title: Subtly altered topological asymmetry of brain structural covariance networks in autism spectrum disorder across 43 datasets from the ENIGMA consortium

abstract: Small average differences in the left-right asymmetry of cerebral cortical thickness have been reported in individuals with autism spectrum disorder (ASD) compared to typically developing controls. Although these alterations affect multiple and widespread cortical regional asymmetries, the extent to which specific structural networks might be affected remains unknown. Inter-regional morphological covariance analysis can capture network connectivity relations between different cortical areas at the macroscale level. Here, we used cortical thickness data from 1,455 individuals with ASD and 1,560 controls, across 43 independent datasets of the ENIGMA consortiums ASD Working Group, to assess hemispheric asymmetries of intra-individual structural covariance networks, using graph theory-based topological metrics. Compared with typical features of small-world architecture in controls, the ASD sample showed significantly altered asymmetry of hemispheric networks involving the fusiform, rostral middle frontal, and medial orbitofrontal cortex, driven by shifts toward higher randomization of the corresponding right-hemispheric networks in ASD. A network involving the superior frontal cortex showed decreased right-hemisphere randomization. Based on comparisons with meta-analyzed functional neuroimaging data, the altered connectivity asymmetry particularly affected networks that subserve working memory, executive functions, language, reading, and sensorimotor processes. Taken together, these findings provide new insights into how altered brain left-right asymmetry in ASD affects specific structural and functional brain networks. Altered asymmetrical brain development in ASD may be partly propagated among spatially distant regions through structural connectivity.


title: Predicting T Cell Quality During Manufacturing Through an Artificial Intelligence-based Integrative Multi-Omics Analytical Platform

abstract: Large-scale, reproducible manufacturing of therapeutic cells with consistently high quality is vital for translation to clinically effective and widely accessible cell therapies. However, the biological and logistical complexity of manufacturing a living product, including challenges associated with their inherent variability and uncertainties of process parameters, currently make it difficult to achieve predictable cell-product quality. Using a degradable microscaffold-based T cell process as an example, we developed an Artificial Intelligence (AI)-driven experimental-computational platform to identify a set of critical process parameters (CPP) and critical quality attributes (CQA) from heterogeneous, high dimensional, time-dependent multi-omics data, measurable during early stages of manufacturing and predictive of end-of-manufacturing product quality. Sequential, Design-of-Experiment (DOE)-based studies, coupled with an agnostic machine-learning framework, were used to extract feature combinations from media assessment that were highly predictive of total live CD4+ and CD8+ naive and central memory (CD63L+CCR7+) T cells and their ratio in the end-product. This computational workflow could be broadly applied to any cell therapy and provide a roadmap for discovering CQAs and CPPs in cell manufacturing.

category: bioengineering


title: Graph neural networks and sequence embeddings enable the prediction and design of the cofactor specificity of Rossmann fold proteins

abstract:
The Rossmann fold enzymes are involved in essential biochemical pathways such as nucleotide and amino acid metabolism. Their functioning relies on interaction with cofactors, small nucleoside-based compounds specifically recognized by a conserved {beta}{beta} motif shared by all Rossmann fold proteins. While Rossmann methyltransferases recognize only a single cofactor type, the S-Adenosylmethionine (SAM), the oxidoreductases, depending on the family, bind nicotinamide (NAD, NADP) or flavin-based (FAD) cofactors. In this study, we show that despite its short length, the {beta}{beta} motif unambiguously defines the specificity towards the cofactor. Following this observation, we trained two complementary deep learning models for the prediction of the cofactor specificity based on the sequence and structural features of the {beta}{beta} motif. A benchmark on two independent test sets, one containing {beta}{beta} motifs bearing no resemblance to those of the training set, and the other comprising 38 experimentally confirmed cases of rational design of the cofactor specificity, revealed the nearly perfect performance of the two methods. The Rossmann-toolbox protocols can be accessed via the webserver at https://lbs.cent.uw.edu.pl/rossmann-toolbox and are available as a Python package at https://github.com/labstructbioinf/rossmann-toolbox.

Key pointsO_LIThe Rossmann fold encompasses a multitude of diverse enzymes involved in most of the essential cellular pathways
C_LIO_LIProteins belonging to the Rossmann fold co-evolved with their nucleoside-based cofactors and require them for the functioning
C_LIO_LIManipulating the cofactor specificity is an important step in the process of enzyme engineering
C_LIO_LIWe developed an end-to-end pipeline for the prediction and design of the cofactor specificity of the Rossmann fold proteins
C_LIO_LIOwing to the utilization of deep learning approaches the pipeline achieved nearly perfect accuracy
C_LI

category: bioinformatics


title: Rossmann-toolbox: a deep learning-based protocol for the prediction and design of cofactor specificity in Rossmann-fold proteins


title: Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins: insight into mechanisms of G protein selectivity

abstract:
G protein-coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here we used cryo-electron microscopy to determine structures of the CCK1R bound to the CCK peptide agonist, CCK-8 and two distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11-GPCR complexes, the Gq-5 helix bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket was essentially equivalent when Gs was bound, with the Gs 5 helix displaying a conformation that arises from "unwinding" of the far C-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein play critical roles in the promiscuous coupling of individual GPCRs.

One-Sentence SummaryCryo-EM structures of the CCK-1R reveal key mechanisms for promiscuous G protein coupling.

category: pharmacology and toxicology


title: Differential Regulation of Rice Transcriptome to Rhizoctonia solani infection

abstract:
Sheath Blight (SB) disease in rice crop caused by the infection of the fungal pathogen Rhizoctonia solani (R. solani) is one of the severe rice diseases that can cause up to 50% yield losses. Naturally occurring rice varieties resistant to SB have not been reported yet. We have performed a Time-Series RNA-Seq analysis on a widely cultivated rice variety BPT-5204 for identifying its transcriptomic response signatures to R. solani infection at 1st, 2nd and 5th day post inoculation (dpi). In total, 428, 3225 and 1225 genes were differentially expressed in the treated rice plants post 1, 2 and 5 dpi, respectively. GO and KEGG enrichment analysis identified significant processes and pathways differentially altered in the rice plant after the fungal infection. Machine learning and network based integrative approach was used to construct Transcriptional Regulatory Networks (TRNs) of the rice plant at the three Time Points. Regulatory network analysis identified SUB1B, MYB30 and CCA1 as important regulatory hub Transcription Factors in rice during R. solani infection. Jasmonic acid signaling pathway was activated and in contrast, photosynthesis and carbon fixation processes were significantly compromised. Involvement of MAPK, CYPs, Peroxidases and PAL genes was observed in response to the fungal infection. Circadian clock was also strongly influenced by R. solani infection. Our integrative analysis identified 7 putative SB resistant genes altered in rice after R. solani infection and provided a better understanding of rice plant response to R. solani infection.

One sentence summaryTime series expression analysis of rice variety BPT-5204 identifies key molecular signatures involved in rice plant response to R. solani infection.

category: systems biology


title: QSMxT: Robust Masking and Artefact Reduction for Quantitative Susceptibility Mapping

abstract:
PurposeQuantitative Susceptibility Mapping (QSM) is a post-processing technique applied to gradient-echo phase data. QSM algorithms require a signal mask to delineate regions with reliable phase signal for subsequent susceptibility estimation. Existing masking techniques used in QSM have limitations that introduce artefacts, exclude anatomical detail, and rely on parameter tuning and anatomical priors that narrow their application. Here, a robust masking and reconstruction procedure is presented to overcome these limitations and enable automated QSM processing for a wide range of use-cases implemented in an open-source software framework: QSMxT.

MethodsA robust masking technique that automatically separates reliable from less reliable phase regions was developed and combined with a two-pass reconstruction procedure that operates on the separated sources before combination, extracting more information while reducing the influence of artefacts.

ResultCompared with standard masking and reconstruction procedures, the two-pass inversion reduces streaking artefacts caused by unreliable phase and high dynamic ranges of susceptibility sources. QSMxT is robust across a range of datasets at 3 T in healthy volunteers and phantoms, at 7 T in tumour patients, and in the QSM challenge 2.0 simulated brain dataset, with significant artefact and error reductions, greater anatomical detail, and minimal parameter tuning.

ConclusionQSMxT generates masks for QSM that separate reliable from less reliable phase regions, enables a more accurate QSM reconstruction that mitigates artefacts, operates without anatomical priors, and requires minimal parameter tuning. QSMxT makes QSM processing more accessible, reliable and reproducible.

category: neuroscience


title: A novel mouse model of focal limbic seizures that reproduces behavioral impairment associated with cortical slow wave activity

abstract: Patients with focal temporal lobe seizures often experience loss of consciousness. In humans, this loss of consciousness has been shown to be positively correlated with EEG neocortical slow waves, similar to those seen in non-REM sleep. Previous work in rat models of temporal lobe seizures suggests that decreased activity of subcortical arousal systems cause depressed cortical function during seizures. However, these studies were performed under light anesthesia, making it impossible to correlate behavior, and therefore consciousness, to electrophysiologic data. Further, the genetic and molecular toolkits allowing for precise study of the underlying neural circuitry are much more developed in mice than in rats. Here, we describe an awake-behaving, head-fixed mouse model of temporal lobe seizures with both spared and impaired behavior reflecting level of consciousness. Water-restricted mice were head-fixed on a running wheel and trained to associate an auditory stimulus to the delivery of a drop of water from a dispenser. To investigate the effect of seizures on behavior, seizures were electrically induced by stimulating either the left or right hippocampus via a chronically-implanted electrode, while mice were performing the task. Behavior was measured by monitoring lick responses to the auditory stimulus and running speed on the wheel. Further, local field potentials (LFP) signals were simultaneously recorded from hippocampus and orbitofrontal cortex (OFC). Induced focal seizures were 5-30s in duration, and repeatable for several weeks (n=20 animals). Behavioral responses showed a decrease in lick rate to auditory stimulus, and decreased running speed during seizures (p<0.01, n=20 animals). Interestingly, licking response to sound could vary from being impaired to normal during seizures. We found that behavioral impairment is correlated with large amplitude cortical slow-wave activity in frontal cortex, as seen in patients with temporal lobe seizures. These results suggest that induced focal limbic seizures in the mouse can impair consciousness and that the impaired consciousness is correlated with depressed cortical function resembling slow wave sleep. This novel mouse model has similar characteristics with previously studied rat models and human temporal lobe seizures. By leveraging the genetic and molecular techniques available in the mouse, this model can be used to further uncover fundamental mechanisms for loss of consciousness in focal seizures.


title: Platform for Incisionless, Focal, and Multisite Brain Interventions

abstract: Transcranial focused ultrasound brings precision healthcare to the human brain, delivering focal interventions to deep brain sites without the need for surgeries or systemic drug administrations. The approaches have included neuromodulation and localized delivery of drugs through an intact or temporarily opened blood brain barrier. Clinical translation of these approaches requires a system that can deliver ultrasound into specific brain targets on command. Here, we demonstrate a platform with these features, and validate its function in nonhuman primates. Specifically, we used the system to flexibly modulate two deep brain nuclei--the left and right lateral geniculate nucleus-during visual choice behavior. We found that ultrasound induced a behavioral bias whose polarity depended on the stimulation waveform. Moreover, repeated applications of ultrasound over 39 consecutive behavioral sessions produced no signs of harm to the stimulated circuits. The system can be be applied to diagnose and possibly treat disorders of brain function with a precision and flexibility previously unattainable.


title: Platform for Remote, Multifocal Brain Interventions


title: Dual-color optical activation and suppression of neurons with high temporal precision

abstract: A fundamental challenge in the optogenetic toolbox is that all opsins, regardless of their excitation spectra, are activated by blue light. We hypothesized that pairing a red-shifted channelrhodopsin with a blue light-sensitive anion channel of appropriately matching kinetics shall render neurons responsive to a red but not blue light. To achieve this, we used a semi-rational mutagenesis strategy to optimize the kinetics and light spectrum of a chloride channelrhodopsin. By pairing optimized variants of blue light-sensitive anion channel ZipACR with vfChrimson, a fast red-shifted channelrhodopsin, we created a system in which red light derives precise and faithful action potentials of high frequencies, while blue light, through shunting inhibition, nullifies the effect of the red-shifted ChR2. Additionally, by a simple switch between red and blue lights, one can effectively excite or inhibit the activity of the same neurons.


title: Simultaneous Brain, Brainstem and Spinal Cord pharmacological-fMRI reveals multilevel opioidergic roles in attentional analgesia in humans

abstract: Pain perception is decreased by shifting attentional focus away from a threatening event. This attentional analgesia engages parallel descending control pathways from anterior cingulate (ACC) to locus coeruleus, and ACC to periaqueductal grey (PAG) - rostral ventromedial medulla (RVM), indicating possible roles for noradrenergic or opioidergic neuromodulators. To determine which pathway modulates nociceptive activity in humans we used simultaneous whole brain-spinal cord pharmacological-fMRI (N=39) across three sessions. Noxious thermal forearm stimulation generated somatotopic-activation of dorsal horn (DH, C6 segment) whose activity mirrored attentional pain modulation. Activity in an adjacent cluster reported the interaction between task and noxious stimulus. Effective connectivity analysis revealed that ACC recruits PAG and RVM to modulate spinal cord activity. Blocking endogenous opioids with Naltrexone impairs attentional analgesia and disrupts RVM-DH and ACC-PAG connectivity. Noradrenergic augmentation with Reboxetine did not alter attentional analgesia. Cognitive pain modulation is mediated by opioidergic ACC-PAG-RVM descending control which supresses spinal nociceptive activity.


title: Simultaneous Brain, Brainstem and Spinal Cord pharmacological-fMRI reveals endogenous opioid network interactions mediating attentional analgesia


title: Dynamics of pollination and seed dispersal mutualisms at low density

abstract: Pollination and seed dispersal mutualisms are critical for biodiversity and ecosystem services yet face mounting threats from anthropogenic perturbations that cause their populations to decline. Characterizing the dynamics of these mutualisms when populations are at low density is important to anticipate consequences of these perturbations. We developed simple population dynamic models detailed enough to distinguish different mechanisms by which plant populations benefit from animal pollination or seed dispersal. We modeled benefits as functions of foraging rate by animals on plant rewards and specified whether they affected plant seed set, germination, or negative density dependence during recruitment. We found that pollination and seed dispersal mutualisms are stable at high density but exhibit different dynamics at low density, depending on plant carrying capacity, animal foraging efficiency, and whether populations are obligate upon their partners for persistence. Under certain conditions, all mutualisms experience destabilizing thresholds in which one population declines because its partner is too rare. Plants additionally experience Allee effects when obligate upon pollinators. Finally, pollination mutualisms can exhibit bistable coexistence at low or high density when plants are facultative upon pollinators. Insights from our models can inform conservation efforts, as mutualist populations continue to decline globally.

category: ecology


title: Simple mechanisms of plant reproductive benefits yield different dynamics in pollination and seed dispersal mutualisms


title: Metamorphosis imposes variable constraints on genome expansion

abstract: Genome size varies [~] 100,000-fold across eukaryotes. Genome size is heavily shaped by transposable element accumulation, the dynamics of which are increasingly well understood. However, given that traits like cell size and rate of development co-vary strongly with genome size, organism-level trait evolution likely shapes genome size diversity as well. Metamorphosis -- a radical transformation of morphology -- has been hypothesized to impact genome size because it can be a vulnerable part of the life cycle. Thus, selection may act to limit metamorphic duration, indirectly constraining the rate of development as well as genome and cell sizes. Salamanders have large and variable genomes -- 3 to 40 times that of humans -- and species exhibit a range of metamorphic and non-metamorphic life histories. Using salamanders, we test the hypothesis that different types of metamorphic repatterning during the life cycle impose different constraints on genome expansion. We show that metamorphosis during which animals are unable to feed imposes the most severe constraint against genome expansion. Other types of metamorphosis that differ in energetic provisioning impose less severe constraints. More generally, our work demonstrates the utility of phylogenetic comparative methods in testing the role of constraint in shaping phenotypic evolution.

category: evolutionary biology


title: Fine-scale quantification of GC-biased gene conversion intensity in mammals.

abstract: GC-biased gene conversion (gBGC) is a molecular evolutionary force that favours GC over AT alleles irrespective of their fitness effect. Quantifying the variation in time and across genomes of its intensity is key to properly interpret patterns of molecular evolution. In particular, the existing literature is unclear regarding the relationship between gBGC strength and species effective population size, Ne. Here we analysed the nucleotide substitution pattern in coding sequences of closely related species of mammals, thus accessing a high resolution map of the intensity of gBGC. Our maximum likelihood approach shows that gBGC is pervasive, highly variable among species and genes, and of strength positively correlated with Ne in mammals. We estimate that gBGC explains up to 60% of the total amount of synonymous AT[->]GC substitutions. We show that the fine-scale analysis of gBGC-induced nucleotide substitutions has the potential to inform on various aspects of molecular evolution, such as the distribution of fitness effects of mutations and the dynamics of recombination hotspots.


title: SWAN Identification of Common Aneuploidy-Based Oncogenic Drivers

abstract:
Haploinsufficiency drives Darwinian evolution. Siblings, while alike in many aspects, differ due to monoallelic differences inherited from each parent. In cancer, solid tumors exhibit aneuploid genetics resulting in hundreds to thousands of monoallelic gene-level copy-number alterations (CNAs) in each tumor. Aneuploidy patterns are heterogeneous, posing a challenge to identify drivers in this high-noise genetic environment. Here, we developed Shifted Weighted Annotation Network (SWAN) analysis to assess biology impacted by cumulative monoallelic changes. SWAN enables an integrated pathway-network analysis of CNAs, RNA expression, and mutations via a simple web platform. SWAN is optimized to best prioritize known and novel tumor suppressors and oncogenes, thereby identifying drivers and potential druggable vulnerabilities within cancer CNAs. Protein homeostasis, phospholipid dephosphorylation, and ion transport pathways are commonly suppressed. An atlas of CNA pathways altered in each cancer type is released. These CNA network shifts highlight new, attractive targets to exploit in solid tumors.

HighlightsO_LICopy-number alteration pathways define solid tumor biology
C_LIO_LISWAN is released as an integrative point-and-click pathway analysis tool
C_LIO_LIModerate impact drivers highlighted by SWAN validated in vitro
C_LIO_LICopy-number altered pathways associate with mutations and survival
C_LI

category: genetics


title: Blood-based liquid biopsy for comprehensive cancer genomic profiling using next-generation sequencing: an emerging paradigm for noninvasive cancer detection and management in dogs

abstract:
This proof-of-concept study demonstrates that blood-based liquid biopsy using next generation sequencing of cell-free DNA can noninvasively detect multiple classes of genomic alterations in dogs with cancer, including alterations that originate from spatially separated tumor sites.

Eleven dogs with a variety of confirmed cancer diagnoses (including localized and disseminated disease) who were scheduled for surgical resection, and 5 presumably cancer-free dogs, were enrolled. Blood was collected from each subject, and multiple spatially separated tumor tissue samples were collected during surgery from 9 of the cancer subjects.

All samples were analyzed using an advanced prototype of a novel liquid biopsy test designed to noninvasively interrogate multiple classes of genomic alterations for the detection, characterization, and management of cancer in dogs.

In 5 of the 9 cancer patients with matched tumor and plasma samples, pre-surgical liquid biopsy testing identified genomic alterations, including single nucleotide variants and copy number variants, that matched alterations independently detected in corresponding tumor tissue samples. Importantly, the pre-surgical liquid biopsy test detected alterations observed in spatially separated tissue samples from the same subject, demonstrating the potential of blood-based testing for comprehensive genomic profiling of heterogeneous tumors. Among the 3 patients with post-surgical blood samples, genomic alterations remained detectable in one patient with incomplete tumor resection, suggesting utility for noninvasive detection of minimal residual disease following curative-intent treatment.

Liquid biopsy allows for noninvasive profiling of cancer-associated genomic alterations with a simple blood draw and has potential to overcome the limitations of tissue-based testing posed by tissue-level genomic heterogeneity.

category: genomics


title: Intracellular and intercellular gene regulatory networks inference from time-course individual RNA-Seq

abstract: Gene regulatory network (GRN) inference is an effective approach to understand the molecular mechanisms underlying biological events. Generally, GRN inference mainly targets intracellular regulatory relationships such as transcription factors and their associated targets. In multicellular organisms, there are both intracellular and intercellular regulatory mechanisms. Thus, we hypothesize that GRNs inferred from time-course individual (whole embryo) RNA-Seq during development can reveal intercellular regulatory relationships (signaling pathways) underlying the development. Here, we conducted time-course bulk RNA-Seq of individual mouse embryos during early development, followed by pseudo-time analysis and GRN inference. The results demonstrated that GRN inference from RNA-Seq with pseudo-time can be applied for individual bulk RNA-Seq similar to scRNA-Seq. Validation using an experimental-source-based database showed that our approach could significantly infer GRN for all transcription factors in the database. Furthermore, the inferred ligand-related and receptor-related downstream genes were significantly overlapped. Thus, the inferred GRN based on whole organism could include intercellular regulatory relationships, which cannot be inferred from scRNA-Seq based only on gene expression data. Overall, inferring GRN from time-course bulk RNA-Seq is an effective approach for understanding the regulatory relationships underlying biological events in multicellular organisms.


title: Inter-laboratory automation of the in vitro micronucleus assay using imaging flow cytometry and deep learning

abstract: The in vitro micronucleus assay is a globally significant method for DNA damage quantification used for regulatory compound safety testing in addition to inter-individual monitoring of environmental, lifestyle and occupational factors. However it relies on time-consuming and user-subjective manual scoring. Here we show that imaging flow cytometry and deep learning image classification represents a capable platform for automated, inter-laboratory operation. Images were captured for the cytokinesis-block micronucleus (CBMN) assay across three laboratories using methyl methanesulphonate (1.25 - 5.0 {micro}g/mL) and/or carbendazim (0.8 - 1.6 {micro}g/mL) exposures to TK6 cells. Human-scored image sets were assembled and used to train and test the classification abilities of the "DeepFlow" neural network in both intra- and inter-laboratory contexts. Harnessing image diversity across laboratories yielded a network able to score unseen data from an entirely new laboratory without any user configuration. Image classification accuracies of 98%, 95%, 82% and 85% were achieved for  mononucleates,  binucleates,  mononucleates with MN and  binucleates with MN, respectively. Successful classifications of  trinucleates (90%) and  tetranucleates (88%) in addition to  other or unscorable phenotypes (96%) were also achieved. Attempts to classify extremely rare, tri- and tetranucleated cells with micronuclei into their own categories were less successful ([≤] 57%). Benchmark dose analyses of human or automatically scored micronucleus frequency data yielded quantitation of the same equipotent dose regardless of scoring method. We conclude that this automated approach offers significant potential to broaden the practical utility of the CBMN method across industry, research and clinical domains. We share our strategy using openly-accessible frameworks.


title: Meta-analytic maps of brain activity evoked by cognitive function diverge from resting state networks

abstract: Spontaneous human brain activity is organized into resting state networks (RSN), complex patterns of synchronized activity that account for the majority of brain metabolism. The correspondence between these patterns and those elicited by task performance suggests that spontaneous brain activity originates from the stream of ongoing cognitive processing. Here we investigate a large number of meta-analytic activation maps obtained from Neurosynth (www.neurosynth.org) to establish that task-rest similarity can be inflated by two potential sources of bias. Applying a hierarchical module detection algorithm to a network representation of activation map similarity, we showed that the correspondence between RSN and task-evoked activity tends to hold only for the largest spatial scales. Second, we established that this correspondence is biased by the inclusion of maps related to neuroanatomical terms (e.g. "parietal", "occipital", "cingulate", etc.). Our results challenge the cognitive origin of spontaneous brain activity, suggesting that anatomically-constrained homeostatic processes could also play an important role in the inception and shaping of human resting state activity fluctuations.


title: Drug distribution along the cochlea is strongly enhanced by low-frequency round window micro vibrations

abstract: The cochleas inaccessibility and complex nature provide significant challenges to delivering drugs and other agents uniformly, safely and efficiently, along the entire cochlear spiral. Large drug concentration gradients are formed along the cochlea when drugs are administered to the middle ear. This undermines the major goal of attaining therapeutic drug concentration windows along the whole cochlea. Here, utilizing a well-known physiological effect of salicylate, we demonstrate a proof of concept in which drug distribution along the entire cochlea is enhanced applying round window membrane low-frequency micro vibrations with a probe that only partially covers the round window. We provide evidence of enhanced drug influx into the cochlea and cochlear apical drug distribution without breaching cochlear boundaries. It is further suggested that ossicular functionality is not required for the effective drug distribution we report. The novel method of local drug delivery to the cochlea presented here could be implemented when ossicular functionality is absent or impeded and can be incorporated in clinically approved auditory protheses for patients who suffer with conductive, sensorineural or mixed hearing loss.


title: Beneficial and detrimental genes in the cellular response to replication arrest

abstract: DNA replication is essential for all living organisms, and a variety of events can perturb replication, including DNA damage (e.g., pyrimidine dimers, crosslinking) and replication arrest due to "roadblocks" such as DNA-binding proteins or transcription. Bacteria have several well-characterized mechanisms for repairing damaged DNA and restoring functional replication forks. However, little is known about the repair of stalled or arrested replication forks in the absence of DNA lesions. Using a library of random transposon insertions in Bacillus subtilis, we identified 35 genes that affect the ability of cells to survive arrest of replication elongation, in the absence of DNA damage. Genes identified included those involved in iron-sulfur homeostasis, cell envelope biogenesis, and DNA repair and recombination. In B. subtilis, and many bacteria, two nucleases (AddAB and RecJ) are involved in early steps in repairing replication forks arrested by DNA damage. Loss of either one causes increased sensitivity to DNA damage. These single-strand nucleases resect DNA ends, leading to assembly of the recombinase RecA onto the single stranded DNA. Notably, we found that disruption of recJ increased survival of cells following replication arrest, indicating that, in the absence of DNA damage, RecJ is detrimental to surviving replication arrest. In contrast, and as expected, disruption of addA decreased survival of cells following replication arrest, indicating that AddA promotes survival. The different phenotypes of addA and recJ mutants appeared to be due to differences in assembly of RecA onto DNA. RecJ promoted too much assembly of RecA, and loss of RecA compensated for the detrimental effects of RecJ. Our results indicate that in the absence of DNA lesions, RecA is dispensable for cells to survive replication arrest and the stable RecA nucleofilaments favored by the RecJ pathway may lead to cell death by preventing proper processing of the arrested replication fork.

category: microbiology


title: Cell-envelope synthesis is required for surface-to-mass coupling, which determines dry-mass density in Bacillus subtilis

abstract: Cells must increase their volumes in response to biomass growth to maintain intracellular mass density, the ratio of dry mass to cell volume, within physiologically permissive bounds. To increase volume, bacteria enzymatically expand their cell envelopes and insert new envelope material. Recently, we demonstrated that the Gram-negative bacterium Escherichia coli expands cell-surface area rather than volume in proportion to mass. Here, we investigate the regulation of cell-volume growth in the evolutionarily distant Bacillus subtilis. First, we demonstrate that the coupling of surface growth to mass growth is conserved in B. subtilis. Therefore, mass density changes with cell shape at the single-cell level. Interestingly, mass density varies by more than 30% when we systematically change cell width by modulation of cell-wall insertion, without any effect on mass-growth rate. Second, we demonstrate that the coupling of surface- and mass growth is broken if peptidoglycan or membrane synthesis are inhibited. Once transient perturbations are relieved, the surface-to-mass ratio is rapidly restored. In conclusion, we demonstrate that surface-to-mass coupling is a conserved principle for volume regulation in bacteria, and that envelope synthesis provides an important link between surface growth and biomass growth in B. subtilis.


title: Ferries and environmental DNA: underway sampling from commercial vessels provides new opportunities for systematic genetic surveys of marine biodiversity

abstract: Marine environmental DNA (eDNA) is an important tool for biodiversity research and monitoring but challenges remain in scaling surveys over large spatial areas, and increasing the frequency of sampling in remote locations at reasonable cost. Here we demonstrate the feasibility of sampling from commercial vessels (Mediterranean ferries) while underway, as a strategy to facilitate replicable, systematic marine eDNA surveys in locations that would normally be challenging and expensive for researchers to access. Sixteen eDNA samples were collected from 4 fixed sampling stations, and in response to 4 cetacean sightings, across three cruises undertaken along the 300 km ferry route between Livorno (Tuscany) and Golfo Aranci (Sardinia) in the Ligurian/Tyrrhenian Seas, June-July 2018. Using 12SrDNA and 16SrDNA metabarcoding markers, we recovered diverse marine vertebrate Molecular Operational Taxonomic Units (MOTUs) from teleost fish, elasmobranchs, and cetaceans. We detected sample heterogeneity consistent with previously known variation in species occurrences, including putative species spawning peaks associated with specific sea surface temperature ranges, and increased night time abundance of bathypelagic species known to undertake diel migrations through the water column. We suggest commercial vessel based marine eDNA sampling using the global shipping network has potential to facilitate broad-scale biodiversity monitoring in the worlds oceans.

category: molecular biology


title: Growth-dependent heterogeneity in the DNA damage response in Escherichia coli

abstract: In natural environments bacteria are frequently exposed to sub-lethal levels of DNA damage which leads to the induction of a stress response (the SOS response in Escherichia coli). Natural environments also vary in nutrient availability, resulting in distinct physiological changes in bacteria which may have direct implications on their capacity to repair their chromosomes. Here, we evaluated the impact of varying the nutrient availability on the expression of the SOS response induced by chronic sub-lethal DNA damage in E. coli. The expression of the SOS regulon was found to be highly heterogeneous at the single-cell level in all growth conditions. Surprisingly, we observed a larger fraction of high SOS-induced cells in slow growth as compared with fast growth, despite a higher rate of SOS induction in fast growth. This counter-intuitive result can be explained by the dynamic balance between the rate of SOS induction and the division rates of cells exposed to DNA damage. Taken together, our data illustrates how cell division and physiology come together to produce growth-dependent heterogeneity in the DNA damage response.

category: cell biology


title: Maternal iron deficiency impacts the placental arterial network.

abstract: Placental vascular gene networks in mammals have been largely unexplored due to a lack of well validated molecular markers to identify them. This is required to study how they form in development, and how they are impacted by embryonic or maternal defects, which in-turn adversely affects the forming heart and vasculature. Such defects are known to be a consequence of maternal iron deficiency (ID), the most common nutrient deficiency world-wide. Here we employed marker analysis to characterise the arterial/arteriole and venous/venule endothelial cells (ECs) during normal placental development, and in the context of maternal ID. We reveal for the first time that placental ECs are unique compared with their embryonic counterparts. In the developing embryo, arterial ECs express Neuropilin1 (Nrp1), Delta-like ligand 4 (Dll4) and Notch1, while developing venous ECs express Neuropilin2 (Nrp2), Apj (Aplnr) and Ephrinb4 (Ephb4). However, in the E15.5 placenta, Nrp1 and Notch1 were restricted to arteries, but not continuing arteriole ECs. The arterial tree exclusively expressed Dll4. Nrp2 showed pan-EC expression at E15.5, while Ephb4 was not present at this stage. However, we found the placental venous vascular tree could be distinguished from the arterial tree by high versus low Endomucin (EMCN) and Apj (Aplnr) expression respectively. Using EMCN, we reveal that the placental arterial, but not venous, vascular tree is adversely impacted by maternal ID, with reduced area, total length and number of junctions of all vessels without affecting the EMCN high vessels. Defects to the embryonic cardiovascular system can therefore have a significant impact on blood flow delivery and expansion of the placental arterial tree.

category: developmental biology


title: Genetic, Genomic and Biophysical Case Study: Familial 46, XY Sex Reversal due to a Novel Inherited Mutation in Human Testis-Determining Factor SRY

abstract:
ObjectiveTo describe the clinical, histopathological and molecular features of a novel inherited SRY allele (p.Met64Val; consensus box position 9) observed within an extensive pedigree: two 46, XY sisters with primary amenorrhea (16 and 14 years of age; probands P1 and P2), their normal father and brother, and an affected paternal XY grandaunt.

Design, Setting, Participants and Outcome MeasurementsFollowing DNA sequencing to identify the SRY mutation, hormonal studies of the probands and histopathological examination of their gonads were performed. Functional consequences of p.Met64Val (and other mutations at this site) were also investigated.

ResultsBreast development in P1 and P2 was Tanner II and IV, respectively. Mullerian structures and gonads resembling ovaries were found in each sister. Histopathology revealed gonadal dysgenesis, gonadablastoma and dysgerminoma. AMH/MIS, P450 SCC, and P450 aromatase were expressed in gonadoblastoma tissues. Genomic sequencing revealed no candidate mutations in other genes related to sexual differentiation. Variant p.Met64Val impaired Sox9 transcriptional activation associated with attenuated occupancy of the testis-specific enhancers Enh13 and TESCO. Biophysical studies indicated that the mutant HMG box retains specific DNA binding and DNA bending but with accelerated rate of protein-DNA dissociation.

ConclusionThe partial biological activity of p.Met64Val SRY, maintained at the threshold of SRY function, rationalizes opposing paternal and proband phenotypes (the "the father-daughter paradox"). Steroidal biosynthesis by gonadoblastoma may delay genetic diagnosis and recognition of gonadal tumors. Quantitative assessment of inherited SRY alleles highlights the tenuous transcriptional threshold of developmental decision-making in the bipotential gonadal ridge.


title: Quantitative assessment of multiple fish species around artificial reefs using environmental DNA metabarcoding

abstract: Since the early 1970s, many artificial reefs (ARs) have been deployed in Japanese coastal waters to create fisheries grounds. Recently, researchers began to use environmental DNA (eDNA) methods for biodiversity monitoring of aquatic species. A metabarcoding approach using internal standard DNAs (i.e., quantitative MiSeq sequencing) makes it possible to monitor eDNA concentrations of multiple species simultaneously. This method can improve the efficiency of monitoring AR effects on fishes. Our study investigated distributions of marine fishes at ARs and surrounding stations in the open oceanographic environment of Tateyama Bay, central Japan, using quantitative MiSeq sequencing and echo sounder survey. Using the quantitative metabarcoding method, we found higher quantities of fish eDNAs at the ARs than at surrounding stations and different fish species compositions between them. Comparisons with eco sounder survey also showed positive correlations between fish eDNA concentration and echo intensity, which indicates a highly localized signal of eDNA at each sampling station. These results suggest that quantitative MiSeq sequencing is a promising technique to complement conventional methods to monitor distributions of multiple fish species.


title: Multiple causal DNA variants in a single gene affect gene expression in trans

abstract: Identifying the specific causal DNA differences in a genome that contribute to variation in phenotypic traits is a key goal of genetic research. Trans-acting DNA variants that alter gene expression are important sources of genetic variation. Several genes are known to carry single causal variants that affect the expression of numerous genes in trans. Whether these single variants are representative of the architecture of trans-acting variation is unknown. Here, we studied the gene IRA2, which carries variants with broad, trans-acting effects on gene expression in two strains of the yeast Saccharomyces cerevisiae. We found that IRA2 contains at least seven causal nonsynonymous variants. The causal variants were located throughout the gene body and included a pair of neighboring variants with opposing effects that largely canceled each other out. The causal variants showed evidence for non-additive epistatic interactions, in particular among variants at the 5 end of the gene. These results show that the molecular basis of trans-acting variation can involve considerable complexity even within a single gene.


title: Spatial and ecological drivers of population structure in Alkanna tinctoria (Boraginaceae), a polyploid medicinal herb

abstract:
Background and AimsQuantifying genetic variation is fundamental to understand a species demographic trajectory and its ability to adapt to future changes. In comparison to diploids, however, genetic variation and factors fostering genetic divergence remains poorly studied in polyploids due to analytical challenges. Here, by employing a ploidy-aware framework, we investigated the genetic structure and its determinants in polyploid Alkanna tinctoria (Boraginaceae), an ancient medicinal herb that is the source of bioactive compounds known as alkannin and shikonin (A/S). From a practical perspective, such investigation can inform biodiversity management strategies.

MethodsWe collected 14 populations of A. tinctoria within its main distribution range in Greece and genotyped them using restriction site-associated DNA sequencing (RAD-seq). As an outgroup, we included two populations of A. sieberi. By using a ploidy-aware genotype calling based on likelihoods, we generated a dataset of 16,107 high quality SNPs. Classical and model-based analysis was done to characterize the genetic structure within and between the sampled populations. Finally, to reveal the drivers of genetic structure, we searched for associations between allele frequencies and spatial and climatic variables.

Key ResultsWe found support for a marked regional structure in A. tinctoria along a latitudinal gradient in line with phytogeographic divisions. Several analyses identified interspecific admixture affecting both mainland and island populations. Modelling of spatial and climatic variables further demonstrated a larger contribution of neutral processes and a lesser albeit significant role of selection in shaping the observed genetic structure in A. tinctoria.

ConclusionsCurrent findings provide evidence of strong genetic structure in A. tinctoria mainly driven by neutral processes. The revealed natural genomic variation in Greek Alkanna can be used to further predict variation in A/S production, whereas our bioinformatics approach should prove useful for the study of other non-model polyploid species.


title: Reinforcement learning modeling reveals a reward-history-dependent strategy underlying reversal learning in squirrel monkeys

abstract: Insight into psychiatric disease and development of therapeutics relies on behavioral tasks that study similar cognitive constructs in multiple species. The reversal learning task is one popular paradigm that probes flexible behavior, aberrations of which are thought to be important in a number of disease states. Despite widespread use, there is a need for a high-throughput primate model that can bridge the genetic, anatomic, and behavioral gap between rodents and humans. Here, we trained squirrel monkeys, a promising preclinical model, on an image-guided deterministic reversal learning task. We found that squirrel monkeys exhibited two key hallmarks of behavior found in other species: integration of reward history over many trials and a side-specific bias. We adapted a reinforcement learning model and demonstrated that it could simulate monkey-like behavior, capture training-related trajectories, and provide insight into the strategies animals employed. These results validate squirrel monkeys as a model in which to study behavioral flexibility.

category: animal behavior and cognition


title: Unbiased integration of single cell transcriptomes using a linear hybrid method

abstract: Single cell transcriptomic approaches are becoming mainstream, with replicate experiments commonly performed with the same single cell technology. Methods that enable integration of these datasets by removing batch effects while preserving biological information are required for unbiased data interpretation. Here we introduce Canek for this purpose. Canek leverages information from mutual nearest neighbor to combine local linear corrections with cell-specific non-linear corrections within a fuzzy logic framework. Using a combination of real and synthetic datasets, we show that Canek corrects batch effects while introducing the least amount of bias compared with competing methods. Canek is computationally efficient and can easily integrate thousands of single-cell transcriptomes from replicated experiments.


title: Unbiased integration of single cell transcriptome replicates


title: Label-free imaging flow cytometry: analysis and sorting of enzymatically dissociated tissues

abstract: Biomedical research often relies on identification and isolation of specific cell types using molecular biomarkers and sorting methods such as fluorescence or magnetic activated cell sorting. Labelling processes potentially alter the cells properties and should be avoided, especially when purifying cells for clinical applications. A promising alternative is the label-free identification of cells based on their physical properties. Sorting real-time deformability and fluorescence cytometry (soRT-FDC) is a microfluidic technique for label-free analysis and sorting of single cells. In soRT-FDC, bright-field images of cells are analyzed by a deep neural net (DNN) to obtain a sorting decision, but sorting was so far only demonstrated for blood cells which show clear morphological differences and are naturally in suspension. Most cells, however, grow in tissues, requiring dissociation before cell sorting which is associated with additional challenges including survival, changes in morphology, or presence of aggregates. Here, we introduce methods for robust analysis and sorting of single cells from mammalian nervous tissue and provide DNNs which are capable of distinguishing visually similar cells. Exemplarily, we employ the DNN for image-based sorting to enrich photoreceptor cells from dissociated retina for transplantation into the mouse eye. Results provide evidence that the combination of machine learning and soRT-FDC allows label-free enrichment of target cells from dissociated tissues.

category: biophysics


title: CBMOS: a GPU-enabled Python framework for the numerical study of center-based models

abstract:
Cell-based models are becoming increasingly popular for applications in developmental biology. However, the impact of numerical choices on the accuracy and efficiency of the simulation of these models is rarely meticulously tested. We present CBMOS, a Python framework for the simulation of the center-based or cell-centered model. Contrary to other implementations, CBMOS focus is on facilitating numerical study of center-based models by providing access to multiple ODE solvers and force functions through a flexible, user-friendly API. We show-case its potential by evaluating the use of the backward Euler method for calculating the trajectories of two-dimensional cell populations. We confirm that although for moderate accuracy levels the backward Euler method allows for larger time step sizes than the commonly used forward Euler method, its additional computational cost due to being an implicit method prohibits its use for practical test cases.

CBMOS is available on GitHub1 and PyPI under an MIT license. It allows for fast prototyping on a CPU for small systems through the use of NumPy. Using CuPy on a GPU, cell populations of up to 10,000 cells can be simulated within a few seconds. As such, we hope it can also be of use to modelers interested in testing preliminary hypotheses before committing to more complex center-based model frameworks.

AMS subject classification65Z05, 92C15, 92-10


title: A Modified Comprehensive Grading System for Murine Knee Osteoarthritis: Scoring the Whole Joint as an Organ

abstract:
ObjectiveThis study aimed to develop a comprehensive but easy to apply histologic grading system to score osteoarthritic changes in the whole knee joint for both spontaneous and posttraumatic osteoarthritis (OA) mouse models.

MethodsThe new OA grading scheme was developed based on extensive literature review and the authors experience in mouse OA models with relatively long periods of observation (up to 24 months of age or 24-week post-surgery). Semi-quantitative assessments of the histopathologic OA changes were applied to all four quadrants of the knee. Grading elements per quadrant were defined as follows: cartilage (0-7) including three new grading elements for early- and late-stage OA, respectively; osteophyte (0-2) covering chondro-osteophytes in both outer and inner joint margins; subchondral bone (0-2) containing subchondral bone thickening and destruction; synovitis (0-2) comprised of both synovial plica and intercondylar notch; and peri-articular tissues (0-2) highlighting ectopic chondrogenesis and ossification in the knee capsule, ligament, and musculature.

ResultsStatistical analyses showed that the new grading system had high intra- and inter-observer reproducibility (Pearsons correlation coefficients r >0.9) for both experienced and novice scorers. Sensitivity and reliability analyses confirmed the ability of the new system to detect minimal OA progression between two timepoints with a two-week interval and to accurately identify tissue-specific OA severity within the knee joint.

ConclusionsThe comprehensive histologic grading system presented here covers all-stage osteoarthritic changes in all major knee joint tissues of mice, which enable us to score OA severity for the whole joint reproducibly and accurately without software-assisted time-consuming measurements.

category: pathology


title: Development of a Cell Surface Display System in Chlamydomonas reinhardtii

abstract: Cell-surface display systems are biotechnological techniques used to express heterologous proteins on the cell surface. Their application depends directly on the cell system used, as well as on the anchoring point for the surface displayed protein. To meet most application demands an inexpensive, safe, and scalable production platform, that reduces the economic barriers for large scale use is needed. Towards this goal, we screened three possible cell surface anchoring points in the green algae Chlamydomonas by fusing mVenus to prospective anchors moieties. The vectors harboring mVenus:anchor were screened for mVenus fluorescence and tested for cellular localization by confocal laser scanning microscopy. This strategy allowed the identification of two functional anchors, one for the cytoplasmic membrane using the MAW8 GPI-anchor signal, and one for the cell wall using the GP1 protein. We also exploited GP1 chemical and biological traits to release the fused proteins efficiently during cell wall shedding. Our work provides a foundation for surface engineering of C reinhardtii supporting both cell biology studies and biotechnology applications.

category: synthetic biology


title: Precision environmental health monitoring by longitudinal exposome and multi-omics profiling

abstract: Conventional environmental health studies primarily focused on limited environmental stressors at the population level, which lacks the power to dissect the complexity and heterogeneity of individualized environmental exposures. Here we integrated deep-profiled longitudinal personal exposome and internal multi-omics to systematically investigate how the exposome shapes an individuals phenome. We annotated thousands of chemical and biological components in the personal exposome cloud and found they were significantly correlated with thousands of internal biomolecules, which was further cross validated using corresponding clinical data. In particular, our results showed that agrochemicals (e.g., carcinogenic pesticides, fungicides, and herbicides) and fungi predominated in the highly diverse and dynamic personal exposome, and the biomolecules and pathways related to the individuals immune system, kidneys, and liver were highly correlated with the personal external exposome. Overall, our findings demonstrate dynamic interactions between the personal exposome and internal multi-omics and provide important insights into the impact of the environmental exposome on precision health.


title: Neural Representations of Food-Related Attributes in the Human Orbitofrontal Cortex in Health and Disease

abstract:
Decisions about what to eat recruit the orbitofrontal cortex (OFC) and involve the evaluation of food-related attributes, such as taste and health. These attributes are utilized differently by healthy individuals and patients with disordered eating behavior, but it is unclear whether these attributes are decodable from activity in the OFC in both groups and whether neural representations of these attributes are differentially related to decisions about food. We used fMRI combined with behavioral tasks to investigate the representation of taste and health attributes in the human OFC and the role of these representations in food choices in healthy individuals and patients with anorexia nervosa (AN). We found that subjective ratings of tastiness and healthiness could be decoded from patterns of activity in the OFC in both groups. However, health-related patterns of activity in the OFC were more related to the magnitude of choice preferences among patients with AN than healthy individuals. These findings suggest that maladaptive decision-making in AN is associated with more consideration of health information represented by the OFC during deliberation about what to eat.

Significance StatementAn open question about the orbitofrontal cortex (OFC) is whether it supports the evaluation of food-related attributes during deliberation about what to eat. We found that healthiness and tastiness information were decodable from patterns of neural activity in the OFC in both patients with anorexia nervosa (AN) and healthy controls. Critically, neural representations of health were more strongly related to choices in patients with AN, suggesting that maladaptive overconsideration of healthiness during deliberation about what to eat is related to activity in the OFC. More broadly, these results show that activity in the human OFC is associated with the evaluation of relevant attributes during value-based decision-making. These findings may also guide future research into the development of treatments for AN.


title: Neural Representations of Food-Related Attributes in the Human Orbitofrontal Cortex During Choice Deliberation in Anorexia Nervosa


title: Somatic genetics analysis of sleep in adult mice

abstract: Classical forward and reverse mouse genetics approaches require germline mutations and, thus, are unwieldy to study sleep functions of essential genes or redundant pathways. It is also time-consuming to conduct electroencephalogram/electromyogram-based mouse sleep screening owning to labor-intensive surgeries and genetic crosses. Here, we describe a highly accurate SleepV (video) system and adeno-associated virus (AAV)-based adult brain chimeric (ABC)- expression/knockout (KO) platform for somatic genetics analysis of sleep in adult mice. A pilot ABC-expression screen identifies CREB and CRTC1, of which constitutive or inducible expression significantly reduces quantity and quality of non-rapid eye movement sleep. Whereas ABC-KO of exon 13 of Sik3 by AAV-Cre injection in Sik3-E13flox/flox adult mice phenocopies Sleepy (Sik3Slp/+) mice, ABC-CRISPR of Slp/Sik3 reverses hypersomnia of Sleepy mice, indicating a direct role of SLP/SIK3 kinase in sleep regulation. Multiplex ABC-CRISPR of both orexin/hypocretin receptors causes narcolepsy-like episodes, enabling one-step analysis of redundant genes in adult mice. Finally, ABC-expression/KO screen identifies Ankrd63 and NR1 as two potentially new sleep regulators. Therefore, this somatic genetics approach should facilitate high-throughput analysis of sleep regulatory genes, especially for essential or redundant genes, in adult mice by skipping the mouse development and genetic crosses.


title: Toward Reliable Network Neuroscience for Mapping Individual Differences

abstract: A rapidly emerging application of network neuroscience in neuroimaging studies has provided useful tools to understand individual differences in complex brain function. However, the variability of methodologies applied across studies - with respect to node definition, edge construction, and graph measurements-makes it difficult to directly compare findings and also challenging for end users to select the optimal strategies for mapping individual differences in brain networks. Here, we aim to provide a benchmark for best practices by systematically comparing the reliability of human brain network measurements of individual differences under different analytical strategies using the test-retest design of the resting-state functional magnetic resonance imaging from the Human Connectome Project. The results uncovered four essential principles to guide reliable network neuroscience of individual differences: 1) use a whole brain parcellation to define network nodes, including subcortical and cerebellar regions, 2) construct functional connectome using spontaneous brain activity in multiple slow bands, 3) optimize topological economy of networks at individual level, 4) characterise information flow with metrics of integration and segregation.


title: Cecropins contribute to Drosophila host defense against fungal and Gram-negative bacterial infection

abstract: Cecropins are small helical secreted peptides with antimicrobial activity that are widely distributed among insects. Genes encoding Cecropins are strongly induced upon infection, pointing to their role in host-defence. In Drosophila, four Cecropin genes clustered in the genome (CecA1, CecA2, CecB and CecC) are expressed upon infection downstream of the Toll and Imd pathways. In this study, we generated a short deletion{Delta} CecA-C removing the whole Cecropin locus. Using the{Delta} CecA-C deficiency alone or in combination with other antimicrobial peptide (AMP) mutations, we addressed the function of Cecropins in the systemic immune response.{Delta} CecA-C flies were viable and resisted challenge with various microbes as wild-type. However, removing{Delta} CecA-C in flies already lacking ten other AMP genes revealed a role for Cecropins in defence against Gram-negative bacteria and fungi. Measurements of pathogen loads confirm that Cecropins contribute to the control of certain Gram-negative bacteria, notably Enterobacter cloacae and Providencia heimbachae. Collectively, our work provides the first genetic demonstration of a role for Cecropins in insect host defence, and confirms their in vivo activity primarily against Gram-negative bacteria and fungi. Generation of a fly line ({Delta}AMP14) that lacks fourteen immune inducible AMPs provides a powerful tool to address the function of these immune effectors in host-pathogen interactions and beyond.

category: immunology


title: Genomic characterization of endemic and ecdemic non-typhoidal Salmonella enterica lineages circulating among animals and animal products in South Africa

abstract:
Non-typhoidal Salmonella enterica imposes a significant burden on human and animal health in South Africa. However, very little is known about lineages circulating among animals and animal products in the country on a genomic scale. Here, we used whole-genome sequencing (WGS) to characterize 63 Salmonella enterica strains (n = 18, 8, 13, and 24 strains assigned to serotypes Dublin, Hadar, Enteritidis, and Typhimurium, respectively) isolated from livestock, companion animals, wildlife, and animal products in South Africa over a 60-year period. Within-serotype phylogenies were constructed using genomes sequenced in this study, as well as publicly available genomes representative of each respective serotypes (i) global (n = 2,802 and 1,569 S. Dublin and Hadar genomes, respectively) and (ii) African (n = 716 and 343 S. Enteritidis and Typhimurium genomes, respectively) population. For S. Dublin, the approaches used here identified a largely antimicrobial-susceptible, endemic lineage circulating among humans, animals, and food in South Africa, as well as a lineage that was likely recently introduced from the United States. For S. Hadar, multiple South African lineages harboring streptomycin and tetracycline resistance-conferring genes were identified. African S. Enteritidis could be primarily partitioned into one largely antimicrobial-susceptible and one largely multidrug-resistant (MDR) clade, with South African isolates confined to the largely antimicrobial-susceptible clade. S. Typhimurium strains sequenced here were distributed across the African S. Typhimurium phylogeny, representing a diverse range of lineages, including numerous MDR lineages. Overall, this study provides insight into the evolution, population structure, and antimicrobial resistome composition of Salmonella enterica in Africa.

IMPORTANCEGlobally, Salmonella enterica is estimated to be responsible for more than 93 million illnesses and 150,000 deaths annually. In Africa, the burden of salmonellosis is disproportionally high; however, WGS efforts are overwhelmingly concentrated in world regions with lower salmonellosis burdens. While WGS is being increasingly employed in South Africa to characterize Salmonella enterica, the bulk of these efforts have centered on characterizing human clinical strains. WGS data derived from non-typhoidal Salmonella enterica serotypes isolated from non-human sources in South Africa is extremely limited. To our knowledge, the genomes sequenced here represent the largest collection of non-typhoidal Salmonella enterica isolate genomes from non-human sources in South Africa to date. Furthermore, this study provides critical insights into endemic and ecdemic non-typhoidal Salmonella enterica lineages circulating among animals, foods, and humans in South Africa and showcases the utility of WGS in characterizing animal-associated strains from a world region with a high salmonellosis burden.


title: Structure-based design of a highly stable, covalently-linked SARS-CoV-2 spike trimer with improved structural properties and immunogenicity

abstract: The continued threat of SARS-CoV-2 to global health necessitates development of improved research tools and vaccines. We present an improved SARS-CoV-2 spike ectodomain, "VFLIP", bearing five proline substitutions, a flexible cleavage site linker, and an inter-protomer disulfide bond. VFLIP displays significantly improved stability, high-yield production and retains its trimeric state without exogenous trimerization motifs. High-resolution cryo-EM and glycan profiling reveal that the VFLIP quaternary structure and glycosylation mimic the native spike on the viral surface. Further, VFLIP has enhanced affinity and binding kinetics relative to other stabilized spike proteins for antibodies in the Coronavirus Immunotherapeutic Consortium (CoVIC), and mice immunized with VFLIP exhibit potent neutralizing antibody responses against wild-type and B.1.351 live SARS-CoV-2. Taken together, VFLIP represents an improved tool for diagnostics, structural biology, antibody discovery, and vaccine design.


title: Discovery of an Antarctic ascidian-associated uncultivated Verrucomicrobia that encodes antimelanoma palmerolide biosynthetic capacity

abstract:
The Antarctic marine ecosystem harbors a wealth of biological and chemical innovation that has risen in concert over millennia since the isolation of the continent and formation of the Antarctic circumpolar current. Scientific inquiry into the novelty of marine natural products produced by Antarctic benthic invertebrates led to the discovery of a bioactive macrolide, palmerolide A, that has specific activity against melanoma and holds considerable promise as an anticancer therapeutic. While this compound was isolated from the Antarctic ascidian Synoicum adareanum, its biosynthesis has since been hypothesized to be microbially mediated, given structural similarities to microbially-produced hybrid non-ribosomal peptide-polyketide macrolides. Here, we describe a metagenome-enabled investigation aimed at identifying the biosynthetic gene cluster (BGC) and palmerolide A-producing organism. A 74 Kbp candidate BGC encoding the multi-modular enzymatic machinery (hybrid Type I-trans-AT polyketide synthase-non-ribosomal peptide synthetase and tailoring functional domains) was identified and found to harbor key features predicted as necessary for palmerolide A biosynthesis. Surveys of ascidian microbiome samples targeting the candidate BGC revealed a high correlation between palmerolide-gene targets and a single 16S rRNA gene variant (R=0.83 - 0.99). Through repeated rounds of metagenome sequencing followed by binning contigs into metagenome-assembled genomes, we were able to retrieve a near-complete genome (10 contigs) of the BGC-producing organism, a novel verrucomicrobium within the Opitutaceae family that we propose here as Candidatus Synoicihabitans palmerolidicus. The refined genome assembly harbors five highly similar BGC copies, along with structural and functional features that shed light on the host-associated nature of this unique bacterium.

ImportancePalmerolide A has potential as a chemotherapeutic agent to target melanoma. We interrogated the microbiome of the Antarctic ascidian, Synoicum adareanum, using a cultivation-independent high-throughput sequencing and bioinformatic strategy. The metagenome-encoded biosynthetic machinery predicted to produce palmerolide A was found to be associated with the genome of a member of the S. adareanum core microbiome. Phylogenomic analysis suggests the organism represents a new deeply-branching genus, Candidatus Synoicihabitans palmerolidicus, in the Opitutaceae family of the Verrucomicrobia phylum. The Ca. S. palmerolidicus 4.29 Mb genome encodes a repertoire of carbohydrate-utilizing and transport pathways enabling its ascidian-associated lifestyle. The palmerolide-producers genome also contains five distinct copies of the large palmerolide biosynthetic gene cluster that may provide structural complexity of palmerolide variants.


title: Discovery of an Antarctic ascidian-associated uncultivated Verrucomicrobia with antimelanoma palmerolide biosynthetic potential


title: A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells

abstract: The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered a novel class of small molecule ketobenzothiazole TMPRSS2 inhibitors with significantly improved activity over existing irreversible inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East Respiratory Syndrome Coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice with a half-life of 8.6 hours in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.


title: Mechanical collaboration between the embryonic brain and the surrounding scalp tissues

abstract: During brain enlargement between embryonic day (E) 11 and E13, within the limited mouse cranial space enclosed by the scalp consisting of epidermal and preosteogenic mesenchymal layers, the angle between the pons and the medulla decreases. This raises the possibility that the scalp, upon being pushed outwardly by the brain and stretched, in return inwardly recoils to confine and fold the brain. Our stress-releasing tests showed that the scalp recoiled to shrink more extensively at E12~13 than at E15~16 and that the in vivo pre-stretch prerequisite for this recoil response depended physically on the brain (pressurization at 77~93 Pa) and on actomyosin and elastin within the scalp layers. Under non-stretched conditions, scalp cell proliferation declined, while re-stretching of the shrunken scalp rescued proliferation. In scalp-removed heads, pons-medulla folding was reduced, and the spreading of ink from the lateral ventricle to the spinal cord that occurred in scalp-intact embryos (with >5 l injection) was lost, suggesting that the scalp plays roles in brain morphogenesis and cerebrospinal fluid homeostasis. Thus, the brain and the scalp mechanically interact and collaborate.


title: Accuracy in near-perfect virus phylogenies

abstract: AO_SCPLOWBSTRACTC_SCPLOWPhylogenetic trees from real-world data often include short edges with very few substitutions per site, which can lead to partially resolved trees and poor accuracy. Theory indicates that the number of sites needed to accurately reconstruct a fully resolved tree grows at a rate proportional to the inverse square of the length of the shortest edge. However, when inferred trees are partially resolved due to short edges, "accuracy" should be defined as the rate of discovering false splits (clades on a rooted tree) relative to the actual number found. Thus, accuracy can be high even if short edges are common. Specifically, in a "near-perfect" parameter space in which trees are large, the tree length{xi} (the sum of all edge lengths), is small, and rate variation is minimal, the expected false positive rate is less than{xi} /3; the exact value depends on tree shape and sequence length. This expected false positive rate is far below the false negative rate for small{xi} and often well below 5% even when some assumptions are relaxed. We show this result analytically for maximum parsimony and explore its extension to maximum likelihood using theory and simulations. For hypothesis testing, we show that measures of split "support" that rely on bootstrap resampling consistently imply weaker support than that implied by the false positive rates in near-perfect trees. The near-perfect parameter space closely fits several empirical studies of human virus diversification during outbreaks and epidemics, including Ebolavirus, Zika virus, and SARS-CoV-2, reflecting low substitution rates relative to high transmission/sampling rates in these viruses.


title: RF-Net 2: Fast Inference of Virus Reassortment and Hybridization Networks

abstract:
MotivationA phylogenetic network is a powerful model to represent entangled evolutionary histories with both divergent (speciation) and convergent (e.g., hybridization, reassortment, recombination) evolution. The standard approach to inference of hybridization networks is to (i) reconstruct rooted gene trees and (ii) leverage gene tree discordance for network inference. Recently, we introduced a method called RF-Net for accurate inference of virus reassortment and hybridization networks from input gene trees in the presence of errors commonly found in phylogenetic trees. While RF-Net demonstrated the ability to accurately infer networks with up to four reticulations from erroneous input gene trees, its application was limited by the number of reticulations it could handle in a reasonable amount of time. This limitation is particularly restrictive in the inference of the evolutionary history of segmented RNA viruses such as influenza A virus (IAV), where reassortment is one of the major mechanisms shaping the evolution of these pathogens.

ResultsHere we expand the functionality of RF-Net that makes it significantly more applicable in practice. Crucially, we introduce a fast extension to RF-Net, called Fast-RF-Net, that can handle large numbers of reticulations without sacrificing accuracy. Additionally, we develop automatic stopping criteria to select the appropriate number of reticulations heuristically and implement a feature for RF-Net to output error-corrected input gene trees. We then conduct a comprehensive study of the original method and its novel extensions and confirm their efficacy in practice using extensive simulation and empirical influenza A virus evolutionary analyses.

AvailabilityRF-Net 2 is available at https://github.com/flu-crew/rf-net-2.


title: DNA methylation calling tools for Oxford Nanopore sequencing: a survey and human epigenome-wide evaluation

abstract:
BackgroundNanopore long-read sequencing technology greatly expands the capacity of long-range single-molecule DNA-modification detection. A growing number of analytical tools have been actively developed to detect DNA methylation from Nanopore sequencing reads. Here, we examine the performance of different methylation calling tools to provide a systematic evaluation to guide practitioners for human epigenome-wide research.

ResultsWe compare five analytic frameworks for detecting DNA modification from Nanopore long-read sequencing data. We evaluate the association between genomic context, CpG methylation-detection accuracy, CpG sites coverage, and running time using Nanopore sequencing data from natural human DNA. Furthermore, we provide an online DNA methylation database (https://nanome.jax.org) with which to display genomic regions that exhibit differences in DNA-modification detection power among different methylation calling algorithms for nanopore sequencing data.

ConclusionsOur study is the first benchmark of computational methods for mammalian whole genome DNA-modification detection in Nanopore sequencing. We provide a broad foundation for cross-platform standardization, and an evaluation of analytical tools designed for genome-scale modified-base detection using Nanopore sequencing.

category: bioinformatics


title: Unbiased lexicometry analyses illuminate plague dynamics during the second pandemic

abstract: Knowledge of the second plague pandemic that swept over Europe during the 14th-19th centuries, mainly relies on the exegesis of contemporary texts, prone to interpretive bias. Leveraging bioinformatic tools routinely used in biology, we here developed a quantitative lexicography of 32 texts describing two major plague outbreaks using contemporary plague-unrelated texts as negative controls. Nested, network and category analyses of a 207-word pan-lexicome over-represented in plague-related texts, indicated that "buboes" and "carbuncles" words were significantly associated with plague, signaling ectoparasite- borne plague. Moreover, plague-related words were associated with the words "merchandise", "movable", "tatters", "bed" and "clothes", while no association was found with rats and fleas. These results support the hypothesis that during the second plague pandemic, human ectoparasites were the major drivers of plague. Analyzing ancient texts using here reported method would certify plague-related historical records and indicate particularities of each plague outbreak, including sources of the causative Yersinia pestis.


title: Diversity and selection of SARS-CoV-2 minority variants in the early New York City outbreak

abstract:
High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. Here we analyzed minority variants within the SARS-CoV-2 data in 12 samples from the early outbreak in New York City, using replicate sequencing for reliable identification. While most minority variants were unique to a single sample, we found several instances of shared variants. We provide evidence that some higher-frequency minority variants may be transmitted between patients or across short transmission chains, while other lower-frequency, more widely shared variants arise independently. Further, our data indicate that even with a small transmission bottleneck, the heterogeneity of intra-host viral populations is enhanced by minority variants present in transmission samples. Our data suggest that analysis of shared minority variants could help identify regions of the SARS-CoV-2 genome that are under increased selective pressure, as well as inform transmission chains and give insight into variant strain emergence.

IMPORTANCEWhen viruses replicate inside a host, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus, nor strongly beneficial, can lead to minority variants. In this study, we analyzed the minority variants in SARS-CoV-2 patient samples from New York City during the early outbreak. We found common minority variants between samples that were closely related and showed that these minority variants may be transmitted from one patient to another. We show that in general, transmission events between individuals likely contain genetically diverse viral particles, and we find signatures of selection governing intra-host evolution. We conclude that the analysis of shared minority variants can help to identify transmission events and give insight into emergence of new viral variants.


title: The Effect of Minnelide against SARS-CoV-2 in a Murine Model

abstract: Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, is the causative agent of coronavirus disease 2019, COVID-19, and the current COVID-19 pandemic. Even as more vaccine candidates are released, more treatment options are critically needed. Here, we investigated the use of Minnelide, a water soluble pro-drug with anti-inflammatory properties, for the treatment of COVID-19. To do this, k18-hACE2 mice were infected with SARS-CoV-2 or given PBS control intranasally. The next day mice were either treated daily with low dose (0.0025mg/day) or high dose Minnelide (0.005mg/day), or given vehicle control intraperitoneal. Mice were weighed daily, and sacrificed at day 6 and 10 post-infection to analyze viral burden, cytokine response, and pathology. We observed a reduction in viral load in the lungs of Minnelide-treated mice infected with SARS-CoV-2 at day 10 post-infection compared to day 6 post-infection. All SARS-CoV-2 infected non-treated mice were moribund six days post-infection while treatment with Minnelide extended survival for both low (60% survival) and high (100% survival) dose treated mice ten days post-infection. Interestingly, cytokine analysis demonstrated a significant reduction in IL-6 (lung and heart) and D-dimer (serum) in high dose treated SARS-CoV-2 infected mice compared to mice infected with SARS-CoV-2 alone at day 6 post-infection. Additionally, histology analysis revealed that Minnelide treatment significantly improved lung pathology ten days post-infection with SARS-CoV-2 with all the mice exhibiting normal lung tissue with thin alveolar septa and no inflammatory cells. Overall, our study exhibits potential for the use of Minnelide to improve survival in COVID-19 patients.


title: Direct comparison of category and spatial selectivity in human occipitotemporal cortex

abstract: Human visual cortex is organised broadly according to two major principles: retinotopy (the spatial mapping of the retina in cortex) and category-selectivity (preferential responses to specific categories of stimuli). Historically, these principles were considered anatomically separate, with retinotopy restricted to the occipital cortex and category-selectivity emerging in the lateral-occipital and ventral-temporal cortex. However, recent studies show that category-selective regions exhibit systematic retinotopic biases, for example exhibiting stronger activation for stimuli presented in the contra- compared to the ipsilateral visual field. It is unclear, however, whether responses within category-selective regions are more strongly driven by retinotopic location or by category preference, and if there are systematic differences between category-selective regions in the relative strengths of these preferences. Here, we directly compare contralateral and category preferences by measuring fMRI responses to scene and face stimuli presented in the left or right visual field and computing two bias indices: a contralateral bias (response to the contralateral minus ipsilateral visual field) and a face/scene bias (preferred response to scenes compared to faces, or vice versa). We compare these biases within and between scene- and face-selective regions and across the lateral and ventral surfaces of visual cortex more broadly. We find an interaction between surface and bias: lateral surface regions show a stronger contralateral than face/scene bias, whilst ventral surface regions show the opposite. These effects are robust across and within subjects, and appear to reflect large-scale, smoothly varying gradients. Together, these findings support distinct functional roles for lateral and ventral visual cortex in terms of the relative importance of the spatial location of stimuli during visual information processing.


title: Direct comparison of contralateral bias and face/scene selectivity in human occipitotemporal cortex


title: Cortical granularity shapes information flow to the amygdala and its striatal targets in nonhuman primate

abstract:
The prefrontal cortex (PFC) and insula, amygdala, and striatum form interconnected networks that drive motivated behaviors. We previously found a connectional trend in which granularity of the ventromedial and orbital PFC/insula predicted connections to the amygdala, and also the breadth of amygdalo-striatal efferents, including projections beyond the classic ventral striatum. To further interrogate connectional relationships among the cortex, amygdala, and striatum, and to further define the limbic (amygdala-recipient) striatum, we conducted tract tracing studies in two cohorts of Macaques (Male n = 14, Female n = 1). We focused on the cortico-amygdalo-striatal (indirect) and cortico- limbic striatal (direct) paths originating in the entire PFC and insula. Larger data sets and a quantitative approach revealed cortical rules in which cortical granularity predicts the complexity and location of projections to both the basal nucleus of the amygdala and striatum. Remarkably, projections from cortical-like basal nucleus to the striatum followed similar patterns. In both direct and indirect paths to the limbic striatum, agranular cortices formed a foundational, broad projection, and were joined by inputs from progressively more differentiated cortices. In amygdalo-striatal paths, the ventral basal nucleus was the  foundational input, with progressively more dorsal basal nucleus regions gradually adding inputs as the limbic striatum extended caudally. Together, the  indirect and  direct paths followed consistent principles in which cortical granularity dictated the strength and complexity of projections at their targets. Cluster analyses independently confirmed these connectional trends, and also highlighted connectional features that predicted termination in specific subregions of the basal nucleus and limbic striatum.

Significance StatementThe limbic system broadly refers to brain circuits that coordinate emotional responses. Here, we investigate circuits of the amygdala, which are involved in coding the emotional value of external cues, and their influence on the striatum. Regions of prefrontal cortex and insula form gradients of overlapping inputs to the amygdalas basal nucleus, which feed forward to the striatum. Direct cortical inputs to these amygdala-recipient striatal areas are surprisingly organized according to similar principles, but subtly shift from the classic ventral striatum to the caudal ventral striatum. Together, these distinct subsystems--cortico-amygdala-striatal circuits and direct cortico-striatal circuits-- provide substantial opportunity for different levels of internal, sensory, and external experiences to be integrated within the striatum, a major motor-behavioral interface.


title: Cortical granularity shapes the organization of afferent paths to the amygdala and its striatal targets in nonhuman primate


title: Identification of DAXX As A Restriction Factor Of SARS-CoV-2 Through A CRISPR/Cas9 Screen

abstract: Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identified DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX was sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricted infection. In contrast with most of its previously described antiviral activities, DAXX-mediated restriction of SARS-CoV-2 was independent of the SUMOylation pathway. SARS-CoV-2 infection triggered the re-localization of DAXX to cytoplasmic sites and promoted its degradation. Mechanistically, this process was mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.


title: Ser/Thr phospho-regulation by PknB and Stp mediates bacterial quiescence and antibiotic persistence in Staphylococcus aureus

abstract:
Staphylococcus aureus colonizes 30 to 50% of healthy adults and can cause a variety of diseases, ranging from superficial to life-threatening invasive infections such as bacteraemia and endocarditis. Often, these infections are chronic and difficult-to-treat despite adequate antibiotic therapy. Most antibiotics act on metabolically active bacteria in order to eradicate them. Thus, bacteria with minimized energy consumption resulting in metabolic quiescence, have increased tolerance to antibiotics. The most energy intensive process in cells - protein synthesis - is attenuated in bacteria entering into quiescence. Eukaryote-like serine/threonine kinases (STKs) and phosphatases (STPs) can fine-tune essential cellular processes, thereby enabling bacteria to quickly respond to environmental changes and to modulate quiescence. Here, we show that deletion of the only annotated functional STP, named Stp, in S. aureus leads to increased bacterial lag-phase and phenotypic heterogeneity under different stress challenges, including acidic pH, intracellular milieu and in vivo abscess environment. This growth delay was associated with reduced intracellular ATP levels and increased antibiotic persistence. Using phosphopeptide enrichment and mass spectrometry-based proteomics, we identified possible targets of Ser/Thr phosphorylation that regulate cellular processes and bacterial growth, such as ribosomal proteins including the essential translation elongation factor EF-G. Finally, we show that acid stress leads to a reduced translational activity in the stp deletion mutant indicating metabolic quiescence correlating with increased antibiotic persistence.

One-sentence summaryPhospho-regulation mediates quiescence and antibiotic persistence in Staphylococcus aureus.


title: Hybridization underlies localized trait evolution in cavefish

abstract: Compared to selection on new mutations and standing genetic variation, the role of gene flow in generating adaptive genetic variation has been subject to much debate. Theory predicts that gene flow constrains adaptive evolution via natural selection by homogenizing allele frequencies among populations and introducing migrant alleles that may be locally maladaptive1. However, recent work has revealed that populations can diverge even when high levels of gene flow are present2-4 and that gene flow may play an underappreciated role in facilitating local adaptation by increasing the amount of genetic variation present for selection to act upon5-8. Here, we investigate how genetic variation introduced by gene flow contributes to adaptive evolution of complex traits using an emerging eco-evolutionary model system, the Mexican tetra (Astyanax mexicanus). The ancestral surface form of the Mexican tetra has repeatedly invaded and adapted to cave environments. The Chica cave is unique in that it contains several pool microenvironments inhabited by putative hybrids between surface and cave populations9, providing an opportunity to investigate the dynamics of complex trait evolution and gene flow on a local scale. Here we conduct high-resolution genomic mapping and analysis of eye morphology and pigmentation in fish from multiple pools within Chica cave. We demonstrate that hybridization between cave and surface populations contributes to highly localized variation in behavioral and morphological traits. Analysis of sleep and locomotor behaviors between individual pools within this cave revealed reduced sleep associated with an increase in ancestry derived from cave populations, suggesting pool-specific ecological differences may drive the highly-localized evolution of sleep and locomotor behaviors. Lastly, our analyses uncovered a compelling example of convergent evolution in a core circadian clock gene in multiple independent cavefish lineages and burrowing mammals, indicating a shared genetic mechanism underlying circadian disruption in subterranean vertebrates. Together, our results provide insight into the evolutionary mechanisms that promote adaptive genetic variation and the genetic basis of complex behavioral phenotypes involved in local adaptation.


title: Genetic Structure and Diversity of Banana Bunchy Top Virus (BBTV) in the Philippines

abstract: Banana bunchy top virus (BBTV) is an important disease of banana in the Philippines and in other banana-producing countries. This study was conducted to investigate the genetic structure and diversity of Philippine BBTV isolates which remain unexplored in the country. BBTV-infected plant tissues were sampled from banana-growing provinces (i.e., Cagayan, Isabela, Quirino, Batangas, Laguna, Rizal, Quezon, Palawan, Cebu, Leyte, and Davao del Sur) and the partial DNA-R gene of BBTV was sequenced. Analysis of all local BBTV isolates showed a nucleotide diversity ({pi}) of 0.00721, average number of nucleotide differences (k) of 5.51984, and haplotype diversity (hd) of 0.971. Neutrality tests using Fus Fs and Tajimas D showed significant and highly negative values which suggest an excess number of rare alleles due to recent population expansion or from genetic hitchhiking. Haplotype network and phylogenetic analyses revealed that the local BBTV isolates were closely related to Southeast Asian (SEA) group and exhibited a monophyletic clade with distinct haplotype grouping from other SEA sequences. However, some Indonesian and Indian reference sequences were also clustered within the Philippine BBTV group suggesting sequence homology. Results also showed that the local BBTV isolates may be categorized into three major haplotype groups (HA, HB, and HC) but only the HC group remained distinct upon comparison with other Philippine and SEA reference sequences. BBTV isolates from Quezon were the most diverse while isolates from Palawan displayed low genetic diversity indices and belonged only in the HC group. The assessment of the degree of variability among Philippine BBTV isolates will provide a reference towards the development of high-throughput BBTV detection systems as well as enable to devise plant breeding strategies to manage the current BBTV spread and variations.


title: LncRNA DLEU2 regulates Sirtuins and mitochondrial respiratory chain complex IV: a novel pathway in obesity and DOHaD

abstract:
Long noncoding RNAs (lncRNAs) are commonly dysregulated in cancer but are mostly unknown for roles in metabolic regulation. Sirtuins, an epigenetic modulator class, regulate metabolic pathways. However, how sirtuins are regulated via lncRNA is unknown. In our findings, DLEU2, a lncRNA involved in primarily blood cancers, and sirtuins were both significantly decreased in the livers of high-fat high-fructose diet (HFD-HF) fed male mouse offspring (F1) whose mothers (F0) were either fed chow diet or HFD-HF during reproductive and pregnancy windows. Confirming this connection, upon silencing DLEU2, transcription levels of SIRT1 through 6 and translational levels of SIRT1, 3, 5, and 6 were significantly downregulated. Knockdown of DLEU2 also significantly decreased the protein level of cytochrome-c oxidase (complex IV, MTCO1) without altering other mitochondrial complexes, leading to increased reactive oxygen species production. Interestingly, in F1 livers, the protein level of MTCO1 was also significantly decreased under an HFD-HF diet or even under chow diet if the mother was exposed to HFD-HF. Our findings therefore reveal for the first time that a lncRNA can regulate sirtuins and a specific mitochondrial complex, furthermore suggesting a potential role of DLEU2 in metabolic disorders over one or more generations.

HIGHLIGHTSMaternal diet can modulate hepatic lncRNA DLEU2 and its downstream regulators in offspring

DLEU2 is required for specific sirtuin regulation and mitochondrial respiration chain complex IV expression

Offspring liver depleted of DLEU2 show decreased mitochondrial respiration chain complex IV and specific sirtuins at transcriptional and translational levels