title: Towards a genome sequence for every animal: where are we now?

abstract:
In less than 25 years, the field of animal genome science has transformed from a discipline seeking its first glimpses into genome sequences across the Tree of Life to a global enterprise with ambitions to sequence genomes for all of Earths eukaryotic diversity (1). As the field rapidly moves forward, it is important to take stock of the progress that has been made to best inform the disciplines future. In this Perspective, we provide a contemporary, quantitative overview of animal genome sequencing. We identified the best available genome assemblies on GenBank, the worlds most extensive genetic database, for 3,278 unique animal species across 24 phyla. We assessed taxonomic representation, assembly quality, and annotation status for major clades. We show that while tremendous taxonomic progress has occurred, stark disparities in genomic representation exist, highlighted by a systemic overrepresentation of vertebrates and underrepresentation of arthropods. In terms of assembly quality, long-read sequencing has dramatically improved contiguity, whereas gene annotations are available for just 34.3% of taxa. Furthermore, we show that animal genome science has diversified in recent years with an ever-expanding pool of researchers participating. However, the field still appears to be dominated by institutions in the Global North, which have been listed as the submitting institution for 77% of all assemblies. We conclude by offering recommendations for how we can collectively improve genomic resource availability and value while also broadening global representation.

Significance statementThe field of animal genome science is rapidly developing, and efforts are underway to sequence genomes for all of Earths eukaryotic biodiversity. Here, we provide an overview of animal genome sequencing, with emphases on taxonomic representation, assembly quality, and geographic representation. We show that while a staggering 3,278 unique animal species have had their genomes sequenced, massive disparities exist in terms of the taxonomic groups receiving attention, the quality of the resources being produced, and the institutions driving the field. We highlight areas where improvements can be made, notably by continuing to increase the quality of genome assemblies, including by improving metadata and voucher specimen associations, and actively developing meaningful collaborations between researchers form the Global North and South.


title: The heterogeneity of the DNA damage response landscape determines patient outcomes in ovarian cancer

abstract:
Defective DNA damage response (DDR) pathways allow cancer cells to accrue genomic aberrations and evade normal cellular growth checkpoints. Defective DDR also determines response to chemotherapy. However, the interaction and overlap between the two double strand repair pathways and the three single strand repair pathways is complex, and has remained poorly understood.

Here we show that, in ovarian cancer, a disease hallmarked by chromosomal instability, explant cultures show a range of DDR abrogation patterns. Defective homologous recombination (HR) and non-homologous end joining (NHEJ) are near mutually exclusive with HR deficient (HRD) cells showing increased abrogation of the single strand repair pathways compared to NHEJ defective cells.

When combined with global markers of DNA damage, including mitochondrial membrane functionality and reactive oxygen species burden, the pattern of DDR abrogation allows the construction of DDR signatures which are predictive of both ex vivo cytotoxicity, and more importantly, patient outcome.

SignificanceHolistic assessment of the DDR is possible, shows improved ability to predict response to chemotherapy over single pathway assessment, and is applicable to a variety of ovarian cancer types. Such an assessment has clinical utility in settings of therapeutic dilemma such as retreatment for relapse.

category: cell biology


title: Catalase protects against non-enzymatic decarboxylations during photorespiration

abstract:
Photorespiration recovers carbon that would be otherwise lost following the oxygenation reaction of rubisco and production of glycolate. Photorespiration is essential in plants and recycles glycolate into usable metabolic products through reactions spanning the chloroplast, mitochondrion, and peroxisome. Catalase in peroxisomes plays an important role in this process by disproportionating H2O2 resulting from glycolate oxidation into O2 and water. We hypothesize that catalase in the peroxisome also protects against non-enzymatic decarboxylations between hydrogen peroxide and photorespiratory intermediates (glyoxylate and/or hydroxypyruvate). We test this hypothesis by detailed gas exchange and biochemical analysis of Arabidopsis thaliana mutants lacking peroxisomal catalase. Our results strongly support this hypothesis, with catalase mutants showing gas exchange evidence for an increased stoichiometry of CO2 release from photorespiration, specifically an increase in the CO2 compensation point, a photorespiratory-dependent decrease in the quantum efficiency of CO2 assimilation, increase in the 12CO2 released in a 13CO2 background and an increase in the post-illumination CO2 burst. Further metabolic evidence suggests this excess CO2 release occurred via the non-enzymatic decarboxylation of hydroxypyruvate. Specifically, the catalase mutant showed an accumulation of photorespiratory intermediates during a transient increase in rubisco oxygenation consistent with this hypothesis. Additionally, end products of alternative hypotheses explaining this excess release were similar between wild type and catalase mutants. Furthermore, the calculated rate of hydroxypyruvate decarboxylation in catalase mutant is much higher than that of glyoxylate decarboxylation. This work provides evidence that these non-enzymatic decarboxylation reactions, predominately hydroxypyruvate decarboxylation, can occur in vivo when photorespiratory metabolism is genetically disrupted.

One Sentence SummaryCatalase guards against additional carbon loss from photorespiration arising from non-enzymatic decarboxylations of photorespiratory intermediates.

category: plant biology


title: Effect of electrical hybrid-frequency water bath stunning on the spontaneous electroencephalogram (EEG) and electrocardiogram (ECG) of broilers

abstract: Concerns about animal welfare and meat quality have encouraged research on new methods for the stunning of broilers during animal slaughter. In this study, the electroencephalogram (EEG) and electrocardiogram (ECG) of broilers were acquired during stunning using an electrical hybrid instead of a single frequency. Considering a square-wave with a current of 220 mA and a frequency of 1100 Hz (duty-cycle 50%), the hybrid-frequency waveform is obtained generating pulses at 6600 Hertz in the pulse-width phase. Sixty broilers aged 42 days were randomly sampled; thirty were used for EEG measurement and thirty for ECG measurement. For EEG measurements, the birds scalps were anaesthetized, and EEG electrode needles were inserted on the subcutaneous part of the occipital scalp. For ECG, the non-invasive surface electrode was used. The electrodes were connected to a digital EEG/ECG system. The results showed that the hybrid-frequency waveform system generated epileptic forms in the birds EEGs. Therefore, a hybrid-frequency system may present better carcass quality results, while preserving the birds welfare, when compared with a single frequency system use.

category: neuroscience


title: Genetic evidence reveals the indispensable role of the rseC gene for autotrophy and the importance of a functional electron balance for nitrate reduction in Clostridium ljungdahlii

abstract:
For Clostridium ljungdahlii, the RNF complex plays a key role for energy conversion from gaseous substrates such as hydrogen and carbon dioxide. In a previous study, a disruption of RNF-complex genes led to the loss of autotrophy, while heterotrophy was still possible via glycolysis. Furthermore, it was shown that the energy limitation during autotrophy could be lifted by nitrate supplementation, which resulted in an elevated cellular growth and ATP yield. Here, we used CRISPR-Cas12a to delete: 1) the RNF complex-encoding gene cluster rnfCDGEAB; 2) the putative RNF regulator gene rseC; and 3) a gene cluster that encodes for a putative nitrate reductase. The deletion of either rnfCDGEAB or rseC resulted in a complete loss of autotrophy, which could be restored by plasmid-based complementation of the deleted genes. We observed a transcriptional repression of the RNF-gene cluster in the rseC-deletion strain during autotrophy and investigated the distribution of the rseC gene among acetogenic bacteria. To examine nitrate reduction and its connection to the RNF complex, we compared autotrophic and heterotrophic growth of our three deletion strains with either ammonium or nitrate. The rnfCDGEAB- and rseC-deletion strains failed to reduce nitrate as a metabolic activity in non-growing cultures during autotrophy but not during heterotrophy. In contrast, the nitrate reductase deletion strain was able to grow in all tested conditions but lost the ability to reduce nitrate. Our findings highlight the important role of the rseC gene for autotrophy and contribute to understand the connection of nitrate reduction to energy metabolism.

Significance StatementAcetogenic bacteria are widely known for their ability to convert gaseous substrates, such as hydrogen, carbon dioxide, and carbon monoxide, into short-chain fatty acids and alcohols, which can be utilized as sustainable platform chemicals and fuels. However, acetogenic bacteria conserve energy at the thermodynamic limit of life during autotrophy, and thus the production of more complex and energy-dense chemicals is limited due to low ATP yields. Therefore, it is key to decipher the interplay of the electron balancing reactions to understand and optimize the acetogenic metabolism. Recent findings with alternative electron acceptors that accelerated the cellular growth and ATP yield during autotrophy, such as nitrate, provide an opportunity to overcome energetic barriers in the acetogenic metabolism. The interrogation of the nitrate metabolism and the interplay between nitrate reduction and energy conservation in C. ljungdahlii, will contribute to fine-tuning of the acetogenic metabolism for biotechnological applications.

category: microbiology


title: The biosynthetic pathway of ubiquinone contributes to pathogenicity of Francisella

abstract:
Francisella tularensis is the causative agent of tularemia. Because of its extreme infectivity and high mortality rate, this pathogen was classified as a biothreat agent. Francisella spp are strict aerobe and ubiquinone (UQ) has been previously identified in these bacteria. While the UQ biosynthetic pathways were extensively studied in Escherichia coli allowing the identification of fifteen Ubi-proteins to date, little is known about Francisella spp. In this study, and using Francisella novicida as a surrogate organism, we first identified UQ8 as the major quinone found in the membranes of this bacterium. Then, we characterized the UQ biosynthetic pathway in F. novicida using a combination of bioinformatics, genetics and biochemical approaches. Our analysis disclosed the presence in Francisella of ten putative Ubi-proteins and we confirmed eight of them by heterologous complementation in E. coli. The UQ biosynthetic pathways from F. novicida and E. coli share a similar pattern. However, differences were highlighted: the decarboxylase remains unidentified in Francisella spp and homologs of the Ubi-proteins involved in the O2-independent UQ pathway are not present. This is in agreement with the strictly aerobic niche of this bacterium. Then, via two approaches, i.e. the use of an inhibitor (3-amino-4-hydroxybenzoic acid) and a transposon mutant, which both strongly impair the synthesis of UQ, we demonstrated that UQ is essential for the growth of F. novicida in a respiratory medium and contributes to its pathogenicity in Galleria mellonella used as an alternative animal model.

ImportanceFrancisella tularensis is the causative bacterium of tularemia and is classified as a biothreat agent. Using multidisciplinary approaches, we investigated the ubiquinone (UQ) biosynthetic pathway that operates in F. novicida used as a surrogate. We showed that UQ8 is the major quinone identified in the membranes of Francisella novicida. We identified a new competitive inhibitor, which strongly decreased the biosynthesis of UQ. Our demonstration of the crucial role of UQ for the respiratory metabolism of F. novicida and for the involving in its pathogenicity in the Galleria mellonella model should stimulate the search for selective inhibitors of bacterial UQ biosynthesis.


title: In-depth analysis of Bacillus anthracis 16S rRNA genes and transcripts reveals intra- and intergenomic diversity and facilitates anthrax detection

abstract: Analysis of 16S ribosomal RNA (rRNA) genes provides a central means of taxonomic classification of bacterial species. Based on presumed sequence identity among species of the Bacillus cereus sensu lato group, the 16S rRNA genes of B. anthracis have been considered unsuitable for diagnosis of the anthrax pathogen. With the recent identification of a single nucleotide polymorphism in some 16S rRNA gene copies, specific identification of B. anthracis becomes feasible. Here, we designed and evaluated a set of in situ-, in vitro- and in silico-assays to assess the yet unknown 16S-state of B. anthracis from different perspectives. Using a combination of digital PCR, fluorescence in situ hybridization, long-read genome sequencing and bioinformatics we were able to detect and quantify a unique 16S rRNA gene allele of B. anthracis (16S-BA-allele). This allele was found in all available B.anthracis genomes and may facilitate differentiation of the pathogen from any close relative. Bioinformatics analysis of 959 B. anthracis genome data-sets inferred that abundances and genomic arrangements of the 16S-BA-allele and the entire rRNA operon copy-numbers differ considerably between strains. Expression ratios of 16S-BA-alleles were proportional to the respective genomic allele copy-numbers. The findings and experimental tools presented here provide detailed insights into the intra- and intergenomic diversity of 16S rRNA genes and may pave the way for improved identification of B. anthracis and other pathogens with diverse rRNA operons.


title: Evolutionary origin of sex differentiation system in insects

abstract: The evolution of the functionality of genes and genetic systems is a major source of animal diversity. Its best example is insect sex differentiation systems: promoting male and female differentiation (dual-functionality) or only male differentiation (single-functionality). However, the evolutionary origin of such functional diversity is largely unknown. Here, we investigate the ancestral functions of doublesex, a key factor of insect sex differentiation system, using the apterygote insect, Thermobia domestica, and reveal that its doublesex is essential for only males at the phenotypic level, but contributes to promoting female-specific vitellogenin expression in females. This functional discordance between the phenotypic and transcription-regulatory levels in T. domestica shows a new type of functionality of animal sex differentiation systems. Then, we examine how the sex differentiation system transited from the single-functionality to the dual-functionality in phenotypes and uncover that a conserved female-specific motif of doublesex is detected in taxa with the dual-functional doublesex. It is estimated that the role of the sex differentiation system for female phenotypes may have evolved through accumulating mutations in the protein motif structures that led to the enhancement of its transcription-regulatory function.

category: evolutionary biology


title: Endothelial Semaphorin 3fb regulates Vegf pathway-mediated angiogenic sprouting

abstract:
Vessel growth integrates diverse extrinsic signals with intrinsic signaling cascades to coordinate cell migration and sprouting morphogenesis. The pro-angiogenic effects of Vascular Endothelial Growth Factor (VEGF) are carefully controlled during sprouting to generate an efficiently patterned vascular network. We identify crosstalk between VEGF signaling and that of the secreted ligand Semaphorin 3fb (Sema3fb), one of two zebrafish paralogs of mammalian Sema3F. The sema3fb gene is expressed by endothelial cells in actively sprouting vessels. Loss of sema3fb results in abnormally wide and stunted intersegmental vessel artery sprouts. Although the sprouts initiate at the correct developmental time, they have a reduced migration speed. These sprouts have persistent filopodia and abnormally spaced nuclei suggesting dysregulated control of actin assembly. sema3fb mutants show simultaneously higher expression of pro-angiogenic (VEGF receptor 2 (vegfr2) and delta-like 4 (dll4)) and anti-angiogenic (soluble VEGF receptor 1 (svegfr1)/ soluble Fms Related Receptor Tyrosine Kinase 1 (sflt1)) pathway components. We show increased phospho-ERK staining in migrating angioblasts, consistent with enhanced Vegf activity. Reducing Vegfr2 kinase activity in sema3fb mutants rescues angiogenic sprouting. Our data suggest that Sema3fb plays a critical role in promoting endothelial sprouting through modulating the VEGF signaling pathway, acting as an autocrine cue that modulates intrinsic growth factor signaling.

Author summaryTo supply tissues with essential oxygen and nutrients, blood vessel development is carefully orchestrated by positive  go and negative  stop growth signals as well as directional cues to shape patterning. Semaphorin proteins are named after the  Semaphore railway signaling system that directed trains along the appropriate tracks. Our work highlights the role of the Semaphorin 3fb protein in providing a pro-growth signal to developing vessels. Semaphorin 3fb is both produced by, and acts on the precursors of blood vessels as they migrate, a process known as autocrine control. We find that losing Semaphorin 3fb leads to stalled blood vessel growth, indicating it normally acts as a positive signal. It acts via modulating the VEGF growth factor signaling pathway that in turn, controls the migration process. We propose that Semaphorin3b fine-tunes vessel growth, thus ensuring a properly patterned network develops.

category: developmental biology


title: Wild Wheat Introgression Promotes Temporal Water fluxes Dynamics under Terminal Drought Stress through Plant-Atmospheric Interrelations

abstract: Drought intensity as experienced by plants depends upon soil moisture status and atmospheric variables such as temperature, radiation, and air vapour pressure deficit (VPD). Although the role of shoot architecture with these edaphic and atmospheric factors is well-characterized, the extent to which shoot and root dynamic interactions as a continuum are controlled by genotypic variation is less known. Here, we targeted these interactions using a wild emmer introgression line (IL20) with a distinct drought-induced shift in the shoot-to-root ratio and its drought-sensitive recurrent parent Svevo. Using a gravimetric platform, we show that IL20 maintained higher root water influx and gas exchange under drought stress, which supported a greater growth. Interestingly, the advantage of IL20 in root water influx and transpiration was expressed earlier during the daily diurnal cycle under lower VPD and therefore supported higher transpiration efficiency. Application of structural equation model indicates that under drought, VPD and radiation are antagonistic to transpiration rate, whereas the root water influx operates as feedback for the higher atmospheric responsiveness of leaves. Collectively, our results suggest that a drought-induced shift in root-to-shoot ratio can improve plant water uptake potential in a short preferable time window determined by both water and atmospheric parameters.


title: Adaptive Wild Introgression Mediate Temporal Wheat Root Water Influxes under Drought Stress through Plant-Atmospheric Interrelations


title: Modifying root/shoot ratios improves root water influxes in wheat under drought stress


title: Allelopathy as an evolutionarily stable strategy

abstract: In plants, most competition is resource competition, where one plant simply pre-empts the resources away from its neighbours. Interference competition, as the name implies, is a form of direct interference to prevent resource access. Interference competition is common among animals who can physically fight, but in plants, one of the main mechanisms of interference competition is Allelopathy. allelopathic plants release of cytotoxic chemicals into the environment which can increase their ability to compete with surrounding organisms for limited resources. The circumstances and conditions favoring the development and maintenance of allelochemicals, however, is not well understood. Particularly, it seems strange that, despite the obvious benefits of allelopathy, it seems to have only rarely evolved. To gain insight into the cost and benefit of allelopathy, we have developed a 2 x 2 matrix game to model the interaction between plants that produce allelochemicals and plants that do not. Production of an allelochemical introduces novel cost associated with synthesis and detoxifying a toxic chemical but may also convey a competitive advantage. A plant that does not produce an allelochemical will suffer the cost of encountering one. Our model predicts three cases in which the evolutionarily stable strategies are different. In the first, the non-allelopathic plant is a stronger competitor, and not producing allelochemicals is the evolutionarily stable strategy. In the second, the allelopathic plant is the better competitor and production of allelochemicals is the more beneficial strategy. In the last case, neither is the evolutionarily stable strategy. Instead, there are alternating stable states, depending on whether the allelopathic or non-allelopathic plant arrived first. The generated model reveals circumstances leading to the evolution of allelochemicals and sheds light on utilizing allelochemicals as part of weed management strategies. In particular, the wide region of alternative stable states in most parameterizations, combined with the fact that the absence of allelopathy is likely the ancestral state, provides an elegant answer to the question of why allelopathy rarely evolves despite its obvious benefits. Allelopathic plants can indeed outcompete non-allelopathic plants, but this benefit is simply not great enough to allow them to go to fixation and spread through the population. Thus, most populations would remain purely non-allelopathic.


title: Influence of internal seiche dynamics on vertical movement of fish

abstract:
O_LIInternal seiches are common in stratified lakes, with significant effects on stratification patterns, hydrodynamics and vertical nutrient transport. In particular, seiche can change the vertical distribution of the thermocline and the cold hypolimnetic and warm epilimnetic water masses by several meters on a timescale of a few hours. The results are rapid and strong changes in temperature profiles and oxygen availability that can have profound effects on vagrant and sessile organisms. Internal seiche dynamics could therefore affect fish communities directly through physiological stress and elevated mortality, and indirectly through prey distribution.
C_LIO_LIThe aim of this study was to analyze the effects of internal seiche dynamics on lacustrine fish behaviour, and to characterize fish reaction patterns, with the main focus on vertical movement of fish in the vicinity of a shifting thermocline, and avoidance of cold hypolimnetic water.
C_LIO_LIThe analysis was based on acoustic telemetry data from Lake Milada, a post-mining lake in the Czech Republic, with a total of 55 tracked individuals of four species: northern pike (Esox lucius), wels catfish (Silurus glanis), tench (Tinca tinca) and rudd (Scardinius erythropthalmus).
C_LIO_LIThe effects of seiche dynamics on the four species studied were weak but significant during the day, but only on rudd during the night. Upward seiche produced stronger reactions in fish than downward seiche, and the effects were manifested only during the strongest seiche events.
C_LIO_LIThermocline shifting during seiche events may induce a transient reduction in habitat for seiche-reacting species, thus potentially affecting predation and other inter- and intra-specific interactions, and probably affecting fish community dynamics.
C_LI

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

category: ecology


title: De novo evolution of macroscopic multicellularity

abstract: The evolution of large organismal size is fundamentally important for multicellularity, creating new ecological niches and opportunities for the evolution of increased biological complexity. Yet little is known about how large size evolves, particularly in nascent multicellular organisms that lack genetically-regulated multicellular development. Here we examine the interplay between biological and biophysical drivers of macroscopic multicellularity using long-term experimental evolution. Over 600 daily transfers (~3,000 generations), multicellular snowflake yeast evolved macroscopic size, becoming ~2{middle dot}104 times larger (~mm scale) and 104-fold more biophysically tough, while remaining clonal. They accomplished this through sustained biophysical adaptation, evolving increasingly elongate cells that initially reduced the strain of cellular packing, then facilitated branch entanglement so that groups of cells stay together even after many cellular bonds fracture. Four out of five replicate populations show evidence of predominantly adaptive evolution, with mutations becoming significantly enriched in genes affecting cell shape and cell-cell bonds. Taken together, this work shows how selection acting on the emergent properties of simple multicellular groups can drive sustained biophysical adaptation, an early step in the evolution of increasingly complex multicellular organisms.


title: Bayesian mixture model for clustering rare-variant effects in human genetic studies

abstract: Rare-variant aggregate analysis from exome and whole genome sequencing data typically summarizes with a single statistic the signal for a gene or the unit that is being aggregated. However, when doing so, the effect profile within the unit may not be easily characterized across one or multiple phenotypes. Here, we present an approach we call Multiple Rarevariants and Phenotypes Mixture Model (MRPMM), which clusters rare variants into groups based on their effects on the multivariate phenotype and makes statistical inferences about the properties of the underlying mixture of genetic effects. Using summary statistic data from a meta-analysis of exome sequencing data of 184,698 individuals in the UK Biobank across 6 populations, we demonstrate that our mixture model can identify clusters of variants responsible for significantly disparate effects across a multivariate phenotype; we study three lipid and three renal traits separately. The method is able to estimate (1) the proportion of non-null variants, (2) whether variants with the same predicted consequence in one gene behave similarly, (3) whether variants across genes share effect profiles across the multivariate phenotype, and (4) whether different annotations differ in the magnitude of their effects. As rare-variant data and aggregation techniques become more common, this method can be used to ascribe further meaning to association results.

category: genetics


title: Principles of sensor-effector organization in six-transmembrane ion channels

abstract: Receptor proteins sense stimuli and generate downstream signals via sensor and effector domains. Presently, the structural constraints on sensor-effector organization across receptor protein superfamilies are not clear. Here, we perform statistical coupling analysis (SCA) on the transient receptor potential (TRP) and voltage-gated potassium (Kv) ion channel superfamilies to characterize the networks of coevolving residues, or protein sectors, that mediate their receptor functions. Comparisons to structural and functional studies reveal a conserved "core" sector that extends from the pore and mediates effector functions, including pore gating and sensor-pore coupling, while sensors correspond to family-specific "accessory" sectors and localize according to three principles: Sensors (1) may emerge in any region with access to the core, (2) must maintain contact with the core, and (3) must preserve the integrity of the core. This sensor-core architecture may represent a conserved and generalizable paradigm for the structure-function relationships underlying the evolution of receptor proteins.

category: biophysics


title: Identifying genomic regions and candidate genes selected during the breeding of rice in Vietnam

abstract:
Background and aimsVietnam harnesses a rich diversity of rice landraces adapted to a broad range of conditions, which constitute a largely untapped source of genetic diversity for the continuous improvement of rice cultivars. We previously identified a strong population structure in Vietnamese rice, which is captured in five Indica and four Japonica subpopulations, including an outlying Indica-5 group. Here, we leveraged on that strong differentiation, and the 672 rice genomes generated, to identify genes within genomic regions putatively selected during domestication and breeding of rice in Vietnam.

MethodologyWe identified significant distorted patterns in allele frequency (XP-CLR method) and population differentiation scores (FST), resulting from differential selective pressures between native subpopulations, and compared them with QTLs previously identified by GWAS in the same panel. We particularly focused on the outlying Indica-5 subpopulation because of its likely novelty and differential evolution.

ResultsWe identified selection signatures in each of the Vietnamese subpopulations and carried out a comprehensive annotation of the 52 regions selected in Indica-5, which represented 8.1% of the rice genome. We annotated the 4,576 genes in these regions, verified the overlap with QTLs identified in the same diversity panel and the comparison with a FST analysis between subpopulations, to select sixty-five candidate genes as promising breeding targets, several of which harboured alleles with non-synonymous substitutions.

ConclusionsOur results highlight genomic differences between traditional Vietnamese landraces, which are likely the product of adaption to multiple environmental conditions and regional culinary preferences in a very diverse country. We also verified the applicability of this genome scanning approach to identify potential regions harbouring novel loci and alleles to breed a new generation of sustainable and resilient rice.

Key MessageWe localised regions in the rice genome selected during breeding by comparing allele frequency patterns among Vietnamese rice subpopulations. We characterised candidate genes in the Indica-5 subpopulation with breeding potential.


title: An interpretable flux-based machine learning model of drug interactions across metabolic space and time

abstract:
AO_SCPLOWBSTRACTC_SCPLOWDrug combinations are a promising strategy to counter antibiotic resistance. However, current experimental and computational approaches do not account for the entire complexity involved in combination therapy design, such as the effect of pathogen metabolic heterogeneity, changes in the growth environment, drug treatment order and time interval. To address these limitations, we present a comprehensive approach that uses genome-scale metabolic modeling and machine learning to guide combination therapy design. Our mechanistic approach (a) accommodates diverse data types, (b) accounts for time- and order-specific interactions, and (c) accurately predicts drug interactions in various growth conditions and their robustness to pathogen metabolic heterogeneity. Our approach achieved high accuracy (AUC = 0.83 for synergy, AUC = 0.98 for antagonism) in predicting drug interactions for E. coli cultured in 57 metabolic conditions based on experimental validation. The entropy in bacterial metabolic response was predictive of combination therapy outcomes across time scales and growth conditions. Simulation of metabolic heterogeneity using population-FBA identified two sub-populations of E. coli cells defined by regulation of cis- to trans-isomerization of unsaturated fatty acids with distinct effects on drug interactions. Analysis of the vast landscape of condition-specific drug interactions revealed a set of 24 robustly synergistic drug combinations with potential for clinical use.

SO_SCPLOWIGNIFICANCEC_SCPLOWWorldwide, 700,000 people die each year from drug-resistant infections. Drug combinations have great potential to reduce the spread of drug-resistant bacteria. However, their potency is impacted by both the pathogen growth environment and the heterogeneity in pathogen metabolism. The metabolic heterogeneity in a pathogen population allows them to survive antibiotic treatment. Here we present a flexible machine-learning framework that utilizes diverse data types to effectively search through the large design space of both sequential and simultaneous combination therapies across hundreds of simulated growth conditions and pathogen metabolic states. Our approach can serve as a useful guide for the selection of robustly synergistic drug combinations.

category: systems biology


title: A flux-based machine learning model to simulate the impact of pathogen metabolic heterogeneity on drug interactions


title: Synaptic plasticity is predicted by spatiotemporal firing rate patterns and robust to in vivo-like variability

abstract: Synaptic plasticity, the experience-induced change in connections between neurons, underlies learning and memory in the brain. Most of our understanding of synaptic plasticity derives from in vitro experiments with precisely repeated stimulus patterns; however, neurons exhibit significant variability in vivo during repeated experiences. Further, the spatial pattern of synaptic inputs to the dendritic tree influences synaptic plasticity, yet is not considered in most synaptic plasticity rules. Here, we address the sensitivity of plasticity to trial-to-trial variability and delineate how spatiotemporal synaptic input patterns produce plasticity with in vivo-like conditions using a data-driven computational model with a calcium-based plasticity rule. Using in vivo spike train recordings as inputs, we show that plasticity is strongly robust to trial-to-trial variability of spike timing, and derive general synaptic plasticity rules describing how spatiotemporal patterns of synaptic inputs control the magnitude and direction of plasticity. Specifically, a high temporal input firing rate to a synapse late in a trial correlated with neighboring synaptic activity produces potentiation, while an earlier, moderate firing rate that is negatively correlated with neighboring synaptic activity produces depression. Together, our results reveal that calcium dynamics can unify diverse plasticity rules and reveal how spatiotemporal firing rate patterns control synaptic plasticity.


title: The decoy SNARE Tomosyn sets tonic versus phasic release properties and is required for homeostatic synaptic plasticity

abstract: Synaptic vesicle release probability (Pr) is a key presynaptic determinant of synaptic strength established by cell intrinsic properties and further refined by plasticity. To characterize mechanisms that generate Pr heterogeneity between distinct neuronal populations, we examined glutamatergic tonic (Ib) and phasic (Is) motoneurons in Drosophila with stereotyped differences in Pr and synaptic plasticity. We found the decoy SNARE Tomosyn is differentially expressed between these motoneuron subclasses and contributes to intrinsic differences in their synaptic output. Tomosyn expression enables tonic release in Ib motoneurons by reducing SNARE complex formation and suppressing Pr to generate decreased levels of synaptic vesicle fusion and enhanced resistance to synaptic fatigue. In contrast, phasic release dominates when Tomosyn expression is low, enabling high intrinsic Pr at Is terminals at the expense of sustained release and robust presynaptic potentiation. In addition, loss of Tomosyn disrupts the ability of tonic synapses to undergo presynaptic homeostatic potentiation (PHP).


title: Deep learning model of somatic hypermutation reveals importance of sequence context beyond targeting of AID and Polη hotspots

abstract: B-cells undergo somatic hypermutation (SHM) of the Immunoglobulin (Ig) variable region to generate high-affinity antibodies. SHM relies on the activity of activation-induced deaminase (AID), which mutates C>U preferentially targeting WRC (W=A/T, R=A/G) hotspots. Downstream mutations at WA Polymerase {eta} hotspots contribute further mutations. Computational models of SHM can describe the probability of mutations essential for vaccine responses. Previous studies using short subsequences (k-mers) failed to explain divergent mutability for the same k-mer. We developed the DeepSHM (Deep learning on SHM) model using k-mers of size 5-21, improving accuracy over previous models. Interpretation of DeepSHM identified an extended DWRCT (D=A/G/T) motif with particularly high mutability. Increased mutability was further associated with lower surrounding G content. Our model also discovered a conserved AGYCTGGGGG (Y=C/T) motif within FW1 of IGHV3 family genes with unusually high T>G substitution rates. Thus, a wider sequence context increases predictive power and identifies novel features that drive mutational targeting.

category: immunology


title: Cytoplasmic labile iron accumulates in aging stem cells perturbing a key rheostat for identity control

abstract: Bone marrow resident and rarely dividing haematopoietic stem cells (HSC) harbour an extensive self-renewal capacity to sustain life-long blood formation;1-5 albeit their function declines during ageing.6, 7 Various molecular mechanisms confer stem cell identity, ensure long-term maintenance and are known to be deregulated in aged stem cells.8, 9 How these programs are coordinated, particularly during cell division, and what triggers their ageing-associated dysfunction has been unknown. Here, we demonstrate that HSC, containing the lowest amount of cytoplasmic chelatable iron (labile iron pool)10 among hematopoietic cells, activate a limited iron response during mitosis. Engagement of this iron homeostasis pathway elicits mobilization and {beta}-oxidation of arachidonic acid and enhances stem cell-defining transcriptional programs governed by histone acetyl transferase Tip60/KAT5. We further find an age-associated expansion of the labile iron pool, along with loss of Tip60/KAT5-dependent gene regulation to contribute to the functional decline of ageing HSC, which can be mitigated by iron chelation. Together, our work reveals cytoplasmic redox active iron as a novel rheostat in adult stem cells; it demonstrates a role for the intracellular labile iron pool in coordinating a cascade of molecular events which reinforces HSC identity during cell division and to drive stem cell ageing when perturbed. As loss of iron homeostasis is commonly observed in the elderly, we anticipate these findings to trigger further studies into understanding and therapeutic mitigation of labile iron pool-dependent stem cell dysfunction in a wide range of degenerative and malignant pathologies.


title: The complex genetics of symbiotic extended phenotypes across environments in a model mutualism

abstract:
A goal of modern biology is to develop the genotype-phenotype (G[->]P) map, a predictive understanding of how genomic information generates trait variation that forms the basis of both natural and managed communities. As microbiome research advances, however, it has become clear that many of these traits are symbiotic extended phenotypes, being governed by genetic variation encoded not only by the hosts own genome, but also by the genomes of myriad cryptic symbionts. Building a reliable G[->]P map therefore requires accounting for the multitude of interacting genes and even genomes involved in symbiosis. Here we use naturally-occurring genetic variation in 191 strains of the model microbial symbiont Ensifer meliloti in four mapping experiments to study the genomic architecture of a key symbiotic extended phenotype - partner quality, or the fitness benefit conferred to a host by a particular symbiont genotype, within and across environmental contexts and host genotypes. We define three novel categories of loci in rhizobium genomes that must be accounted for if we want to build a reliable G[->]P map of partner quality; namely, 1) loci whose identities depend on the environment, 2) those that depend on the host genotype with which rhizobia interact, and 3) universal loci that are likely important in all or most environments.

IMPORTANCEGiven the rapid rise of research on how microbiomes can be harnessed to improve host health, understanding the contribution of microbial genetic variation to host phenotypic variation is pressing, and will better enable us to predict the evolution of (and select more precisely for) symbiotic extended phenotypes that impact host health. We uncover extensive context-dependency in both the identity and functions of symbiont loci that control host growth, which makes predicting the genes and pathways important for determining symbiotic outcomes under different conditions more challenging. Despite this context-dependency, we also resolve a core set of universal loci that are likely important in all or most environments, and thus, serve as excellent targets both for genetic engineering and future coevolutionary studies of symbiosis.


title: A Calcium/Palmitoylation Switch Interfaces the Signaling Networks of Stress Response and Transition to Flowering

abstract:
The precise timing of flowering in adverse environments is critical for plants to secure reproductive success. We report a novel mechanism controlling the time of flowering by which the palmitoylation-dependent nuclear import of protein SOS3/CBL4, a Ca2+-signaling intermediary in the plant response to salinity, results in the selective stabilization of the flowering time regulator GIGANTEA inside the nucleus under salt stress, while degradation of GIGANTEA in the cytosol releases the protein kinase SOS2 to achieve salt tolerance. S-acylation of SOS3 was critical for its nuclear localization and the promotion of flowering, but dispensable for salt tolerance. SOS3 interacted with the photoperiodic flowering components GIGANTEA and FKF1 on the CONSTANS gene promoter to sustain the transcription of CO and FT under salinity. Thus, SOS3 acts as a Ca2+- and palmitoylation-dependent molecular switch that fine-tunes flowering in a saline environment through the shared spatial separation and selective stabilization of GIGANTEA. The SOS3 protein connects two signaling networks to co-regulate stress adaptation and time of flowering.

Short summaryS-acylation promoted the nuclear import of SOS3/CBL4 for the selective stabilization of the photoperiodic floral regulator GIGANTEA to fine-tune flowering time in a saline environment. Spatial separation of SOS3 acts as a molecular switch co-regulating stress adaptation and time of flowering.


title: A pathogenic DYT-THAP1 dystonia mutation causes hypomyelination and loss of YY1 binding

abstract: Dystonia is a disabling disease that manifests as prolonged involuntary twisting movements. DYT-THAP1 is an inherited form of isolated dystonia caused by mutations in THAP1 encoding the transcription factor THAP1. The phe81leu (F81L) missense mutation is representative of a category of poorly understood mutations that do not occur on residues critical for DNA binding. Here, we demonstrate that the F81L mutation (THAP1F81L) impairs THAP1 transcriptional activity and disrupts CNS myelination. Strikingly, THAP1F81L exhibits normal DNA binding but causes a significantly reduced DNA binding of YY1, its transcriptional partner that also has an established role in oligodendrocyte lineage progression. Our results suggest a model of molecular pathogenesis whereby THAP1F81L normally binds DNA but is unable to efficiently organize an active transcription complex.


title: University-wide chronotyping shows late-type students have lower grades, shorter sleep, poorer well-being, lower self-regulation, and more absenteeism

abstract: A persons preferred timing of nocturnal sleep (chronotype) has important implications for cognitive performance. Students who prefer to sleep late may have a selective learning disadvantage for morning classes due to inadequate sleep and circadian desynchrony. Here, (1) we tested whether late-type students perform worse only for morning classes, and (2) we investigated factors that may contribute to their poorer academic achievement. Chronotype was determined objectively in 33,645 university students (early, n=3,965; intermediate, n=23,787; late, n=5,893) by analyzing the diurnal distribution of their logins on the universitys Learning Management System (LMS). Late-type students had lower grades than their peers for courses held at all different times of day, and during semesters when they had no morning classes. Actigraphy studies (n=261) confirmed LMS-derived chronotype was associated with students sleep patterns. Nocturnal sleep on school days was shortest in late-type students because they went to bed the latest and woke up early compared with non-school days. Surveys showed that late-type students had lower self-rated health and mood (n=357), and lower metacognitive self-regulation (n=752). Wi-Fi connection data for classrooms (n=17,356) revealed that late-type students had lower lecture attendance than their peers for classes held in both the morning and the afternoon. Our findings suggest that multiple factors converge to impair learning in late-type students. Shifting classes later can improve sleep and circadian synchrony in late-type students but is unlikely to eliminate the performance gap. Interventions that focus on improving students well-being and learning strategies may be important for addressing the late-type academic disadvantage.


title: Mathematical Relationships between Motoneuron Properties Derived by Empirical Data Analysis: Size Determines All Motoneuron Properties

abstract:
Our understanding of the behaviour of spinal alpha-motoneurons (MNs) in mammals partly relies on our knowledge of the relationships between MN membrane properties, such as MN size, resistance, rheobase, capacitance, time constant, axonal conduction velocity and afterhyperpolarization period. Qualitative relations between some of these properties are often assumed based on scattered but converging evidence from experimental studies and review papers.

We reprocessed the data from 40 experimental studies in adult cat, rat and mouse MN preparations in vivo to empirically derive precise mathematical relationships between MN electrophysiological and anatomical properties. This validated mathematical framework, which demonstrates that the MN membrane properties are all related to each other, expands our knowledge in neural electrophysiology beyond available literature data. For example, it extends the Hennemans size principle by the association between MN size and all other MN properties.

The derived mathematical relationships provide a convenient tool for neuroscientists and experimenters to estimate non-measurable MN properties, to explore relationships between pairs of MN properties never concurrently observed in previous experiments, to support future experimental investigations, or to investigate inter-mammalian-species variations in MN membrane properties. Using this mathematical framework, which supports the classic description of a MN as a membrane equivalent electrical circuit, modelers can build profiles of inter-consistent MN-specific properties to scale advanced models of MNs, with consequences on our understanding of the neural control of movement.


title: Mathematical Relationships between Spinal Motoneuron Properties


title: An open MRI dataset for multiscale neuroscience

abstract: AO_SCPLOWBSTRACTC_SCPLOWMultimodal neuroimaging grants a powerful window into the structure and function of the human brain at multiple scales. Recent methodological and conceptual advances have enabled investigations of the interplay between large-scale spatial trends (also referred to as gradients) in brain microstructure and connectivity, offering an integrative framework to study multiscale brain organization. Here, we share a multimodal MRI dataset for Microstructure-Informed Connectomics (MICA-MICs) acquired in 50 healthy adults (23 women; 29.54{+/-}5.62 years) who underwent high-resolution T1-weighted MRI, myelin-sensitive quantitative T1 relaxometry, diffusion-weighted MRI, and resting-state functional MRI at 3 Tesla. In addition to raw anonymized MRI data, this release includes brain-wide connectomes derived from i) resting-state functional imaging, ii) diffusion tractography, iii) microstructure covariance analysis, and iv) geodesic cortical distance, gathered across multiple parcellation scales. Alongside, we share large-scale gradients estimated from each modality and parcellation scale. Our dataset will facilitate future research examining the coupling between brain microstructure, connectivity, and macroscale function. MICA-MICs is available on the Canadian Open Neuroscience Platforms data portal (https://portal.conp.ca).


title: Distributed Patterns of Functional Connectivity Underlie Individual Differences in Long-Term Memory Forgetting

abstract: Despite the importance of memories in everyday life and the progress made in understanding how they are encoded and retrieved, the neural processes by which declarative memories are maintained or forgotten remain elusive. Part of the problem is that it is empirically difficult to measure the rate at which memories fade and, without such a measure, it is hard to identify the corresponding neural correlates. This study addresses this problem using a combination of individual differences, model-based inferences, and resting-state functional connectivity. The individual-specific values of rate of forgetting in long-term memory (LTM) were estimated for 33 participants using a formal model fit to data from an adaptive fact learning task. Individual rates of forgetting were then used to examine participant-specific patterns of resting-state fMRI connectivity, using machine-learning techniques to identify the most predictive and generalizable features. Consistent with the existing literature, our results identified a sparse, distributed network of cortical and subcortical regions that underlies forgetting in LTM. Cross-validation showed that individual rates of forgetting were predicted with high accuracy (r = .96) from this connectivity pattern alone. These results open up new opportunities for the study of individual differences in LTM function and dysfunction.


title: Flexible recruitments of fundamental muscle synergies in the trunk and lower limbs for highly variable movements and postures

abstract: The extent to which muscle synergies represent the neural control of human behavior remains unknown. Here, we tested whether certain sets of muscle synergies that are fundamentally necessary across behaviors exist. We measured the electromyographic activities of 26 muscles including bilateral trunk and lower limb muscles during 24 locomotion, dynamic and static stability tasks, and extracted the muscle synergies using non-negative matrix factorization. Our results showed that 13 muscle synergies that may have unique functional roles accounted for almost all 24 tasks by combinations of single and/or merging of synergies. Therefore, our results may support the notion of the low dimensionality in motor outputs, in which the central nervous system flexibly recruits fundamental muscle synergies to execute diverse human behaviors. Further studies using manipulations of the central nervous system and/or neural recording are required the neural representation with such fundamental components of muscle synergies.


title: Harmonisation of multi-site MRS data with ComBat

abstract:
Magnetic resonance spectroscopy (MRS) is a non-invasive neuroimaging technique used to measure brain chemistry in vivo and has been used to study the healthy brain as well as neuropathology in numerous neurological disorders. The number of multi-site studies using MRS are increasing; however, non-biological variability introduced during data collection across multiple sites, such as differences in scanner vendors and site-specific acquisition implementations for MRS, can obscure detection of biological effects of interest. ComBat is a data harmonisation technique that can remove non-biological sources of variance in multisite studies. It has been validated for use with structural and functional MRI metrics but not for MRS metabolites. This study investigated the validity of using ComBat to harmonize MRS metabolites for vendor and site differences. Analyses were performed using data acquired across 20 sites and included edited MRS for GABA+ (N=218) and macromolecule-suppressed GABA data (N=209), as well as standard PRESS data to quantify NAA, creatine, choline, and glutamate (N=195). ComBat harmonisation successfully mitigated vendor and site differences for all metabolites of interest. Moreover, significant associations were detected between sex and choline levels and between age and glutamate and GABA+ levels that were not detectable prior to harmonisation, confirming the importance of removing site and vendor effects in multi-site data. In conclusion, ComBat harmonisation can be successfully applied to MRS data in multi-site MRS studies.

HighlightsO_LIMulti-site MRS data contains sources of non-biological variance that can mask biological effects of interest.
C_LIO_LIComBat data harmonisation successfully removes variance contributed by scanner and site differences.
C_LIO_LIRemoval of this variance revealed biological effects that were previously not detected.
C_LIO_LIComBat harmonisation can be successfully applied to MRS data in multi-site MRS studies
C_LI


title: Dynamic configuration of large-scale cortical networks during an inhibitory task accounts for heterogeneity in attention-deficit/hyperactivity disorder traits

abstract: The heterogeneity of attention-deficit/hyperactivity disorder (ADHD) traits (inattention vs. hyperactivity/impulsivity) complicates diagnosis and intervention. Identifying how the configuration of large-scale functional brain networks during cognitive processing correlate with this heterogeneity could help us understand the neural mechanisms altered across ADHD presentations. Here, we recorded high-density EEG while 62 non-clinical participants (ages 18-24; 32 male) underwent an inhibitory control task (Go/No-Go). Functional EEG networks were created using sensors as nodes and across-trial phase-lag index values as edges. Using cross-validated LASSO regression, we examined whether graph-theory metrics applied to both static networks (averaged across time-windows: -500-0ms, 0-500ms) and dynamic networks (temporally layered with 2ms intervals), were associated with hyperactive/impulsive and inattentive traits. Network configuration during response execution/inhibition was associated with hyperactive/impulsive (mean R2 across test sets = .20, SE = .02), but not inattentive traits. Post-stimulus results at higher frequencies (Beta, 14-29Hz; Gamma, 30-90Hz) showed the strongest association with hyperactive/impulsive traits, and predominantly reflected less burst-like integration between modules in oscillatory beta networks during execution, and increased integration/small-worldness in oscillatory gamma networks during inhibition. We interpret the beta network results as reflecting weaker integration between specialized pre-frontal and motor systems during motor response preparation, and the gamma results as reflecting a compensatory mechanism used to integrate processing between less functionally specialized networks. This research demonstrates that the neural network mechanisms underlying response execution/inhibition might be associated with hyperactive/impulsive traits, and that dynamic, task-related changes in EEG functional networks may be useful in disentangling ADHD heterogeneity.


title: Learning graph networks: sleep targets highly connected global and local nodes for consolidation

abstract: Much of our long-term knowledge is organised in complex networks. Sleep is thought to be critical for abstracting knowledge and enhancing important item memory for long-term retention. Thus, sleep should aid the development of memory for networks and the abstraction of their structure for efficient storage. However, this remains unknown because past sleep studies have focused on discrete items. Here we explored the impact of sleep (night-sleep/day-wake within-subject paradigm) on memory for graph-networks where some items were important due to dense local connections (degree centrality) or, independently, important due to greater global connections (closeness / betweenness centrality). A network of 27 planets (nodes) sparsely interconnected by 36 teleporters (edges) was learned via discrete associations without explicit indication of any network structure. Despite equivalent exposure to all connections in the network, we found that memory for the links between items with high local centrality or high global centrality were better retained after sleep. These results highlight that sleep has the capacity for strengthening both global and local structure from the world and abstracting over multiple experiences to efficiently form internal networks of knowledge.


title: Petascale neural circuit reconstruction: automated methods

abstract: 3D electron microscopy (EM) has been successful at mapping invertebrate nervous systems, but the approach has been limited to small chunks of mammalian brains. To scale up to larger volumes, we have built a computational pipeline for processing petascale image datasets acquired by serial section EM, a popular form of 3D EM. The pipeline employs convolutional nets to compute the nonsmooth transformations required to align images of serial sections containing numerous cracks and folds, detect neuronal boundaries, label voxels as axon, dendrite, soma, and other semantic categories, and detect synapses and assign them to presynaptic and postsynaptic segments. The output of neuronal boundary detection is segmented by mean affinity agglomeration with semantic and size constraints. Pipeline operations are implemented by leveraging distributed and cloud computing. Intermediate results of the pipeline are held in cloud storage, and can be effortlessly viewed as images, which aids debugging. We applied the pipeline to create an automated reconstruction of an EM image volume spanning four visual cortical areas of a mouse brain. Code for the pipeline is publicly available, as is the reconstructed volume.


title: Transcranial Ultrasound Stimulation in Anterior Cingulate Cortex Impairs Information Sampling and Learning in Loss Contexts

abstract:
Neuronal subgroups in anterior cingulate cortex (ACC) and the anterior striatum (STR) encode the reward structure of a given environment. But whether or how this reward information is used to guide information sampling, optimize decision making, or motivate behavior in cognitively challenging situations has remained elusive. Here, we causally tested these scenarios by transiently disrupting ACC and STR of rhesus monkeys with transcranial ultrasound with a learning task that independently varied cognitive and motivational demands. We found that disrupting the ACC, but not the STR, prolonged information sampling and reduced learning efficiency whenever the motivational payoff was low. These impairments were most pronounced at high cognitive demands and based on an inability to use loss experiences to improve performance. These results provide causal evidence that the ACC is necessary for motivation, to overcome anticipated costs from negative (loss) outcomes, and for cognition, to enhance visual information sampling during adaptive behavior.

HIGHLIGHTSO_LITranscranial ultrasound stimulation of the anterior cingulate cortex disrupts learning after loss experience.
C_LIO_LIThe ultrasound-induced learning deficit is exacerbated at high cognitive load.
C_LIO_LIThe ultrasound-induced learning deficit is accompanied by inefficient fixational information sampling.
C_LIO_LIAnterior cingulate cortex causally supports credit assignment of aversive outcomes to visual features.
C_LI


title: Scaling up to meet new challenges of brain and behaviour: Deep brain electrophysiology in freely moving sheep

abstract: While rodents are arguably the easiest animals to use for studying brain function, relying on them as model species for translational research comes with its own sets of limitations. Here, we propose sheep as a practical large animal species for in vivo brain function studies performed in naturalistic settings. To demonstrate their experimental usefulness, we performed proof-of-principle deep brain electrophysiological recording experiments from unrestrained sheep. Recordings were made from cortex and hippocampus both whilst sheep performed goal-directed behaviours (two-choice discrimination tasks), and across states of vigilance that included natural sleep. Hippocampal and cortical oscillatory rhythms were consistent with those seen in rodents and non-human primates, and included cortical alpha oscillations during immobility, hippocampal theta oscillations (5-6Hz) during locomotion and hippocampal sharp wave ripple oscillations ([~]150 Hz) during immobility. Moreover, we found clear examples of neurons whose activity was modulated by task, speed of locomotion, spatial position, reward and vigilance states. Recordings were conducted over a period of many months. Due to the exceptional stability of individual electrodes we were able to record from some neurons repeatedly across the span of a month. Together these experiments demonstrate that sheep are an excellent experimental animal model to use in longitudinal electrophysiological and imaging studies, particularly those requiring a large brained mammal, large scale recordings across distributed neuronal networks, experimentation outside the confounds of the traditional laboratory, or all the above concomitantly.


title: Scaling up to meet new challenges of brain and behaviour through deep brain electrophysiology in freely moving sheep


title: Trafficking of the glutamate transporter is impaired in LRRK2-related Parkinson's disease

abstract: The Excitatory Amino Acid Transporter 2 (EAAT2) accounts for 80 % of brain glutamate clearance and is mainly expressed in astrocytic perisynaptic processes. EAAT2 function is finely regulated by endocytic events, recycling to the plasma membrane and degradation. Noteworthy, deficits in EAAT2 have been associated with neuronal excitotoxicity and neurodegeneration. In this study, we show that EAAT2 trafficking is impaired by the leucine-rich repeat kinase 2 (LRRK2) pathogenic variant G2019S, a common cause of late-onset familial Parkinsons disease (PD). In LRRK2 G2019S human brains and experimental animal models, EAAT2 protein levels are significantly decreased, which is associated with elevated gliosis. The decreased expression of the transporter correlates with its reduced functionality in mouse LRRK2 G2019S purified astrocytic terminals and in Xenopus laevis oocytes expressing human LRRK2 G2019S. In LRRK2 G2019S knockin mouse brain, the correct surface localization of the endogenous transporter is impaired, resulting in its interaction with a plethora of endo-vesicular proteins. Mechanistically, we report that pathogenic LRRK2 kinase activity delays the recycling of the transporter to the plasma membrane via Rabs inactivation, causing its intracellular relocalization and degradation. Taken together, our results demonstrate that pathogenic LRRK2 interferes with the physiology of EAAT2, pointing to extracellular glutamate overload as a possible contributor to neurodegeneration in PD.


title: Single particle cryo-EM structure of the outer hair cell motor protein prestin

abstract: The mammalian outer hair cell (OHC) protein prestin (Slc26a5), a member of the solute carrier 26 (Slc26) family of membrane proteins, differs from other members of the family owing to its unique piezoelectric-like property that drives OHC electromotility. OHCs require prestin for cochlear amplification, a process that enhances mammalian hearing. Despite substantial biophysical characterization, the mechanistic basis for the prestins electro-mechanical behavior is not fully understood. To gain insight into such behavior, we have used cryo-electron microscopy at subnanometer resolution (overall resolution of 4.0 [A]) to investigate the three-dimensional structure of prestin from gerbil (Meriones unguiculatus). Our studies show that prestin dimerizes with a 3D architecture strikingly similar to the dimeric conformation observed in the Slc26a9 anion transporter in an inside open/intermediate state, which we infer, based on patch-clamp recordings, to reflect the contracted state of prestin. The structure shows two well-separated transmembrane (TM) subunits and two cytoplasmic sulfate transporter and anti-sigma factor antagonist (STAS) domains forming a swapped dimer. The dimerization interface is defined by interactions between the domain-swapped STAS dimer and the transmembrane domains of the opposing half unit, further strengthened by an antiparallel beta-strand at its N terminus. The structure also shows that each one of its two transmembrane subunits consists of 14 transmembrane segments organized in two inverted 7-segment repeats with a topology that was first observed in the structure of bacterial symporter UraA (Lu F, et al., Nature 472, 2011). Finally, the solved anion binding site structural features of prestin are quite similar to that of SLC26a9 and other family members. Despite this similarity, we find that SLC26a9 lacks the characteristic displacement currents (or NonLinear Capacitance(NLC)) found with prestin, and we show that mutation of prestins Cl- binding site removes salicylate competition with anions in the face of normal NLC, thus refuting the yet accepted extrinsic voltage sensor hypothesis and any associated transport-like requirements for voltage-driven electromotility.


title: The kpc-1 (furin) 3'UTR promotes dendritic transport and local translation of mRNAs to regulate dendrite branching and self-avoidance of a nociceptive neuron

abstract: Mechanical stimuli on the skin of C. elegans are detected by the dendritic arbors of PVD nociceptive neurons, which provide uniform sensory coverage outside the head region across the entire animal. Through genetic screens, we isolate three mutants that display profound dendrite self-avoidance defects in PVD neurons. Studying dendrite self-avoidance in C. elegans is likely to provide new mechanistic insight into the process as the well-known self-avoidance molecule Dscam is absent from the C. elegans genome. Through whole genome sequencing, we identify the responsible mutations in the kpc-1 gene. Compared to wild-type animals, a strong kpc-1 mutant allele exhibits secondary dendrite branching defects whereas a weak kpc-1 mutant allele displays tertiary dendrite self-avoidance defects. Here, we show that the kpc-1 3UTR is required for kpc-1s functions in both dendrite branching and dendrite self-avoidance. The kpc-1 3UTR facilitates kpc-1 RNA localization to branching points and contact points between sibling dendrites. Using fluorescence recovery after photoconversion, we show that the kpc-1 3UTR promotes local protein synthesis in the distal segment of PVD dendrites. We identify an important secondary structural motif in the kpc-1 3UTR required for tertiary dendrite self-avoidance. We demonstrate that over-expression of kpc-1 leads to greater self-avoidance without limiting initial dendrite outgrowth, supporting a direct role of kpc-1 in self-avoidance. Animals with dma-1 receptor over-expression display similar secondary dendrite branching and tertiary dendrite self-avoidance defects that are suppressed with kpc-1 over-expression, which suggests that DMA-1 is a potential KPC-1 target that is down-regulated by KPC-1. Our results support a model where KPC-1 proteins are synthesized at branching points and contact points between neighboring dendrites to locally down-regulate DMA-1 receptors to promote dendrite branching and self-avoidance. A recently reported Schizophrenia-associated genetic variant in the 3UTR of the human furin gene, a homolog of kpc-1, highlights the important role of the kpc-1 (furin) 3UTR in neuronal development, which is further demonstrated by this mechanistic study.


title: Specific telomere protection ensured by FOXO3a upon genotoxic stress and during aging

abstract: Longevity is determined by diverse signaling pathways including telomere protection and homeostasis master regulators like FOXO3a. We previously showed that the telomeric repeat binding factor 2 (TRF2) expression decreases with age in human skeletal muscle and that, surprisingly, its loss in myofibers does not trigger telomere deprotection. We reveal here that in TERF2-compromised myotubes, FOXO3a is recruited to telomeres where it acts as a protective factor against ATM-dependent DNA damage activation. Moreover, we show that FOXO3a-telomere association increases with age in human skeletal muscle biopsies. In mitotic fibroblasts, the telomere protective properties of FOXO3a are operative if the cells are treated with bleomycin. The telomere function of FOXO3a does not require its Forkhead DNA binding domain but the CR2C. Overall, these findings demonstrate a direct connection between two key longevity pathways, FOXO3a and telomere protection. This unveils an unexpected higher level of integration in the regulation of longevity signaling pathway.


title: Local regulation of extracellular vesicle traffic by the synaptic endocytic machinery

abstract: Neuronal extracellular vesicles (EVs) can be locally released from presynaptic terminals, carrying cargoes that are important in intercellular signaling and disease. EVs are derived from endosomes, but it remains unclear how synaptic cargoes are directed to the EV pathway, rather than undergoing conventional retrograde endosomal transport and degradation. Here, we find that the clathrin-mediated endocytic machinery plays an unexpected role in maintaining a release-competent pool of synaptic EV cargoes. Endocytic mutants, including nervous wreck (nwk), Shibire/Dynamin, and AP-2, exhibit local depletion of multiple cargoes in EV donor terminals. Accordingly, nwk mutants phenocopy synaptic plasticity defects associated with loss of the EV cargo Synaptotagmin-4, and suppress lethality upon overexpression of the EV cargo Amyloid Precursor Protein. These EV defects are genetically separable from canonical functions of endocytic proteins in synaptic vesicle recycling and synaptic growth. This endocytic pathway opposes the endosomal retromer complex to regulate EV cargo levels, and acts upstream of synaptic cargo removal by retrograde axonal transport. Our data suggest a novel molecular mechanism that protects EV cargoes from local depletion at synapses.


title: AATF/Che-1, a new component of paraspeckles, controls R-loops formation and Interferon activation in Multiple Myeloma.

abstract: Multiple myeloma (MM) is a hematological neoplasm of plasma cells characterized by abnormal production of immunoglobulins. Che-1/AATF (Che-1) is an RNA binding protein involved in transcription regulation and is highly expressed in this malignancy. Here we experimentally show that Che-1 interacts with paraspeckle components, including the lncRNA NEAT1_2 (NEAT1), which serves as the seed for the maintenance of these structures. Che-1 and NEAT1 localize on R-loops, three-stranded RNA:DNA hybrids structures involved in DNA transcription and repair. Depletion of Che-1 produces a marked accumulation of RNA:DNA hybrids sustaining activation of a systemic inflammatory response. We provide evidence that high levels of Unfolded Protein Response (UPR) in MM cells induces RNA:DNA hybrids and an interferon (IFN) gene signature. We found that MM patients exhibit elevated R-loops levels and paraspeckle genes mRNAs increase linearly to MM progression. Strikingly, patients showing elevated IFN genes signature are associated with a marked poor prognosis. Overall, these findings delineate that elevated R-loops accumulation and inflammatory signaling may contribute to MM progression and that Che-1/NEAT1 plays an essential role in maintaining R-loops homeostasis by preventing excessive inflammatory signaling.


title: Autophagy suppression by TORC1 maintains epithelial plasma membrane integrity and inhibits syncytium formation

abstract:
Epithelial wound healing in Drosophila involves the formation of multinucleate cells surrounding the wound. We show that autophagy, a cellular degradation process often deployed in stress responses, is required for the formation of a multinucleated syncytium during wound healing. In addition, uncontrolled autophagy in the unwounded epidermis leads to the degradation of endo-membranes and the lateral plasma membrane, while the apical and basal membranes and the epithelial barrier function remain intact. Proper functioning of TORC1 is needed to prevent autophagy from destroying the larval epidermis, which depends on membrane isolation and phagophore expansion, but does not require the fusion of autophagosomes to lysosomes. Our findings reveal a function for TORC1-mediated regulation of autophagy in maintaining membrane integrity and homeostasis in the epidermis and during wound healing. Finally, autophagy can counteract experimentally induced nuclear defects resembling laminopathies.

Key findingsO_LIA novel role for TORC1/autophagy pathway to control plasma membrane integrity and homeostasis.
C_LIO_LIAutophagy as the only known necessary and sufficient inducer of syncytium formation in the epithelium and during wound healing.
C_LI


title: A study of spatio-temporal dynamics of emotion processing usingDENS dataset

abstract: The emotion research with artificial stimuli does not represent the dynamic processing of emotions in real-life situations. The lack of data on emotion with the ecologically valid naturalistic paradigm hinders the knowledge of emotion mechanism in a real-world interaction. To this aim, we collected the emotional multimedia clips, validated them with the university students, recorded the neuro-physiological activities and self-assessment ratings for these stimuli. Participants localized their emotional feelings (in time) and were free to choose the best emotion for describing their feelings with minimum distractions and cognitive load. The obtained electrophysiological and self-assessment responses were analyzed with functional connectivity, machine learning and source localization techniques. We observed that the connectivity patterns in the theta and beta band could differentiate emotions better. Using machine learning, we observed that the classification of affective self-assessment features, namely dominance, familiarity, and self-relevance, involves midline brain regions responsible for mentalization and event construction activity compared to valence and arousal, which were mainly associated with lateral brain regions. This finding advocates the need for more than two dimensions for emotion representation. In addition, the channels with high predictability were source localized to the brain regions in default-mode, sensorimotor and salience networks. Hence, in this naturalistic study, we find that the domain-general systems contribute to emotion construction.


title: Naturalistic paradigm reveals multi-component emotion dynamics in theta and beta bands using DENS dataset


title: Chronic and progressive deficits after a single closed head injury in mice

abstract:
BackgroundAcute injury following brain trauma may evolve into a chronic and progressive disorder. Assessment of chronic consequences of TBI must distinguish between effects of age and injury.

MethodsC57BL/6 mice receive single closed head injury (CHI) and are analyzed at 14DPI or 180DPI for cortical atrophy and 7DPI or 180DPI for behavioral outcomes.

ResultsCHI induces ipsilesional atrophy at 14DPI that increases 180 DPI due to an effect of age. On open field, injured mice develop a turn bias at 180DPI not present at 7DPI. On rotarod, injured mice have shorter latencies at 7DPI, but not at 180DPI due to worsening performance of aging uninjured mice. On beam walk, both groups at 180DPI more slowly traverse a 2cm and 1cm beam than at 7DPI. Foot-faults show no significant effects of age or injury. Limb position was assessed using Deeplabcut markerless tracking followed by computation of absition (integral of limb displacement over time) using custom Python scripts. On the 2cm beam, age increased absition in all limbs of uninjured mice and both forelimbs of injured mice. Injury increased left hindlimb absition at 7DPI. On the 1cm beam both forelimbs and the left hindlimb of injured mice at 180DPI have larger absition than uninjured mice at 180DPI or injured mice at 7DPI. These data suggest chronic and progressive motor deficits of injured mice at 180DPI.

ConclusionsA single impact produces ipsilesional cortical atrophy and chronic and progressive motor deficits. Quantitative behavioral analysis reveals deficits not seen using standard outcomes.


title: Chronic behavioral manipulation via orally delivered chemogenetic actuator in macaques

abstract: The chemogenetic technology referred to as designer receptors exclusively activated by designer drugs (DREADDs) offers reversible means to control neuronal activity for investigating its functional correlation with behavioral action. Deschloroclozapine (DCZ), a recently-developed highly potent and selective DREADDs actuator, displays a capacity to expand the utility of DREADDs for chronic manipulation without side-effects in nonhuman primates, which has not yet been validated. Here we investigated the pharmacokinetics and behavioral effects of orally administered DCZ in macaque monkeys. Pharmacokinetic analysis and positron emission tomography (PET) occupancy examination demonstrated that oral administration of DCZ yielded slower and prolonged kinetics, and that its bioavailability was 10-20% of that in the case of systemic injection. Oral DCZ (300-1000 g/kg) induced significant working memory impairments for at least 4 h in monkeys with hM4Di expressed in the prefrontal cortex. Repeated daily oral doses of DCZ consistently caused similar impairments over two weeks without discernible desensitization. Our results indicate that orally delivered DCZ affords a less invasive strategy for chronic but reversible chemogenetic manipulation of neuronal activity in nonhuman primates, and this has potential for clinical application.


title: Biophysical network models of phase-synchronization in MEG resting-state

abstract:
Magnetoencephalography (MEG) is used extensively to study functional connectivity (FC) networks of phase-synchronization, but the relationship of these networks to their biophysical substrates is poorly understood. Biophysical Network Models (BNMs) have been used to produce networks corresponding to MEG-derived networks of phase-synchronization, but the roles of inter-regional conduction delays, the structural connectome and dynamics of model of individual brain regions, in obtaining this correspondence remain unknown. In this study, we investigated the roles of conduction delays, the structural connectome, and dynamics of models of individual regions, in obtaining a correspondence between model-generated and MEG-derived networks between left-hemispheric regions. To do this, we compared three BNMs, respectively comprising Wilson-Cowan oscillators interacting with diffusion Magnetic Resonance Imaging (MRI)-based patterns of structural connections through zero delays, constant delays and distance-dependent delays respectively. For the BNM whose networks corresponded most closely to the MEG-derived network, we used comparisons against null models to identify specific features of each model component, e.g. the pattern of connections in the structure connectome, that contributed to the observed correspondence. The Wilson-Cowan zero delays model produced networks with a closer correspondence to the MEG-derived network than those produced by the constant delays model, and the same as those produced by the distance-dependent delays model. Hence, there is no evidence that including conduction delays improves the correspondence between model-generated and MEG-derived networks. Given this, we chose the Wilson-Cowan zero delays model for further investigation. Comparing the Wilson-Cowan zero delays model against null models revealed that both the pattern of structural connections and Wilson-Cowan oscillatory dynamics contribute to the correspondence between model-generated and MEG-derived networks. Our investigations yield insight into the roles of conduction delays, the structural connectome and dynamics of models of individual brain regions, in obtaining a correspondence between model-generated and MEG-derived networks. These findings result in a parsimonious BNM that produces networks corresponding closely to MEG-derived networks of phase-synchronization.

HighlightsO_LISimple biophysical model produces close match ({rho}=0.49) between model and MEG networks
C_LIO_LINo evidence for conduction delays improving match between model and MEG networks
C_LIO_LIPattern of structural connections contributes to match between model and MEG networks
C_LIO_LIWilson-Cowan oscillatory dynamics contribute to match between model and MEG networks
C_LI


title: A toolbox for studying cortical physiology in primates

abstract: Lesioning and neurophysiological studies have facilitated the elucidation of cortical functions and mechanisms of functional recovery following injury. Clinical translation of such studies is contingent on their employment in non-human primates (NHPs), yet tools for monitoring and modulating cortical physiology are incompatible with conventional NHP lesioning techniques. To address these challenges, we developed a toolbox demonstrated in seven macaques. We introduce the photothrombotic method to induce focal cortical lesions alongside a quantitative model for the design of lesion profiles based on experimental needs. Large-scale ([~]5 cm2) vascular and neural dynamics can be monitored and lesion induction can be validated in vivo with optical coherence tomography angiography and our electrocorticographic array, the latter of which also enables testing stimulation-based interventions. By combining optical and electrophysiological techniques in NHPs, we can enhance our understanding of cortical functions, investigate functional recovery mechanisms, integrate physiological and behavioral findings, and develop treatments for neurological disorders.


title: Dataset on Emotions using Naturalistic Stimuli (DENS)

abstract: Emotion is a constructed phenomenon that emerges from the dynamic interaction of multiple components neurologically, physiologically and behaviorally. Such dynamics can not be captured by static and controlled experiments. Hence, the study of emotion with a naturalistic paradigm is needed. In this dataset, multimedia naturalistic stimuli are used to acquire the emotional dynamics using EEG, ECG, EMG and behavioural scales. The stimuli are multimedia videos collected from youtube for 372 affective words, analyzed with multimedia content analysis to filter out non-emotional stimuli and then validated with university students. The validated stimuli had the least variance in subjective ratings on self-assessment scales. The stimuli are then used to acquire neurological dynamics along with peripheral channels and subjective ratings-valence, arousal, dominance, liking, familiarity, relevance and emotion category. Both the raw data and pre-processed data is provided along with the pre-processing pipeline. This data can be utilized to study dynamic activation and connectivity in the whole brain source localization study, understand the mutual interaction between the central and autonomic nervous system, understand temporal hierarchy using multiresolution tools, and perform machine learning-based classification and complex networks analysis. The data is accessible at 10.18112/openneuro.ds003751.v1.0.0


title: Retinal degeneration: Multilevel protection of photoreceptor and ganglion cell viability and function with the novel PKG inhibitor CN238.

abstract: Hereditary retinal degeneration (RD) is often associated with excessive cGMP-signaling in photoreceptors. Previous research has shown that inhibition of cGMP-dependent protein kinase G (PKG) can slow down the loss of photoreceptors in different RD animal models. In this study, we identified a novel PKG inhibitor, the cGMP analogue CN238, with strong protective effects on photoreceptors in retinal degeneration rd1 and rd10 mutant mice. In long-term organotypic retinal explants, CN238 preserved rd1 and rd10 photoreceptor viability and function. Surprisingly, in explanted retinae CN238 also protected retinal ganglion cells from axotomy induced retrograde degeneration and preserved their functionality. Together, these results confirm the strong neuroprotective capacity of PKG inhibitors for both photoreceptors and retinal ganglion cells, thereby significantly broadening their potential applications for the treatment of retinal diseases and possibly neurodegenerative diseases in general.


title: Membrane remodeling and matrix dispersal intermediates during mammalian acrosomal exocytosis

abstract:
To become fertilization-competent, mammalian sperm must undergo a complex series of biochemical and morphological changes in the female reproductive tract. These changes, collectively called capacitation, culminate in the exocytosis of the acrosome, a large vesicle overlying the nucleus. Acrosomal exocytosis is not an all-or-nothing event, but rather a regulated process in which vesicle cargo disperses gradually. However, the structural mechanisms underlying this controlled release remain undefined. In addition, unlike other exocytotic events, fusing membranes are shed as vesicles; the cell thus loses the entire anterior two-thirds of its plasma membrane and yet remains intact while the remaining non-vesiculated plasma membrane becomes fusogenic. Precisely how cell integrity is maintained through-out this drastic vesiculation process is unclear, as is how it ultimately leads to the acquisition of fusion competence. Here, we use cryo-electron tomography to visualize these processes in unfixed, unstained, fully-hydrated sperm. We show that crystalline structures within the acrosome disassemble during capacitation and acrosomal exocytosis, representing a plausible mechanism for gradual dispersal of the acrosomal matrix. We find that the architecture of the sperm head supports an atypical membrane fission-fusion pathway that maintains cell integrity. Finally, we detail how the acrosome reaction transforms both the micron-scale topography and the nano-scale protein landscape of the sperm surface, thus priming the sperm for fertilization.

SignificanceMammalian sperm must undergo a complex series of biochemical and morphological changes in the female reproductive tract in order to become fertilization-competent. These changes culminate in acrosomal exocytosis, during which multiple membrane fusions destabilize the acrosomal vesicle and liberate its contents, which include proteins implicated in penetrating and binding to the egg vestments. Here, we use cryo-electron tomography to visualize acrosomal exocytosis intermediates in unfixed, unstained sperm. Our results suggest structural bases for how gradual dispersal of acrosome contents is regulated, as well as for how the cell remains intact after losing much of its plasma membrane, We also show that acrosomal exocytosis transforms both the micron-scale topography and the nano-scale molecular landscape of the sperm surface, thus priming it for interaction and fusion with the egg. These findings yield important insights into sperm physiology and contribute to our understanding of the fundamental yet enigmatic process of mammalian fertilization.


title: The role of Musashi-1 in CEP290 c.2991+1655A>G cryptic exon splicing in Leber Congenital Amaurosis

abstract:
Human photoreceptors maximise alternative exon splicing to generate a unique set of gene isoforms. Conversely, the inclusion of a cryptic exon caused by the c.2991+1655A>G deep intronic change in CEP290 occurs in the human retina leading to Leber Congenital Amaurosis (LCA10). The RNA-binding protein Musashi-1 (MSI1) is a key component of alternative splicing in the developing mouse retina. Here we investigated the role of MSI1 in human photoreceptor-specific splicing and its potential role in CEP290 aberrant splicing disease. Alternative splicing was studied using human induced pluripotent stem cell derived 3D retinal organoid and RPE RNA-seq datasets and several photoreceptor gene isoforms were identified. Their temporal expression was resolved in control 3D retinal organoids in comparison to development and differentiation markers. Morpholino knockdown of MSI1 in control retinal organoids reduced the expression of several photoreceptor differentiation markers and the inclusion of photoreceptor-specific exons. Nonetheless, MSI1 knockdown in homozygous CEP290 c.2991+1655A>G LCA10 retinal organoids did not affect the inclusion of the LCA10-associated cryptic exon. These results show that while MSI1 is important for photoreceptor alternative splicing and homeostasis, it is not a major driver of the recognition of the CEP290 cryptic splice site and the manifestation of LCA10.

HIGHLIGHTS{blacksquare} The human retina expresses a unique set of gene isoforms
{blacksquare}Musashi-1 regulates alternative splicing in 3D human retinal organoids
{blacksquare}Musashi-1 knockdown in 3D retinal organoids affects gene splicing and homeostasis in photoreceptors
{blacksquare}Musashi-1 may regulate alternative splicing of cryptic exons in retina but not in LCA10


title: Size-scaling promotes senescence-like changes in proteome and organelle content

abstract: Senescent cells typically have an enlarged cell size but the reason for this has not been fully elucidated. As abnormal cell size may alter protein concentrations and cellular functionality, we used proteomic data from 59 unperturbed human cell lines to systematically characterize cell-size dependent changes in intracellular protein concentrations and organelle content. Increase in cell size leads to ubiquitous transcriptionally and post-transcriptionally regulated reorganization and dilution of the proteome. Many known senescence proteins display disproportionate size-scaling consistent with their altered expression in senescent cells, while lysosomes and the endoplasmic reticulum expand in larger cells contributing to the senescence phenotype. Analysis of organelle proteome expression identifies p53 and retinoblastoma pathways as mediators of size-scaling, consistent with their role in senescence. Taken together, cell size can alter cellular fitness and function through cumulative reorganization of the proteome and organelle content. An extreme consequence of this pervasive size-scaling appears to be senescence.


title: An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus

abstract: In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and -219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively  drop out a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. miRNA-219 and miR-196a KOs both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.


title: A Self-Limiting Circuit Regulates Mammary Cap Cell Plasticity Through TGF-beta Signaling

abstract: The organization and maintenance of complex tissues requires emergent properties driven by self-organizing and self-limiting cell-cell interactions. We examined these interactions in the murine mammary gland. Luminal and myoepithelial subpopulations of the postnatal mammary gland arise from unipotent progenitors, but the destiny of cap cells, which enclose terminal end buds (TEB) in pubertal mice, remains controversial. Using a transgenic strain (Tg11.5kb-GFP) that specifically marks cap cells, we found ~50% of these cells undergo divisions perpendicular to the TEB surface, suggesting they might contribute to the underlying luminal cell population. To address their stemness potential we developed a lineage tracing mouse driven from the s-SHIP (11.5 kb) promoter. Induction of tdTomato (tdTom) from this promoter in vivo demonstrated that all cap cell progeny are myoepithelial, with no conversion to luminal lineage. Organoid cultures also exhibited unipotency. However, isolated cap cells cultured as mammospheres generated mixed luminal/myoepithelial spheres. Moreover, ablation of luminal cells in vivo using diphtheria toxin triggered repopulation by progeny of tdTom+ cap cells. A signaling inhibitor screen identified the TGF{beta} pathway as a potential regulator of multipotency. TGF{beta}R inhibitors or gene deletion blocked conversion to the luminal lineage, consistent with an autocrine loop in which cap cells secrete TGF{beta} to activate the receptor and promote luminal transdifferentiation. Ductal tree regeneration in vivo from isolated cap cells was much more efficient when they were pre-treated with inhibitor, consistent with more cells retaining cap cell potential prior to transplantation. Notably, in vitro transdifferentiation of cap cells was blocked by co-culture with luminal cells. Overall, these data reveal a self-limiting cell circuit through which mammary luminal cells suppress cap cell conversion to the luminal lineage.


title: The CD8+ T cell landscape of human brain metastases

abstract: Despite improved outcomes with checkpoint blockade immunotherapy, patients with brain metastases have the worst prognosis among patients with metastatic cancer. Immune checkpoint blockade agents target inhibitory receptors, such as PD-1, on exhausted CD8+ T cells to restore their anti-cancer function. Many patients, however, either do not respond or progress after an initial response to immune checkpoint blockade, and distant intracranial failure is common despite excellent options for local treatment of brain metastasis. To develop more effective therapeutic strategies for the treatment of brain metastases, an understanding of the phenotype of brain metastasis-infiltrating CD8+ T cells is essential. Here we performed a detailed characterization of the CD8+ T cells contained in brain metastases. Brain metastases were densely infiltrated by CD8+ T cells; blood contamination of tumor samples was rare. Compared to patient-matched circulating cells, brain metastasis-infiltrating CD8+ T cells had a distinct phenotype characterized by more frequent expression of PD-1, with subpopulations defined by expression of additional co-inhibitory molecules and the residence marker CD69. Single cell RNA-sequencing identified four phenotypic subpopulations within brain metastasis-infiltrating PD-1+ CD8+ T cells. Two of these populations -- a terminally-differentiated and a dividing population -- were characterized by high expression of co-inhibitory molecules and lacked expression of progenitor markers such as TCF-1. There was significant T cell receptor (TCR) overlap between the terminally-differentiated and dividing populations, suggesting that the dividing cells give rise to the terminally-differentiated cells. There was minimal TCR overlap between these two populations and other brain metastasis-infiltrating PD-1+ CD8+ T cells. T cell clones from brain metastasis-infiltrating CD8+ T cells were rare in circulation, particularly clones from the terminally-differentiated and dividing populations. We systematically identified bystander CD8+ T cells specific for microbial antigens; these cells infiltrated brain metastases and expressed genes shared with exhausted progenitor CD8+ T cells, such as TCF7 and IL7R. We performed spatial transcriptomics on brain metastases and used a novel method to obtain TCR sequences from spatial transcriptomics data. These data revealed distinct niches within the TME defined by their gene expression patterns and cytokine profiles. Terminally-differentiated CD8+ T cells preferentially occupied niches within the tumor parenchyma. Together, our results show that antigen-specificity restricts the spatial localization, phenotypic states, and differentiation pathways available to CD8+ T cells within the brain metastasis TME.