfamilies, accounting for 27-32% of the genomes. We calculated Simpsons and Shannons diversity indices to quantify overall TE diversity. In both pairwise comparisons, the diversity index values for the smaller and larger genome were almost identical. This result indicates that, when genomes reach extremely large sizes, they maintain high levels of TE diversity at the superfamily level, in contrast to predictions made by previous studies on smaller genomes.


title: Stress Evaluation of Various Breeding Environments in Mouse

abstract: Laboratory animals are raised in a fixed space during the study period and are environmentally bound. Laboratory animal may be under stress on the constrained environment, which changes physiological indicators, affecting the reproducibility and accuracy of animal study. Therefore, reducing animal stress by providing proper breeding environment and environmental enrichment can be the basis for animal study. In this study, the stress level was assessed according to the mouse breeding environment. According to the results of the experiment, it was determined that the individual ventilation cage had less cortisol concentration in serum and body weight increased in the individual ventilation cage than individual isolated cage, when providing environmental enrichment rather than group breeding or not providing environmental enrichment. The results will provide appropriate guidelines for laboratory animal welfare.

category: animal behavior and cognition


title: Discriminating predation attempt outcomes during natural foraging using the post-buzz pause in Japanese large-footed bat Myotis macrodactylus

abstract:
Bats emit a series of echolocation calls with an increasing repetition rate (the terminal buzz), when attempting to capture prey. This is often used as an acoustic indicator of prey-capture attempts. However, because it is directly linked to foraging efficiency, predation success is a more useful measure than predation attempts in ecological research. The characteristics of echolocation calls that consistently signify predation success across different situations have not been identified. Due to additional influencing factors, identification of these characteristics is particularly challenging for wild bats foraging in their natural environment compared to those in flight chambers. This study documented the natural foraging behavior of wild Japanese large-footed bat Myotis macrodactylus using synchronized acoustic and video recordings. From the video recordings, we could assign 137 attacks to three outcome categories: prey captured (51.8%), prey dropped (29.2%), and failed attempt (19%). Based on previous indications from laboratory studies that the length of the silent interval following the terminal buzz (post-buzz pause) might reflect the prey capture outcome, we compared post-buzz pause durations among categories of attack outcomes. The post-buzz pause was longest in the case of successful capture, suggesting that the length of the post-buzz pause is a useful acoustic indicator of predation success during natural foraging in M. macrodactylus. Our finding will advance the study of bat foraging behavior using acoustic data, including estimations of foraging efficiency and analyses of feeding habitat quality.

Summary statementWe investigated the natural foraging behavior of wild Myotis macrodactylus and found that the length of the post-buzz pause is a useful acoustic indicator of predation success.


title: Comparative kinetic analysis of ascorbate (Vitamin-C) recycling dehydroascorbate reductases from plant and human.

abstract: Ascorbate, a primary antioxidant, gets readily oxidized to dehydroascorbate (DHA). Hence, recycling by dehydroascorbate reductase (DHAR) enzymes is vital for protection from cellular oxidative stress in eukaryotes. However, a detailed kinetic analysis of plant DHARs and their human orthologs; chloride intracellular channels (HsCLICs) is lacking. We demonstrate that DHAR from stress adapted pearl millet Pennisetum glaucum (PgDHAR) shows the highest turnover rate whereas HsCLIC1, 3, and 4 reduce DHA, albeit at lower rates. We further show that the catalytic cysteine is susceptible to varying levels of oxidation, supported by crystal structures and mass-spectrometry analysis. The differences in kinetic parameters among plant and human DHA reductases corroborate with the levels of reactive oxygen species H2O2 encountered in their respective intracellular environment. Our findings may have broader implications in crop improvement using pearl millet DHAR, and anti-cancer therapeutics targeting Vitamin-C recycling capability of human CLICs.


title: A base-resolution panorama of the in vivo impact of cytosine methylation on transcription factor binding

abstract:
While methylation of CpG dinucleotides is traditionally considered antagonistic to the DNA-binding activity of most transcription factors (TFs), recent in vitro studies have revealed a more complex picture, suggesting that over a third of TFs may preferentially bind to methylated sequences. Expanding these in vitro observations to in vivo TF binding preferences, however, is challenging, as the effect of methylation of individual CpG sites cannot be easily isolated from the confounding effects of DNA accessibility and regional DNA methylation. As a result, the in vivo methylation preferences of most TFs remain uncharacterized.

Here, we introduce joint accessibility-methylation-sequence (JAMS) models, which connect the strength of the binding signal observed in ChIP-seq to the DNA accessibility of the binding site, regional methylation level, DNA sequence, and base-resolution cytosine methylation. We show that JAMS models quantitatively explain the TF binding strength, recapitulate cell type-specific TF binding, and have high precision for inferring intra-motif methylation effects. Analysis of 2209 ChIP-seq experiments resulted in high-confidence JAMS models for 260 TFs, revealing that 45% of TFs are inhibited by intra-motif methylation in vivo. In contrast, only 16 TFs (6%) preferentially bind to methylated sites, including 11 novel methyl-binding TFs that are mostly from the multi-zinc finger family of TFs.

Our study substantially expands the repertoire of in vivo methyl-binding TFs, but also suggests that most TFs that prefer methylated CpGs in vitro present themselves as methylation agnostic in vivo, potentially due to the balancing effect of competition with other methyl-binding proteins.


title: A Wide-Field Micro-Computed Tomography Detector: Micron Resolution at Half-centimeter Scale

abstract:
Ideal 3-dimensional imaging of many complex samples, such as biological tissues made up of micro-scale structures extending over millimeter- to centimeter-scale tissue samples and organisms, requires both a wide field-of-view and high resolution. With existing optics and detectors used for micro-CT imaging, sub-micron pixel resolution can only be achieved for fields-of-view of <2 mm. This manuscript presents a unique detector system with a 6-mm field-of-view image circle and 0.5 m pixel size that can be used in both synchrotron facilities and tabletop micro-CT units. A resolution-test pattern with linear microstructures and whole adult Daphnia magna were imaged on Beamline 8.3.2 of the Advanced Light Source. Volumes of 10,000 x 10,000 x 7,096 isotropic 0.5 m voxels were reconstructed over a 5.0 x 3.5 mm field-of-view. Measurements in the projection domain confirmed a 1.182 m measured spatial resolution that is largely Nyquist-limited. This unprecedented combination of field-of-view and resolution dramatically reduces the need for sectional scans and computational stitching for large samples, ultimately offering the means to elucidate change in tissue and cellular morphology in the context of larger whole, intact model organisms and specimens. This development is also anticipated to benefit micro-CT imaging in materials science, microelectronics, agricultural science, and biomedical engineering.

SynopsisA custom wide-field lens and a new-generation megapixel camera enabled microCT scanning over a 3.5 x 5 mm field-of-view at a 1 m resolution / 0.5 m pixel size at the Berkeley Lawrence Advanced Light Source using a phantom with micron scale features. This novel combination of resolution and field-of-view will be broadly applicable to any setting in which micron-scale structures need to be characterized comprehensively in 3 dimensions over mm to cm scale.


title: Gene regulatory network analysis identifies key genes and regulatory mechanisms involved in acute myocardial infarction using bulk and single cell RNA-seq data

abstract: Cardiovascular and cerebrovascular diseases are leading causes of death worldwide, accounting for more than 40% of all deaths in China. Acute myocardial infarction (AMI) is a common cardiovascular disease and traditionally divided into ST-segment (STEMI) and non-ST-segment elevation myocardial infarction (NSTEMI), which are known with different prognoses and treatment strategies. However, key regulatory genes and pathways involved in AMI that may be used as potential biomarker for prognosis are unknown. In this study, we constructed weighted gene co-expression networks for differential expressed genes between STEMI and NSTEMI patients based on whole-blood RNA-seq transcriptomics. Network topological attributes (e.g., node degree, betweenness) were analyzed to identify key genes involved in different functional network modules. Furthermore, we used single-cell RNA-seq data to construct multilayer signaling network to infer regulatory mechanisms of the above key genes. PLAUR (receptor for urokinase plasminogen activator) was found to play a vital role in transducing inter-cellular signals from endothelial cells and fibroblast cells to intra-cellular pathways of myocardial cells, leading to gene expression involved in cellular response to hypoxia. Our study sheds lights on identifying molecular biomarkers for diagnosis and prognosis of AMI, and provides candidate key regulatory genes for further experimental validation.


title: A large-sample crisis? Exaggerated false positives by popular differential expression methods

abstract: We report a surprising phenomenon about identifying differentially expressed genes (DEGs) from population-level RNA-seq data: two popular bioinformatics methods, DESeq2 and edgeR, have unexpectedly high false discovery rates (FDRs). Via permutation analysis on an immunotherapy RNA-seq dataset, we observed that DESeq2 and edgeR identified even more DEGs after samples condition labels were randomly permuted. Motivated by this, we evaluated six DEG identification methods (DESeq2, edgeR, limma-voom, NOISeq, dearseq, and the Wilcoxon rank-sum test) on population-level RNA-seq datasets. We found that the FDR control was often failed by the three popular parametric methods--DESeq2, edgeR, and limma-voom-- and the new non-parametric method dearseq. In particular, the actual FDRs of DESeq2 and edgeR sometimes exceeded 20% when the target FDR threshold was only 5%. Although NOISeq, a non-parametric method used by GTEx, controlled the FDR better than the other four methods did, its power was much lower than that of the Wilcoxon rank-sum test, a classic nonparametric test that consistently controlled the FDR and achieved good power in our evaluation. Based on these results, for population-level RNA-seq studies, we recommend the Wilcoxon rank-sum test.


title: Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding

abstract: The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N-(NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we showed that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that was consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.

category: biophysics


title: Development and Characterization of a Weaned Pig Model of Shiga Toxin-Producing E. coli-Induced Gastrointestinal Disease

abstract:
Post-weaning enteropathies in swine caused by pathogenic E. coli, such as post-weaning diarrhea (PWD) or edema disease (ED), remain a significant problem for the swine industry. Reduction in the use of antibiotics over concerns of antibiotic resistance and public health concerns, necessitate the evaluation of effective antibiotic alternatives to prevent significant loss of livestock and/or reductions in swine growth performance. For this purpose, an appropriate piglet model of enterotoxigenic E. coli enteropathy is required. In this study, we attempted to induce clinical signs of post-weaning disease in a piglet model using a one-time acute or lower daily chronic dose of a Shiga toxin-producing and enterotoxigenic E. coli strain. The induced disease state was monitored by determining fecal shedding and colonization of the challenge strain, animal growth performance, cytokine levels, fecal calprotectin, histology, fecal metabolomics, and fecal microbiome shifts. The most informative analyses were colonization and shedding of the pathogen, serum cytokines, metabolomics, and targeted metagenomics to determine dysbiosis. Histopathological changes of the gastrointestinal (GI) tract and tight junction leakage as measured by fecal calprotectin concentrations were not observed. Chronic dosing was similar to the acute regimen suggesting that a high dose of pathogen, as used in many studies, may not be necessary. The piglet disease model presented here can be used to evaluate alternative PWD treatment options. Furthermore, this relatively mild disease model presented here may be informative for modeling human chronic gastrointestinal diseases, such as inflammatory bowel disease, which otherwise require invasive procedures for study.

ImportancePost-weaning diarrhea remains a significant problem in swine production and appropriate models of pathogenesis are needed to test alternative treatment options. In this study, we present an E. coli induced piglet model for post-weaning diarrhea, and also explore its translational potential as a model for human intestinal inflammation. Our study here presents two firsts to our knowledge. 1) The first simultaneous analysis of the intestinal microbiome and metabolome through fecal sampling of piglets challenged with Shiga toxin-producing E. coli. This is valuable given the limited metabolomics data from swine in various disease states. 2) A comparison of the clinical signs caused by a daily chronic vs one-time dosing regimen of E. coli. This comparison is key as infection by pathogenic E. coli in real-world settings likely occurs from chronic exposure to contaminated food, water, or environment rather than the highly concentrated dose of pathogen that is commonly given in the literature.

category: pathology