title: A Humanized Antibody against LRG1 that Inhibits Angiogenesis and Reduces Retinal Vascular Leakage

abstract: Pathological angiogenesis contributes to morbidity in a number of diseases including cancer, diabetic retinopathy and the neovascular form of age-related macular degeneration, leading to significant efforts to develop effective anti-angiogenic therapeutics for these conditions. The field is dominated by inhibitors of vascular endothelial growth factor (VEGF), yet angiogenesis can also be driven and modified by other factors. We have previously demonstrated that leucine-rich alpha-2-glycoprotein 1 (LRG1) contributes to abnormal vessel growth by activating a TGF{beta} switch. Here we report the development and characterisation of a function-blocking fully humanised IgG4 and its Fab fragment, that bind to LRG1 with high affinity and specificity and inhibit vascular leakage in the mouse model of laser-induced choroidal neovascularisation. In summary, we have developed a therapeutic antibody that targets a VEGF-independent signalling axis, which may be effective in a number of conditions either as monotherapy or in combination with other vascular targeted therapies.

category: pathology


title: Surface-bound antigen induces B-cell permeabilization and lysosome exocytosis facilitating antigen uptake and presentation to T-cells

abstract: B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral memory immune responses. Antigen encountered by B-cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane (PM) permeabilization, a process that requires BCR signaling and non-muscle myosin II activity. B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently presented to T-cells. Thus, PM permeabilization by surface-associated antigen triggers a lysosome-mediated B-cell resealing response, providing the extracellular hydrolases that facilitate antigen internalization and presentation.

category: cell biology


title: Surface-associated antigen induces B-cell permeabilization and lysosome exocytosis facilitating antigen uptake and presentation to T-cells


title: Human Beta Cell Mass Expansion In Vivo With A Harmine and Exendin-4 Combination: Quantification and Visualization By iDISCO+ 3D Imaging

abstract: 463 million people globally suffer from diabetes. The majority are deficient in insulin-producing pancreatic beta cells, although beta cells remain in most people with diabetes. Unfortunately, although many diabetes drugs exist, none is able to increase adult human beta cell numbers. Recently, small molecules that inhibit the kinase, DYRK1A, have been suggested to induce human beta cell replication in vitro and in vivo as assessed using proliferation markers, and this is enhanced by drugs that stimulate the GLP1 receptor (GLP1R) on beta cells. DYRK1A inhibitors also enhance human beta cell differentiation and function. However, it is unknown whether any drug can actually increase human beta cell mass in vivo, reflecting: 1) the intrinsic resistance of human beta cells to regeneration; and, 2) the current technical inability to accurately assess human beta cell mass in vivo. Here, we demonstrate for the first time that combining a DYRK1A inhibitor with a GLP1R agonist increases actual human beta cell numbers and overall mass in vivo by 400-700% in diabetic and non-diabetic mice over three months. We further describe a novel application of tissue-clearing and 3D imaging for quantification of human beta cell mass. These findings should be transformative for diabetes treatment.


title: Analysis and modeling of cancer drug responses using cell cycle phase-specific rate effects

abstract: A major challenge to improving outcomes for patients with cancer is the identification of effective therapeutic strategies that can prevent tumor cell proliferation. Here we sought to gain a deeper understanding of how anti-cancer agents modulate cell cycle progression in HER2+ breast cancer, a disease subtype that accounts for 20% of all breast cancers. We treated HER2+ breast cancer cells with a panel of drugs and tracked changes in cell number and cell cycle phase, which revealed drug-specific cell cycle effects that varied across time. This suggested that a computational model that could account for cell cycle phase durations would provide a framework to explore drug-induced changes in cell cycle changes. Toward that goal, we developed a linear chain trick (LCT) computational model, in which the cell cycle is partitioned into subphases that faithfully captured drug-induced dynamic responses. The model inferred phase-specific drug effects and independent modulation of cell cycle phases, which we confirmed experimentally. We then used our LCT model to predict the effect of unseen drug combinations that target cells in different cell cycle phases. Experimental testing confirmed several model predictions and identified combination treatment strategies that may improve therapeutic response in patients with HER2+ breast cancer. Overall, this integrated experimental and modeling approach opens new avenues for assessing drug responses, predicting effective drug combinations, and identifying optimal drug sequencing strategies.

category: cancer biology


title: Benchmarking challenging small variants with linked and long reads

abstract: Genome in a Bottle (GIAB) benchmarks have been widely used to help validate clinical sequencing pipelines and develop new variant calling and sequencing methods. Here, we use accurate linked reads and long reads to expand the prior benchmarks in 7 samples to include difficult-to-map regions and segmental duplications that are not readily accessible to short reads. Our new benchmark adds more than 300,000 SNVs, 50,000 indels, and 16 % new exonic variants, many in challenging, clinically relevant genes not previously covered (e.g., PMS2). For HG002, we include 92% of the autosomal GRCh38 assembly, while excluding problematic regions for benchmarking small variants (e.g., copy number variants and reference errors) that should not have been in the previous version, which included 85% of GRCh38. By including difficult-to-map regions, this benchmark identifies eight times more false negatives in a short read variant call set relative to our previous benchmark.We have demonstrated the utility of this benchmark to reliably identify false positives and false negatives across technologies in more challenging regions, which enables continued technology and bioinformatics development.

category: genomics


title: Branched-Chain Amino Acid Metabolism is Regulated by ERRα in Primary Human Myotubes and is Further Impaired by Glucose Loading in Type 2 Diabetes

abstract:
Aims/hypothesisIncreased levels of branched-chain amino acids (BCAAs) are associated with type 2 diabetes pathogenesis. However, most metabolomic studies are limited to an analysis of plasma metabolites under fasting conditions, rather than the dynamic shift in response to a metabolic challenge. Moreover, metabolomic profiles of peripheral tissues involved in glucose homeostasis are scarce and the transcriptomic regulation of genes involved in BCAA catabolism is partially unknown. This study aimed to identify differences in circulating and skeletal muscle BCAA levels in response to an OGTT in individuals with normal glucose tolerance (NGT) or type 2 diabetes. Additionally, transcription factors involved in the regulation of the BCAA gene set were identified.

MethodsPlasma and vastus lateralis muscle biopsies were obtained from individuals with NGT or type 2 diabetes before and after an OGTT. Plasma and quadriceps muscles were harvested from skeletal muscle-specific PGC-1 knockout and transgenic mice. BCAA-related metabolites and genes were assessed by LC-MS/MS and RT-PCR, respectively. Small interfering RNA and adenovirus-mediated overexpression techniques were used in primary human skeletal muscle cells to study the role of PGC-1A and ESRRA in the expression of the BCAA gene set. Radiolabeled leucine was used to analyze the impact of ERR knockdown on leucine oxidation.

ResultsImpairments in BCAA catabolism in people with type 2 diabetes under fasting conditions were exacerbated after a glucose load. Branched-chain keto acids were reduced 37-56% after an OGTT in the NGT group, whereas no changes were detected in individuals with T2D. These changes were concomitant with a stronger correlation with glucose homeostasis biomarkers and downregulated expression of BCAT2, BCKDH complex subunits and 69% of downstream BCAA-related genes in skeletal muscle. In primary human myotubes overexpressing PGC-1, 61% of the analyzed BCAA genes were upregulated, while 67% were downregulated in the quadriceps of skeletal muscle-specific PGC-1 knockout mice. ESRRA (encoding estrogen-related receptor , ERR) silencing completely abrogated the PGC-1-induced upregulation of BCAA-related genes in primary human myotubes.

Conclusions/interpretationMetabolic inflexibility in type 2 diabetes impacts BCAA homeostasis and attenuates the decrease of circulating and skeletal muscle BCAA-related metabolites after a glucose challenge. Transcriptional regulation of BCAA genes in primary human myotubes via a PGC-1 is ERR-dependent.

Research in contextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LICirculating levels of BCAA are elevated in type 2 diabetes.
C_LIO_LIPGC-1 is involved in the transcription of the BCAA gene set.
C_LI

What is the key question?O_LIDoes metabolic inflexibility associated with type 2 diabetes encompass BCAA homeostasis and PGC-1 mediated transcription of the BCAA gene set?
C_LI

What are the new findings?O_LIBCAA homeostasis is further compromised by a glucose challenge in type 2 diabetes.
C_LIO_LIAn OGTT reveals coordinated regulation between BCAA metabolites, blood glucose, and HbA1c levels.
C_LIO_LIERR is essential for PGC-1-mediated BCAA gene expression in primary human myotubes.
C_LI

How might this impact on clinical practice in the foreseeable future?O_LIAn OGTT can be used to underscore impairments in BCAA metabolism. These findings suggest that interventions targeting the PGC-1/ ERR axis may improve BCAA homeostasis.
C_LI

category: physiology


title: SARS-CoV-2 and SARS-CoV spike-mediated cell-cell fusion differ in the requirements for receptor expression and proteolytic activation

abstract:
The severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with Angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and the sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S(SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by Batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector-target-cell fusion when ACE2 or TMPRSS2 were limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target-effector-cell fusion was unaltered compared to wt SARS2-S, but syncytia remained smaller. Mutation of the S2 site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor Bromhexine was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S as opposed to the inhibitor Camostat. Paradoxically, Bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite Ambroxol exhibited inhibitory activity in some conditions. On Calu-3 lung cells, Ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend towards weak inhibition of authentic SARS-CoV-2.

IMPORTANCECell-cell fusion allows the virus to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2 cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently tested in clinical trials against coronavirus disease 2019. Our results indicate that Bromhexine enhances fusion in some conditions. We therefore caution against use of Bromhexine in higher dosage until its effects on SARS-CoV-2 spike activation are better understood. The related compound Ambroxol, which similarly to Bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for Ambroxol.

category: microbiology


title: The neuropeptides VIP and PACAP inhibit SARS-CoV-2 replication in monocytes and lung epithelial cells, decrease the production of proinflammatory cytokines, and VIP levels are associated with survival in severe COVID-19 patients.

abstract: Infection by SARS-CoV-2 may elicit uncontrolled and damaging inflammatory responses. Thus, it is critical to identify compounds able to inhibit virus replication and thwart the inflammatory reaction. Here, we show that the plasma levels of the immunoregulatory neuropeptide VIP are elevated in patients with severe COVID-19, correlating with reduced inflammatory mediators and with survival on those patients. In vitro, VIP and PACAP, highly similar neuropeptides, decreased the SARS-CoV-2 genome replication in human monocytes and viral production in lung epithelial cells, also reducing cell death. Both neuropeptides inhibited the production of proinflammatory mediators in lung epithelial cells and in monocytes. VIP and PACAP prevented in monocytes the SARS-CoV-2-induced activation of NF-kB and SREBP1 and SREBP2, transcriptions factors involved in proinflammatory reactions and lipid metabolism, respectively. They also promoted CREB activation, a transcription factor with antiapoptotic activity and negative regulator of NF-kB. Specific inhibition of NF-kB and SREBP1/2 reproduced the anti-inflammatory, antiviral and cell death protection effects of VIP and PACAP. Our results support further clinical investigations of these neuropeptides against COVID-19.


title: Protective effects of VIP and PACAP in SARS-CoV-2 infection


title: VIP plasma levels associate with survival in severe COVID-19 patients, correlating with protective effects in SARS-CoV-2-infected cells.

category: immunology


title: Interneuron Specific Gamma Synchronization Indexes Cue Uncertainty and Prediction Errors in Lateral Prefrontal and Anterior Cingulate Cortex

abstract: Inhibitory interneurons are believed to realize critical gating functions in cortical circuits, but it has been difficult to ascertain the content of gated information for well characterized interneurons in primate cortex. Here, we address this question by characterizing putative interneurons in primate prefrontal and anterior cingulate cortex while monkeys engaged in attention demanding reversal learning. We find that subclasses of narrow spiking neurons have a relative suppressive effect on the local circuit indicating they are inhibitory interneurons. One of these interneuron subclasses showed prominent firing rate modulations and (35-45 Hz) gamma synchronous spiking during periods of uncertainty in both, lateral prefrontal cortex (LPFC) and in anterior cingulate cortex (ACC). In LPFC this interneuron subclass activated when the uncertainty of attention cues was resolved during flexible learning, whereas in ACC it fired and gamma-synchronized when outcomes were uncertain and prediction errors were high during learning. Computational modeling of this interneuron-specific gamma band activity in simple circuit motifs suggests it could reflect a soft winner-take-all gating of information having high degree of uncertainty. Together, these findings elucidate an electrophysiologically-characterized interneuron subclass in the primate, that forms gamma synchronous networks in two different areas when resolving uncertainty during adaptive goal-directed behavior.

category: neuroscience


title: Eyes wide open: regulation of arousal by temporal expectations

abstract:
To perform a task continuously over an extended period of time, it is necessary to maintain an adequate level of arousal. In cognitive research, traditional studies have used repetitive, monotonous tasks to learn about the dynamics of arousal in tasks that require sustained vigilance, such as driving or monitoring a factory line. To date, studies have rarely considered whether observers use task-embedded regularities in such continuous contexts to anticipate events and regulate arousal accordingly. In the current study, we explored whether arousal is an adaptive process that reacts to temporal stimulus predictability. Across two experiments, we used pupillometry as a proxy measure of arousal in human observers performing continuous tasks. Within the tasks, we manipulated the temporal predictability of relevant events by presenting stimuli either in a fixed rhythm or at varied intervals. Temporal predictability led to the lowering of tonic levels of arousal. Trial-wise temporal structures led to short-scale changes in pupil size related to temporal anticipation. Accordingly, we suggest that arousal is sensitive to varying levels of temporal predictability and dynamically adapts to task demands to bring about performance benefits as well as energetic efficiencies overall.

Statement of RelevancePeople often have to sustain focus and high levels of performance on extended and non-stimulating tasks (e.g., driving, sowing, monitoring data acquisition). A critical factor to the success (or failure) in sustained performance is arousal - the  energetic state of the cognitive system. Here we used pupil dilation as a proxy to study levels of arousal during sustained performance on monotonous tasks. We reveal that arousal is dynamically regulated to support performance according to an important fundamental property of any task: its temporal structure. When the timing of task-relevant events is predictable, arousal levels fluctuate accordingly, saving energy overall while also optimally guiding performance. Our study and findings add ecological validity to the study of temporal expectations, by moving investigations beyond typical trial-by-trial designs. They also carry significant implications for clinical studies relying on sustained performance tasks.


title: RapidoPGS: A rapid polygenic score calculator for summary GWAS data without validation dataset

abstract:
MotivationPolygenic scores (PGS) aim to genetically predict complex traits at an individual level. PGS are typically trained on genome-wide association summary statistics and require an independent test dataset to tune parameters. More recent methods allow parameters to be tuned on the training data, removing the need for independent test data, but approaches are computationally intensive. Based on fine-mapping principles, we present RapidoPGS, a flexible and fast method to compute PGS requiring summary-level GWAS datasets only, with little computational requirements and no test data required for parameter tuning.

ResultsWe show that RapidoPGS performs slightly less well than two out of three other widely-used PGS methods (LDpred2, PRScs, and SBayesR) for case-control datasets, with median r2 difference: -0.0092, -0.0042, and 0.0064, respectively, but up to 17,000-fold faster with reduced computational requirements. RapidoPGS is implemented in R and can work with user-supplied summary statistics or download them from the GWAS catalog.

Availability and implementationOur method is available with a GPL license as an R package from GitHub.

category: genetics


title: RapidoPGS: A rapid polygenic score calculator for summary GWAS data without a test dataset


title: Temporally and Spatially Partitioned Neuropeptide Release from Individual Clock Neurons

abstract:
Neuropeptides control rhythmic behaviors, but the timing and location of their release within circuits is unknown. Here imaging in the brain shows that synaptic neuropeptide release by Drosophila clock neurons is diurnal, peaking at times of day that were not anticipated by prior electrical and Ca2+ data. Furthermore, hours before peak synaptic neuropeptide release, neuropeptide release occurs at the soma, a neuronal compartment that has not been implicated in peptidergic transmission. The timing disparity between release at the soma and terminals results from independent and compartmentalized mechanisms for daily rhythmic release: consistent with conventional electrical activity-triggered synaptic transmission, terminals require Ca2+ influx, while somatic neuropeptide release is triggered by the biochemical signal IP3. Upon disrupting the somatic mechanism, the rhythm of terminal release and locomotor activity period are unaffected, but the number of flies with rhythmic behavior and sleep-wake balance are reduced. These results support the conclusion that somatic neuropeptide release controls specific features of clock neuron dependent behaviors. Thus, compartment specific mechanisms within individual clock neurons produce temporally and spatially partitioned neuropeptide release to expand the peptidergic connectome underlying daily rhythmic behaviors.

Significance StatementIt is believed that electrical activity simultaneously stimulates widespread release sites in single neurons to elicit neuropeptide dependent behaviors. However, optically detecting neuropeptide release in the intact brain shows that clock neurons release neuropeptides from different sites at different times of the day. This is possible because one neuronal compartment, the soma, uses biochemical signaling instead of electrical activity to evoke release. Disrupting somatic release affects specific features of circadian locomotor activity and sleep. Thus, neuropeptide release is elicited by independent triggers from distinct parts of clock neurons to engage different regions of behavior regulating circuitry. This strategy for expanding the connectome may be used for other neuropeptide dependent behaviors, such as feeding and pain perception.


title: Specific changes in sleep oscillations after blocking human metabotropic glutamate receptor 5 in the absence of altered memory function

abstract:
BackgroundSleep consolidates declarative memory by repeated replay linked to the cardinal oscillations of NonREM sleep. However, there is so far little evidence of classical glutamatergic plasticity induced by this replay. Rather, we have previously reported that blocking NMDA or AMPA receptors does not affect sleep-dependent consolidation of declarative memory.

AimsInvestigate the role of metabotropic glutamate receptor 5 (mGluR5) on memory processing during sleep.

MethodsIn two placebo-controlled within-subject cross-over experiments with 20 healthy humans each, we used fenobam to block mGluR5 during sleep. In Experiment I, participants learned word-pairs (declarative task) and a finger sequence (procedural task) in the evening, then received the drug and recall was tested in the next morning. To cover possible effects on synaptic renormalization processes during sleep, in Experiment II, participants learned new word-pairs in the morning after sleep.

Results/OutcomesSurprisingly, fenobam neither reduced retention of memory across sleep nor new learning after sleep, although it severely altered sleep architecture and memoryrelevant EEG oscillations. In NonREM sleep, fenobam suppressed 12-15 Hz spindles but augmented 2-4 Hz delta waves, whereas in REM sleep it suppressed 4-8 Hz theta and 16-22 Hz beta waves. Notably, under Fenobam NonREM spindles became more consistently phase-coupled to the slow oscillation.

Conclusions/InterpretationsOur findings indicate that mGluR5-related plasticity is not essential for memory processing during sleep, even though mGlurR5 are strongly implicated in the regulation of the cardinal sleep oscillations.

Declaration of interest/FundingThe authors have nothing to disclose and funders had no influence on the research presented here.


title: A rapid ecological assessment for necrophagous flies (Diptera, Calyptratae) in a mosaic landscape of the Colombian Andes

abstract: A necrophagous flies ensemble (Diptera, Calyptratae) was rapidly assessed in four coverages of an anthropogenic landscape in the east range of the Colombian Andes. Ninetyseven individuals belonging to eight species were collected in only two hours of sampling. The highest diversity values and the occurrence of asynanthropic flies in the forest under conservation management may reflect a positively natural restoration process in the area assessed. Grassland, rural and urban coverages were similarly low in diversity and composition. A brief discussion about the flies bionomy and their environment association is offered. Necrophagous flies ensembles by coverage behave as an indicator of the anthropic impact on the landscape.

category: ecology


title: Global dynamics of microbial communities emerge from local interaction rules

abstract: Interactions between cells drive biological processes across all of life, from microbes in the environment to cells in multicellular organisms. Interactions often arise in spatially structured settings, where cells mostly interact with their neighbors. A central question is how local interactions shape the properties of biological systems. This question is very relevant in the context of microbial communities, such as biofilms, where cells live close by in space and are connected via a dense network of biochemical interactions. To understand and control the functioning of these communities, it is essential to uncover how community-level properties, such as the community composition, spatial arrangement, and growth rate, arise from these interactions. Here, we develop a mathematical framework that can predict community-level properties from the molecular mechanisms underlying the cell-cell interactions for systems consisting of two cell types. Our predictions can qualitatively reproduce measurements from an experimental cross-feeding community. For these cross-feeding communities, the community growth rate is reduced when cells interact only with few neighbors; as a result, some communities can co-exist in a well-mixed system, where cells can interact with all other cells, but not in systems where cells can interact only with close by neighbors. In general, our framework shows that key molecular parameters underlying the cell-cell interactions (e.g. the uptake and leakage rates of molecules) determine community-level properties. Our framework can be extended to a variety of systems of two interacting cell types, within and beyond the microbial world, and contributes to our understanding of how community-level dynamics and biological functions emerge from microscopic interactions between single cells.

category: systems biology


title: Exploring Efficient Linear Mixed Models to Detect Quantitative Trait Locus-by-environment Interactions

abstract: Genotype-by-environment interactions (GxE) are important for understanding genotype-phenotype relationships. To date, various statistical models have been proposed to account for GxE effects, especially in genomic selection (GS) studies. Generally, GS does not focus on the detection of each quantitative trait locus (QTL), while the genome-wide association study (GWAS) was designed for QTL detection. GxE modeling methods in GS can be included as covariates in GWAS using unified linear mixed models (LMMs). However, the efficacy of GxE modeling methods in GS studies has not been evaluated for GWAS. In this study, we performed a comprehensive comparison of LMMs that integrate the GxE modeling methods to detect both QTL and QTL-by-environment interaction (QxE) effects. Model efficacy was evaluated using simulation experiments. For the fixed effect terms representing QxE effects, simultaneous scoring of specific and non-specific environmental effects was recommended because of the higher recall and improved genomic inflation factor value. For random effects, it was necessary to account for both GxE and genotype-by-trial (GxT) effects to control genomic inflation factor value. Thus, the recommended LMM includes fixed QTL effect terms that simultaneously score specific and non-specific environmental effects and random effects accounting for both GxE and GxT. The LMM was applied to real tomato phenotype data obtained from two different cropping seasons. We detected not only QTLs with persistent effects across the cropping seasons but also QTLs with QxE effects. The optimal LMM identified in this study successfully detected more QTLs with QxE effects.


title: The sleep-wake distribution contributes to the peripheral rhythms in PERIOD-2

abstract: In the mouse, Period-2 (Per2) expression in tissues peripheral to the suprachiasmatic nuclei (SCN) increases during sleep deprivation and at times of the day when animals are predominantly awake spontaneously, suggesting that the circadian sleep-wake distribution directly contributes to the daily rhythms in Per2. We found support for this hypothesis by recording sleep-wake state alongside PER2 bioluminescence in freely behaving mice, demonstrating that PER2 increases during spontaneous waking and decreases during sleep. The temporary reinstatement of PER2 rhythmicity in behaviorally arrhythmic SCN-lesioned mice submitted to daily recurring sleep deprivations substantiates our hypothesis. Mathematical modelling revealed that PER2 dynamics can be described by a damped harmonic oscillator driven by two forces: a sleep-wake-dependent force and a SCN-independent circadian force. Our work underscores the notion that in peripheral tissues the clock gene circuitry integrates sleep-wake information and could thereby contribute to behavioral adaptability to respond to homeostatic requirements.


title: A mitochondrial mutational signature of temperature in ectothermic and endothermicvertebrates.

abstract: Mutational spectrum of the mitochondrial genome (mtDNA) fluctuates between species, however, factors responsible for this variation are mainly unknown. Recently, we demonstrated that the mammalian mtDNA mutational spectrum is associated with age-related oxidative damage by means of A>G substitutions on a heavy chain (hereafter Ah>Gh). Here we extend this logic hypothesizing that oxidative damage can also depend on a species-specific level of aerobic metabolism. Using body temperature of endotherms and the environmental temperature of ectotherms as a proxy for their levels of aerobic metabolism, we reconstructed and analyzed 1350 species-specific mtDNA mutational spectra of vertebrate species. First, within ray-finned fishes, we observed that temperature is associated with increased asymmetry of Ah>Gh substitution, estimated as a ratio Ah>Gh/Th>Ch. Second, comparing within-species geographically distinct clades of widely distributed European anchovy, we observed similarly increased Ah>Gh/Th>Ch in the tropical clade. Third, analysing nucleotide composition in the most neutral synonymous sites of fishes, we demonstrated that warm-versus cold-water fishes are expectedly more AC poor and GT rich. Fourth, within mammals we observed an increased Ah>Gh/Th>Ch in warmer species as compared to colder ones (hybernators, daily torpors, naked mole rat, etc.) Fifth, comparing mtDNA mutational spectra between five classes of vertebrates, we observed an increased Ah>Gh/Th>Ch in warm- (mammals and birds) versus cold- (actinopterygii, amphibia, reptilia) blooded classes. Altogether, we conclude that temperature, through the level of the metabolism and oxidative damage, shapes the properties of the mitochondrial genome.

category: evolutionary biology


title: Integration of phenomics and transcriptomics data to reveal drivers of inflammatory processes in the skin

abstract: Chronic inflammatory diseases are characterized by complex interactions between genetic predisposition and tissue-specific immune responses. This heterogeneity complicates diagnoses and the ability to exploit omics approaches to improve disease management, develop more effective therapeutics, and apply precision medicine. Using skin inflammation as a model, we developed a method that integrates deep clinical phenotyping information (phenomics) with transcriptome data of lesional and non-lesional skin (564 samples) to identify clinically-relevant gene signatures. It led us to discover so-far unexplored factors, including CCAAT Enhancer-Binding Protein Beta (CEBPB) in neutrophil invasion, and Pituitary Tumor-Transforming 2 (PTTG2) in the pathogenic epithelial response to inflammation. These factors were validated using genetically-modified human skin equivalents, migration assays, and in situ imaging. Thus, by meaningful integration of deep clinical phenotyping and omics data we reveal hidden drivers of clinically-relevant biological processes.


title: De novo designed transmembrane domains tune engineered receptor functions

abstract: De novo designed receptor transmembrane domains (TMDs) present opportunities for precise control of cellular receptor functions. We developed a de novo design strategy for generating programmed membrane proteins (proMPs): single-pass -helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces. We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor (CAR) and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD, from monomers up to tetramers. All programmed CARs (proCARs) stimulated substantially lower T cell cytokine release relative to the commonly used CD28 TMD, which we show elevated cytokine release through lateral recruitment of the endogenous T cell costimulatory receptor CD28. Precise design using orthogonal and modular TMDs thus provides a new way to program receptor structure and predictably tune activity for basic or applied synthetic biology.

category: synthetic biology


title: partR2: Partitioning R2 in generalized linear mixed models

abstract: The coefficient of determination R2 quantifies the amount of variance explained by regression coefficients in a linear model. It can be seen as the fixed-effects complement to the repeatability R (intra-class correlation) for the variance explained by random effects and thus as a tool for variance decomposition. The R2 of a model can be further partitioned into the variance explained by a particular predictor or a combination of predictors using semi-partial (part) R2 and structure coefficients, but this is rarely done due to a lack of software implementing these statistics. Here, we introduce partR2, an R package that quantifies part R2 for fixed effect predictors based on (generalized) linear mixed-effect model fits. The package iteratively removes predictors of interest from the model and monitors the change in the variance of the linear predictor. The difference to the full model gives a measure of the amount of variance explained uniquely by a particular predictor or a set of predictors. partR2 also estimates structure coefficients as the correlation between a predictor and fitted values, which provide an estimate of the total contribution of a fixed effect to the overall prediction, independent of other predictors. Structure coefficients can be converted to the total variance explained by a predictor, here called  inclusive R2, as the square of the structure coefficients times total R2. Furthermore, the package reports beta weights (standardized regression coefficients). Finally,partR2 implements parametric bootstrapping to quantify confidence intervals for each estimate. We illustrate the use of partR2 with real example datasets for Gaussian and binomial GLMMs and discuss interactions, which pose a specific challenge for partitioning the explained variance among predictors.

category: bioinformatics


title: Modeling the complete kinetics of coxsackievirus B3 reveals human determinants of host-cell feedback

abstract: Complete kinetic models are pervasive in chemistry but lacking in biological systems. We encoded the complete kinetics of infection for coxsackievirus B3 (CVB3), a compact and fast-acting RNA virus. The kinetics are built from detailed modules for viral binding-delivery, translation-replication, and encapsidation. Specific module activities are dampened by the type I interferon response to viral double-stranded RNAs (dsRNAs), which is itself disrupted by viral proteinases. The validated kinetics uncovered that cleavability of the dsRNA transducer mitochondrial antiviral signaling protein (MAVS) becomes a stronger determinant of viral outcomes when cells receive supplemental interferon after infection. Cleavability is naturally altered in humans by a common MAVS polymorphism, which removes a proteinase-targeted site but paradoxically elevates CVB3 infectivity. These observations are reconciled with a simple nonlinear model of MAVS regulation. Modeling complete kinetics is an attainable goal for small, rapidly infecting viruses and perhaps viral pathogens more broadly.


title: Structural and Genetic Determinants of Convergence in the Drosophila tRNA Structure-Function Map

abstract: The evolution of tRNA multigene families remains poorly understood, exhibiting unusual phenomena such as functional conversions of tRNA genes through anticodon shift substitutions. We improved FlyBase tRNA gene annotations from twelve Drosophila species, incorporating previously identified ortholog sets to compare substitution rates across tRNA bodies at single-site and base-pair resolution. All rapidly evolving sites fell within the same metal ion-binding pocket, that lies at the interface of the two major stacked helical domains. We applied our tRNA Structure-Function Mapper (tSFM) method independently to each Drosophila species and one outgroup species Musca domestica and found that, although predicted tRNA structure-function maps are generally highly conserved in flies, one tRNA Class-Informative Feature (CIF) within the rapidly-evolving ion-binding pocket -- Cytosine 17 (C17), ancestrally informative for lysylation identity -- independently gained asparaginylation identity and substituted in parallel across tRNAAsn paralogs at least once, possibly multiple times, during evolution of the genus. In D. melanogaster, most tRNALys and tRNAAsn genes are co-arrayed in one large heterologous gene cluster, suggesting that heterologous gene conversion as well as structural similarities of tRNA-binding interfaces in the closely related asparaginyl-tRNA synthetase (AsnRS) and lysyl-tRNA synthetase (LysRS) proteins may have played a role in these changes. A previously identified Asn-to-Lys anticodon shift substitution in D. ananassae may have arisen to compensate for the convergent and parallel gains of C17 in tRNAAsn paralogs in that lineage. Our results underscore the functional and evolutionary relevance of our tRNA structure-function map predictions and illuminate multiple genomic and structural factors contributing to rapid, parallel and compensatory evolution of tRNA multigene families.

category: molecular biology


title: Weak catch bonds make strong networks

abstract: Molecular catch bonds are ubiquitous in biology and well-studied in the context of leukocyte extravasion1, cellular mechanosensing2,3, and urinary tract infection4. Unlike normal (slip) bonds, catch bonds strengthen under tension. The current paradigm is that this remarkable ability enables cells to increase their adhesion in fast fluid flows1,4, and hence provides  strength-on-demand. Recently, cytoskeletal crosslinkers have been discovered that also display catch bonding5-8. It has been suggested that they strengthen cells, following the strength-on-demand paradigm9,10. However, catch bonds tend to be weaker compared to regular (slip) bonds because they have cryptic binding sites that are often inactive11-13. Therefore, the role of catch bonding in the cytoskeleton remains unclear. Here we reconstitute cytoskeletal actin networks to show that catch bonds render them both stronger and more deformable than slip bonds, even though the bonds themselves are weaker. We develop a model to show that weak binding allows the catch bonds to mitigate crack initiation by moving from low- to high-tension areas in response to mechanical loading. By contrast, slip bonds remain trapped in stress-free areas. We therefore propose that the mechanism of catch bonding is typified by dissociation-on-demand rather than strength-on-demand. Dissociation-on-demand can explain how both cytolinkers5-8,10,14,15 and adhesins1,2,4,12,16-20 exploit continuous redistribution to combine mechanical strength with the adaptability required for movement and proliferation21. Our findings provide a new perspective on diseases where catch bonding is compromised11,12 such as kidney focal segmental glomerulosclerosis22,23, caused by the -actinin-4 mutant studied here. Moreover, catch bonds provide a route towards creating life-like materials that combine strength with deformability24.

category: biophysics


title: Including tree spatial extension in the evaluation of neighbourhood competition effects in Bornean rain forest

abstract: Classical tree neighbourhood models use size variables acting at point distances. In a new approach here, trees were spatially extended as a function of their crown sizes, represented impressionistically as points within crown areas. Extension was accompanied by plasticity in the form of crown removal or relocation under the overlap of taller trees. Root systems were supposedly extended in a similar manner. For the 38 most abundant species in the focal size class (10 - <100 cm stem girth) in two 4-ha plots at Danum (Sabah), for periods P1 (1986-1996) and P2 (1996-2007), stem growth rate and tree survival were individually regressed against stem size, and neighbourhood conspecific (CON) and heterospecific (HET) basal areas within incremented steps in radius. Model parameters were critically assessed, and statistical robustness in the modelling set by randomization testing. Classical and extended models differed importantly in their outcomes. Crown extension weakened the relationship of CON effect on growth versus plot species abundance, showing that models without plasticity over-estimated negative density dependence. A significant negative trend of difference in CON effects on growth (P2 - P1) versus CON or HET effect on survival in P1 was strongest with crown extension. Model outcomes did not then support an explanation of CON and HET effects being due to (asymmetric) competition for light alone. An alternative hypothesis is that changes in CON effects on small trees, largely incurred by a drought phase (relaxing light limitation) in P2, and following the more shaded (suppressing) conditions in P1, were likely due to species-specific (symmetric) root competition and mycorrhizal processes. The very high variation in neighbourhood composition and abundances led to a strong  neighbourhood stochasticity, and hence to largely idiosyncratic species responses. A need to much better understand the roles of rooting structure and processes at the individual tree level was highlighted.


title: The Mouse Action Recognition System (MARS) software pipeline for automated analysis of social behaviors in mice

abstract: The study of naturalistic social behavior requires quantification of animals interactions. This is generally done through manual annotation--a highly time consuming and tedious process. Recent advances in computer vision enable tracking the pose (posture) of freely-behaving animals. However, automatically and accurately classifying complex social behaviors remains technically challenging. We introduce the Mouse Action Recognition System (MARS), an automated pipeline for pose estimation and behavior quantification in pairs of freely interacting mice. We compare MARSs annotations to human annotations and find that MARSs pose estimation and behavior classification achieve human-level performance. We also release the pose and annotation datasets used to train MARS, to serve as community benchmarks and resources. Finally, we introduce the Behavior Ensemble and Neural Trajectory Observatory (BENTO), a graphical user interface for analysis of multimodal neuroscience datasets. Together, MARS and BENTO provide an end-to-end pipeline for behavior data extraction and analysis, in a package that is user-friendly and easily modifiable.


title: A small, computationally flexible network produces the phenotypic diversity of song recognition in crickets

abstract: How neural networks evolved to generate the diversity of species-specific communication signals is unknown. For receivers of the signals one hypothesis is that novel recognition phenotypes arise from parameter variation in computationally flexible feature detection networks. We test this hypothesis in crickets, where males generate and females recognize the mating songs with a species-specific pulse pattern, by investigating whether the song recognition network in the cricket brain has the computational flexibility to recognize different temporal features. Using electrophysiological recordings from the network that recognizes crucial properties of the pulse pattern on the short timescale in the cricket Gryllus bimaculatus, we built a computational model that reproduces the neuronal and behavioral tuning of that species. An analysis of the models parameter space reveals that the network can provide all recognition phenotypes for pulse duration and pause known in crickets and even other insects. Phenotypic diversity in the model is consistent with known preference types in crickets and other insects, and arise from computations that likely evolved to increase energy efficiency and robustness of pattern recognition. The models parameter to phenotype mapping is degenerate--different network parameters can create similar changes in the phenotype--which likely supports evolutionary plasticity. Our study suggests that computationally flexible networks underlie the diverse pattern recognition phenotypes and we reveal network properties that constrain and support behavioral diversity.


title: Transcription factor binding activity is the primary driver of noise in gene expression

abstract: Noise in expression of individual genes gives rise to variations in activity of cellular pathways and generates phenotypic heterogeneity. Phenotypic heterogeneity has important implications for antibiotic persistence, mutation penetrance, cancer growth and therapy resistance. Even though the role of specific factors such as presence of TATA box sequence in the promoter of a gene and promoter nucleosome occupancy in noise regulation have been studied, the ability of these factors to predict noise remains unknown. Here we build an integrated quantitative statistical model for gene expression noise in yeast and show that the number of regulating transcription factors and their binding activity are the primary drivers of noise. Combining our model with stochastic simulations, we specifically show that the binding activity arising from competition and cooperation among TFs for binding to the promoter region can drive noise. Our work uncovers a general process of noise regulation that arises out of the dynamic nature of gene regulation that is not dependent on specific transcription factor or specific promoter sequence and thus can be extended across biological systems.


title: Bro1 stimulates Vps4 activity to promote Intralumenal Vesicle Formation during Multivesicular Body biogenesis

abstract:
Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homolog of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT-driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologs in licensing ESCRT-III-dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1/Vps4/ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.

SummaryCargo sorting is coordinated with intralumenal vesicle budding during ESCRT-mediated multivesicular body biogenesis. Bro1 V domain stimulates Vps4 to promote ESCRT-III-driven intralumenal vesicle formation in a manner required for this coordinated process.


title: The ABCD Stop Signal Data: Response to Bissett et al.

abstract: This paper responds to a recent critique by Bissett and colleagues (Bissett et al., eLife, In Press) of the fMRI Stop task being used in the Adolescent Brain Cognitive DevelopmentSM Study (ABCD Study(R)). The critique focuses primarily on a design feature of the task that the authors contend lead to a violation of race model assumptions (i.e., that the Go and Stop processes are fully independent) which are relevant to the calculation of the Stop Signal Reaction Time, a measure of the inhibition process. Bissett and colleagues also raise a number of secondary concerns. In this response we note that satisfying race model assumptions is a pernicious challenge for Stop task designs but also that the race model is quite robust against violations of its assumptions. Most importantly, while Bissett et al. raise conceptual concerns with the task we focus here on analyses of both the performance and the neuroimaging data and we conclude that the concerns appear to have minimal impact on the neuroimaging data (the validity of which do not rely on race model assumptions) and have far less of an impact on the performance data than the critique suggests. We note that Bissett et al. did not apply any performance-based exclusions to the data they analyzed, that a number of the trial coding errors that they flagged were already identified and corrected in the ABCD annual data releases, that a number of the secondary concerns reflect sensible design decisions and, indeed, that their own computational modeling of the ABCD Stop task suggests the problems they identify have just a modest impact on the rank ordering of individual differences in subject performance. In this paper, we list some adjustments that have been made to the task and some new flags that are now added to the annual, curated data releases. We stress that the ABCD data are fully available to the scientific community who are empowered to apply whatever inclusion and exclusion criteria they deem appropriate for their analyses and we conclude that the ABCD Stop task yields valuable data that researchers can use to track adolescent neurodevelopment.


title: Cortical Silent Period reflects individual differences in action stopping performance.

abstract: Inhibitory control is the ability to suppress inappropriate movements and unwanted actions, allowing to regulate impulses and responses. This ability can be measured via the Stop Signal Task, which provides a temporal index of response inhibition, the stop signal reaction time (SSRT). At the neural level, Transcranial Magnetic Stimulation (TMS) allows to investigate of motor inhibition within the primary motor cortex (M1), such as the Cortical Silent period (CSP). CSPs length is an index of GABAB-mediated intracortical inhibition within M1. Although there is strong evidence that intracortical inhibition varies during action stopping, it is still not clear whether differences in the neurophysiological markers of intracortical inhibition contribute to behavioral differences in actual inhibitory capacities. Hence, we here explored the relationship between intracortical inhibition within M1 and behavioral response inhibition. GABABergic-mediated inhibition in M1 was determined by the length of CSP, while behavioral inhibition was assessed by the SSRT. We found a significant positive correlation between CSPs length and SSRT, namely that individuals with greater levels of GABABergic-mediated inhibition seem to perform overall worse in inhibiting behavioral responses. These results support the assumption that individual differences in intracortical inhibition are mirrored by individual differences in action stopping abilities.


title: Minimizing ion competition boosts volatile metabolome analysis by secondary electrospray ionization orbitrap mass spectrometry

abstract: Secondary electrospray ionization high-resolution mass spectrometry (SESI-HR-MS) is an emerging technique for the detection of volatile metabolites. However, sensitivity and reproducibility of SESI-HRMS have limited its applications in untargeted metabolomics profiling. Ion suppression in the SESI source has been considered to be the main cause. Here, we show that besides ion suppression, ion competition in the C-trap of Orbitrap instruments is another important factor that influences sensitivity and reproducibility of SESI-MS. Instead of acquiring the full mass-to-charge ratio (m/z) range, acquisition of consecutive m/z windows to minimize the ion competition effect allows the detection of more features. m/z window ranges are optimized to fill the C-trap either with an equal number of features or an equal cumulative intensity per window. Considering a balance between maximizing scanning speed and minimizing ion competition, splitting the m/z = 50-500 range into 4 windows is selected for measuring human breath and bacterial culture samples on SESI-Orbitrap MS, corresponding to a duty cycle of 2.3 s at a resolution of 140000. In a small cohort of human subjects, the proposed splitting into 4 windows allows three times more features to be detected compared to the classical full m/z range method.


title: coronaSPAdes: from biosynthetic gene clusters to RNA viral assemblies

abstract:
MotivationThe COVID-19 pandemic has ignited a broad scientific interest in viral research in general and coronavirus research in particular. The identification and characterization of viral species in natural reservoirs typically involves de novo assembly. However, existing genome, metagenome and transcriptome assemblers often are not able to assemble many viruses (including coronaviruses) into a single contig. Coverage variation between datasets and within dataset, presence of close strains, splice variants and contamination set a high bar for assemblers to process viral datasets with diverse properties.

ResultsWe developed coronaSPAdes, a novel assembler for RNA viral species recovery in general and coronaviruses in particular. coronaSPAdes leverages the knowledge about viral genome structures to improve assembly extending ideas initially implemented in biosyntheticSPAdes. We have shown that coronaSPAdes outperforms existing SPAdes modes and other popular short-read metagenome and viral assemblers in the recovery of full-length RNA viral genomes.

AvailabilitycoronaSPAdes version used in this article is a part of SPAdes 3.15 release and is freely available at http://cab.spbu.ru/software/spades.

Contacta.korobeynikov@spbu.ru

Supplementary informationSupplementary data are available at Bioinformatics


title: Systematic investigation of imprinted gene expression and enrichment in the mouse brain explored at single-cell resolution

abstract: Although a number of imprinted genes are known to be highly expressed in the brain, and in certain brain regions in particular, whether they are truly over-represented in the brain has never been formally tested. Using fifteen single-cell RNA sequencing datasets we systematically investigated imprinted gene over-representation at the organ, brain region, and cell-specific levels. We established that imprinted genes are indeed over-represented in the adult brain, and in neurons particularly compared to other brain cell-types. We then examined brain-wide datasets to test enrichment within distinct brain regions and demonstrated over-representation of imprinted genes in the hypothalamus, ventral midbrain, pons and medulla. Finally, using datasets focusing on these regions of enrichment, we identified hypothalamic neuroendocrine populations and the monoaminergic hindbrain neurons as specific hotspots of imprinted gene expression. These analyses provide the first robust assessment of the neural systems on which imprinted genes converge. Moreover, the unbiased approach, with each analysis informed by the findings of the previous level, permits highly informed inferences about the functions on which imprinted gene expression converges. Our findings indicate the neuronal regulation of motivated behaviours such as feeding, parental behaviour and sleep as functional hotspots for imprinting, thus adding statistical rigour to prior assumptions and providing testable predictions for novel neural and behavioural phenotypes associated with specific genes and imprinted gene networks. In turn, this work sheds further light on the potential evolutionary drivers of genomic imprinting in the brain.


title: Breathing Behaviors in Common Marmoset (Callithrix jacchus)

abstract: The respiratory system maintains homeostatic levels of oxygen (O2) and carbon dioxide (CO2) in the body through rapid and efficient regulation of frequency and depth (tidal volume) of breathing. Many studies on homeostatic control of breathing have been done on rodent animal models, in which they have relatively reduced chemosensitivities when compared with primates. The use of common marmoset (Callithrix jacchus), a New World non-human primate model, in neuroscience is increasing, however, the data on their breathing is limited and their respiratory behaviors have yet to be characterized. Using Whole-body Plethysmography in room air as well as in hypoxic (low O2) and hypercapnic (high CO2) conditions, we defined breathing behaviors in awake, freely behaving marmosets. Additionally, we optimized an analysis toolkit for unsupervised analysis of respiratory activities in common marmoset. Our data indicate that marmosets exposure to hypoxia decreased metabolic rate and increased sigh rate. However, the hypoxic condition did not augment the ventilatory response. Hypercapnia, on the other hand, increased both the frequency and tidal volume. In this study, we described breathing behaviors of common marmosets in a variety of O2 and CO2 conditions.


title: Unsupervised analysis of multi-experiment transcriptomic patterns with SegRNA identifies unannotated transcripts

abstract:
BackgroundExploratory analysis of complex transcriptomic data presents multiple challenges. Many methods often rely on preexisting gene annotations, impeding identification and characterization of new transcripts. Even for a single cell type, comprehending the diversity of RNA species transcribed at each genomic region requires combining multiple datasets, each enriched for specific types of RNA. Currently, examining combinatorial patterns in these data requires time-consuming visual inspection using a genome browser.

MethodWe developed a new segmentation and genome annotation (SAGA) method, SegRNA, that integrates data from multiple transcriptome profiling assays. SegRNA identifies recurring combinations of signals across multiple datasets measuring the abundance of transcribed RNAs. Using complementary techniques, SegRNA builds on the Segway SAGA framework by learning parameters from both the forward and reverse DNA strands. SegRNAs unsupervised approach allows exploring patterns in these data without relying on pre-existing transcript models.

ResultsWe used SegRNA to generate the first unsupervised transcriptome annotation of the K562 chronic myeloid leukemia cell line, integrating multiple types of RNA data. Combining RNA-seq, CAGE, and PRO-seq experiments together captured a diverse population of RNAs throughout the genome. As expected, SegRNA annotated patterns associated with gene components such as promoters, exons, and introns. Additionally, we identified a pattern enriched for novel small RNAs transcribed within intergenic, intronic, and exonic regions. We applied SegRNA to FANTOM6 CAGE data characterizing 285 lncRNA knockdowns. Overall, SegRNA efficiently summarizes diverse multi-experiment data.


title: Stem girth changes in response to soil water potential in lowland dipterocarp forest in Borneo: a time-series analysis

abstract: Time series data offer a way of investigating the causes driving ecological processes as phenomena. To test for possible differences in water relations between species of different forest structural guilds at Danum (Sabah, NE Borneo), daily stem girth increments (gthi), of 18 trees across six species were regressed individually on soil moisture potential (SMP) and temperature (TEMP), accounting for temporal autocorrelation (in GLS-arima models), and compared between a wet and a dry period. The best-fitting significant variables were SMP the day before and TEMP the same day. The first resulted in a mix of positive and negative coefficients, the second largely positive ones. An adjustment for dry-period showers was applied. Interactions were stronger in dry than wet period. Negative relationships for overstorey trees can be interpreted in a reversed causal sense: fast transporting stems depleted soil water and lowered SMP. Positive relationships for understorey trees meant they took up most water at high SMP. The unexpected negative relationships for these small trees may have been due to their roots accessing deeper water supplies (if SMP was inversely related to that of the surface layer), and this was influenced by competition with larger neighbour trees. A tree-soil flux dynamics manifold may have been operating. Patterns of mean diurnal girth variation were more consistent among species, and time-series coefficients were negatively related to their maxima. Expected differences in response to SMP in the wet and dry periods did not clearly support a previous hypothesis differentiating drought and non-drought tolerant understorey guilds. Trees within species showed highly individual responses when tree size was standardized. Data on individual root systems and SMP at several depths are needed to get closer to the mechanisms that underlie the tree-soil water phenomena in these tropical forests. Neighborhood stochasticity importantly creates varying local environments experienced by individual trees.


title: Stem girth changes in response to soil water potential in lowland dipterocarp forest in Borneo: a phenomenological and individualistic time-series analysis


title: Evaluation of Polygenic Prediction Methodology within a Reference-Standardized Framework

abstract:
BackgroundThe predictive utility of polygenic scores is increasing, and many polygenic scoring methods are available, but it is unclear which method performs best. This study evaluates the predictive utility of polygenic scoring methods within a reference-standardized framework, which uses a common set of variants and reference-based estimates of linkage disequilibrium and allele frequencies to construct scores.

MethodsEight polygenic score methods were tested: p-value thresholding and clumping (pT+clump), SBLUP, lassosum, LDPred1, LDPred2, PRScs, DBSLMM and SBayesR, evaluating their performance to predict outcomes in UK Biobank and the Twins Early Development Study (TEDS). Strategies to identify optimal p-value threshold and shrinkage parameters were compared, including 10-fold cross validation, pseudovalidation and infinitesimal models (with no validation sample), and multi-polygenic score elastic net models.

ResultsLDPred2, lassosum and PRScs performed strongly using 10-fold cross-validation to identify the most predictive p-value threshold or shrinkage parameter, giving a relative improvement of 16-18% over pT+clump in the correlation between observed and predicted outcome values. Using pseudovalidation, the best methods were PRScs and DBSLMM, with a relative improvement of >10% over other pseudovalidation and infinitesimal methods (lassosum, SBLUP, SBayesR, LDPred1, LDPred2). PRScs pseudovalidation was only 3% worse than the best polygenic score identified by 10-fold cross validation. Elastic net models containing polygenic scores based on a range of parameters consistently improved prediction over any single polygenic score.

ConclusionWithin a reference-standardized framework, the best polygenic prediction was achieved using LDPred2, lassosum and PRScs, modeling multiple polygenic scores derived using multiple parameters. This study will help researchers performing polygenic score studies to select the most powerful and predictive analysis methods.


title: Biogeochemical and omic evidence for paradoxical methane production via multiple co-occurring mechanisms in aquatic ecosystems

abstract: Aquatic ecosystems are globally significant sources of the greenhouse gas methane (CH4) to the atmosphere. However, CH4 is produced  paradoxically in oxygenated water via poorly understood mechanisms, fundamentally limiting our understanding of overall CH4 production. Here we resolve paradoxical CH4 production mechanisms through CH4 measurements,{delta} 13CH4 analyses, 16S rRNA sequencing, and metagenomics/metatranscriptomics applied to freshwater incubation experiments with multiple time points and treatments (addition of a methanogenesis inhibitor, dark, high-light). We captured significant paradoxical CH4 production, as well as consistent metabolism of methylphosphonate by abundant bacteria--resembling observations from marine ecosystems. Metatranscriptomics and{delta} 13CH4 analyses applied to experimental treatments identified an additional CH4 production mechanism associated with (bacterio)chlorophyll metabolism and photosynthesis by Cyanobacteria, and especially by Proteobacteria. Both mechanisms occured together within metagenome-assembled genomes, and appear widespread in freshwater. Our results indicate that multiple, co-occurring, and broadly-distributed bacterial groups and metabolic pathways produce CH4 in aquatic ecosystems.


title: From plants to ants: Fungal modification of leaf lipids for nutrition and communication in the leaf-cutter ant fungal garden ecosystem

abstract:
Lipids are essential to all living organisms, as an energy source, as an important cellular structural component, and as a communication tool. In this study, we used global lipidomic methods to evaluate the lipids in leaf-cutter ant fungal gardens. Leaf-cutter ants and their coevolved fungal cultivar, Leucoagaricus gongylophorus, are a model mutualistic system. The fungus enzymatically digests fresh plant material that the ants cut and deliver, converting energy and nutrients from plants, and providing them to the ants through specialized hyphal swellings called gongylidia. Using combined liquid chromatography, ion mobility spectrometry, and tandem mass spectrometry we evaluated differences between the molecular speciation of lipids in the leaf-cutter ant fungal garden ecosystem. This lipidomic study characterized leaves that are fed to the gardens, gongylidia that are produced by the fungus to feed the ants, and spatially resolved regions of the fungal garden through stages of leaf degradation. Lipids containing alpha-linolenic acid (18:3) were enriched in leaves and the top of the gardens, but not dominant in the middle or bottom regions. Gongylidia were dominated by lipids containing linoleic acid (18:2). To evaluate the communicative potential of the lipids in fungal gardens we conducted a behavioral experiment that showed Atta leaf-cutter ants responded differently to 18:3 and 18:2 fatty acids, with aggression towards 18:3 and attraction for 18:2. This work demonstrates the role of lipids in both the transfer of energy and as an inter-kingdom communication tool in leaf-cutter ant fungal gardens.

ImportanceIn this work we examined the role of lipids in the mutualism between leaf-cutter ants and fungus. These ants cut fresh leaf material, which they provide to their fungal cultivar, that converts energy and nutrients from the plants and provides it to the ants in specialized hyphal swellings called gongylidia. This work constitutes the first example of a global lipidomics study of a symbiotic system and provides insights as to how the fungus modifies plant lipids into a usable source for the ants. Through a behavioral experiment, this work also demonstrates how lipids can be used as an inter-kingdom communication tool, in this case an attractant, rather than as a repellant, which is more often seen.


title: Estradiol Action at the Median Preoptic Nucleus is Necessary and Sufficient for Sleep Suppression in Female Rats

abstract:
To further our understanding of how gonadal steroids impact sleep biology, we sought to address the mechanism by which proestrus levels of cycling ovarian steroids, particularly estradiol (E2), suppress sleep in female rats. We showed that steroid replacement of proestrus levels of E2 to ovariectomized female rats, suppressed sleep to similar levels as those reported by endogenous ovarian hormones. We further showed that this suppression is due to the high levels of E2 alone, and that progesterone did not have a significant impact on sleep behavior. We found that E2 action within the Median Preoptic Nucleus (MnPO), which contains estrogen receptors (ERs), is necessary for this effect; antagonism of ERs in the MnPO attenuated the E2-mediated suppression of both non-Rapid Eye Movement (NREM) and Rapid Eye Movement (REM) sleep. Finally, we found E2 action at the MnPO is also sufficient for sleep suppression, as direct infusion of E2 into the MnPO suppressed sleep. Based on our findings, we predict proestrus levels of E2 alone, acting at the MnPO, mediate sex-hormone driven suppression of sleep in female rats.

Competing Interest StatementMDS is now an employee of Jazz Pharmaceuticals; the views expressed in this manuscript are solely the authors and do not reflect the views, policies or procedures of Jazz Pharmaceuticals.


title: ExBoX: a simple Boolean exclusion strategy to drive expression in neurons

abstract:
The advent of modern single-cell biology has revealed the striking molecular diversity of cell populations once thought to be more homogeneous. This newly appreciated complexity has made intersectional genetic approaches essential to understanding and probing cellular heterogeneity at the functional level. Here we build on previous knowledge to develop a simple AAV-based approach to define specific subpopulations of cells by Boolean exclusion logic (AND NOT). This Expression by Boolean Exclusion (ExBoX) system encodes for a gene of interest which is turned On by a particular recombinase (Cre or FlpO) and turned Off by another. ExBoX allows for the specific transcription of a gene of interest in cells expressing only the activating recombinase, but not in cells expressing both. We show the ability of the ExBoX system to tightly regulate expression of fluorescent reporters in vitro and in vivo, and further demonstrate the adaptability of the system by achieving expression of a variety of virally-delivered coding sequences in the mouse brain. This simple strategy will expand the molecular toolkit available for cell- and time-specific gene expression in a variety of systems.

Summary statementUbina et al. describe the generation of a novel AAV-based intersectional approach to define and target specific subpopulations of cells in time and space via Expression by Boolean Exclusion (ExBoX).


title: Predictive visuo-motor communication through neural oscillations

abstract: The mechanisms coordinating action and perception over time are poorly understood. The sensory cortex needs to prepare for upcoming changes contingent on action, and this requires temporally precise communication that takes into account the variable delays between sensory and motor processing. Several theorists1,2 have proposed synchronization of the endogenous oscillatory activity observed in most regions of the brain3 as the basis for an efficient and flexible communication protocol between distal brain areas2,4, a concept known as "communication through coherence". Synchronization of endogenous oscillations5,6 occurs after a salient sensory stimulus, such as a flash or a sound7-11, and after a voluntary action12-18, and this impacts directly on perception, causing performance to oscillate rhythmically over time. Here we introduce a novel fMRI paradigm to probe the neural sources of oscillations, based on the concept of perturbative signals, which overcomes the low temporal resolution of BOLD signals. The assumption is that a synchronized endogenous rhythm will modulate cortical excitability rhythmically, which should be reflected in the BOLD responses to brief stimuli presented at different phases of the oscillation cycle. We record rhythmic oscillations of V1 BOLD synchronized by a simple voluntary action, in phase with behaviourally measured oscillations in visual sensitivity in the theta range. The functional connectivity between V1 and M1 also oscillates at the same rhythm. By demonstrating oscillatory temporal coupling between primary motor and sensory cortices, our results strongly implicate communication through coherence to achieve precise coordination and to encode sensory-motor timing.


title: Global drivers of obligate mycorrhizal symbionts diversification

abstract: Analyzing diversification dynamics is key to understanding the past evolutionary history of clades that led to present-day biodiversity patterns. While such analyses are widespread in well-characterized groups of species, they are much more challenging in groups which diversity is mostly known through molecular techniques. Here, we use the largest global database on the small subunit (SSU) rRNA gene of Glomeromycotina, a subphylum of microscopic arbuscular mycorrhizal fungi that provide mineral nutrients to most land plants by forming one of the oldest terrestrial symbioses, to analyze the diversification dynamics of this clade in the past 500 million years (Myr). We perform a range of sensitivity analyses and simulations to control for potential biases linked to the nature of the data. We find that Glomeromycotina tend to have low speciation rates compared to other eukaryotes. After a peak of speciations between 200 and 100 Myr ago, they experienced an important decline in speciation rates toward the present. Such a decline could be at least partially related to a shrinking of their mycorrhizal niches and to their limited ability to colonize new niches. Our analyses identify patterns of diversification in a group of obligate symbionts of major ecological and evolutionary importance and illustrate that short molecular markers combined with intensive sensitivity analyses can be useful for studying diversification dynamics in microbial groups.


title: Nonlinear effects of intrinsic dynamics on temporal encoding in a model of avian auditory cortex

abstract:
Neurons exhibit diverse intrinsic dynamics, which govern how they integrate synaptic inputs to produce spikes. Intrinsic dynamics are often plastic during development and learning, but the effects of these changes on stimulus encoding properties are not well known. To examine this relationship, we simulated auditory responses to zebra finch song using a linear-dynamical cascade model, which combines a linear spectrotemporal receptive field with a dynamical, conductance-based neuron model, then used generalized linear models to estimate encoding properties from the resulting spike trains. We focused on the effects of a low-threshold potassium current (KLT) that is present in a subset of cells in the zebra finch caudal mesopallium and is affected by early auditory experience. We found that KLT affects both spike adaptation and the temporal filtering properties of the receptive field. The direction of the effects depended on the temporal modulation tuning of the linear (input) stage of the cascade model, indicating a strongly nonlinear relationship. These results suggest that small changes in intrinsic dynamics in tandem with differences in synaptic connectivity can have dramatic effects on the tuning of auditory neurons.

Author summaryExperience-dependent developmental plasticity involves changes not only to synaptic connections, but to voltage-gated currents as well. Using biophysical models, it is straightforward to predict the effects of this intrinsic plasticity on the firing patterns of individual neurons, but it remains difficult to understand the consequences for sensory coding. We investigated this in the context of the zebra finch auditory cortex, where early exposure to a complex acoustic environment causes increased expression of a low-threshold potassium current. We simulated responses to song using a detailed biophysical model and then characterized encoding properties using generalized linear models. This analysis revealed that this potassium current has strong, nonlinear effects on how the model encodes the songs temporal structure, and that the sign of these effects depend on the temporal tuning of the synaptic inputs. This nonlinearity gives intrinsic plasticity broad scope as a mechanism for developmental learning in the auditory system.


title: APOBEC2 is a Transcriptional Repressor 1 required for proper Myoblast Differentiation

abstract: The activation induced cytidine deaminase/apolipoprotein B editing complex (AID/APOBEC) family comprises several nucleic acid editors with roles ranging from antibody diversification to mRNA editing. APOBEC2, an evolutionarily conserved member of this family, has neither an established substrate nor a mechanism of action, however genetic evidence suggests functional relevance in tissues such as muscle. Here, we demonstrate that in muscle, APOBEC2 does not have any of the attributed molecular functions of the AID/APOBEC family, such as RNA editing, DNA demethylation, or DNA mutation. Instead, we show that APOBEC2 occupies chromatin at promoter regions of certain genes, whose expression is repressed during muscle cell differentiation. We further demonstrate that APOBEC2 on one hand binds promoter region DNA directly and in a sequence specific fashion, while on the other it interacts with HDAC transcriptional corepressor complexes. Therefore, APOBEC2, by actively repressing the expression of non-myogenesis pathway genes, plays a key role in enforcing the proper establishment of muscle cell fate.


title: The ciliary protein IFT88 controls post-natal cartilage thickness and influences development of osteoarthritis.

abstract: Mechanical and biological cues drive cellular signalling in cartilage development, health, and disease. Proteins of the primary cilium, implicated in transduction of biophysiochemical signals, control cartilage formation during skeletal development, but their influence in post-natal cartilage remains unknown. Ift88fl/fl and AggrecanCreERT2 mice were crossed to create a cartilage-specific, inducible knockout mouse AggrecanCreERT2;Ift88fl/fl. Tibial articular cartilage (AC) thickness was assessed, through adolescence and adulthood, by histomorphometry and integrity by OARSI score. In situ mechanisms were investigated by immunohistochemistry (IHC), RNA scope and qPCR of micro-dissected cartilage. OA was induced by surgical destabilisation (DMM). Mice voluntarily exercised using wheels. Deletion of IFT88 resulted in progressive reductions in medial AC thickness during adolescence, and marked atrophy in adulthood. At 34 weeks of age, medial thickness was reduced from 104.00m, [100.30-110.50, 95% CI] in Ift88fl/fl to 89.42m [84.00-93.49, 95% CI] in AggrecanCreERT2;Ift88fl/fl (p<0.0001), associated with reductions in calcified cartilage. Occasionally, atrophy was associated with complete, spontaneous, medial cartilage degradation. Following DMM, AggrecanCreERT2;Ift88fl/fl mice had increased OA scores. Atrophy in mature AC was not associated with obvious increases in aggrecanase-mediated destruction or chondrocyte hypertrophy. Ift88 expression positively correlated with Tcf7l2, connective tissue growth factor (Ctgf) and Enpp1. RNA scope revealed increased hedgehog (Hh) signalling (Gli1), associated with reductions in Ift88, in AggrecanCreERT2;Ift88fl/fl cartilage. Wheel exercise restored both AC thickness and levels of Hh signalling in AggrecanCreERT2;Ift88fl/fl. Our results demonstrate that IFT88 is chondroprotective, regulating AC thickness, potentially by thresholding a Hh response to physiological loading that controls cartilage calcification.


title: Intrinsic excitability mechanisms of neuronal ensemble formation

abstract:
Neuronal ensembles are coactive groups of cortical neurons, found in spontaneous and evoked activity, that can mediate perception and behavior. To understand the mechanisms that lead to the formation of ensembles, we co-activated optogenetically and electrically layer 2/3 pyramidal neurons in brain slices from mouse visual cortex, in animals from both sexes, replicating in vitro an optogenetic protocol to generate ensembles in vivo. Using whole-cell and perforated patch-clamp pair recordings we find that, after optogenetic or electrical stimulation, coactivated neurons increase their correlation in spontaneous activity, a hallmark of ensemble formation. Coactivated neurons showed small biphasic changes in presynaptic plasticity, with an initial depression followed by a potentiation after a recovery period. Unexpectedly, optogenetic and electrical stimulation-induced significant increases in frequency and amplitude of spontaneous EPSPs, even after single-cell stimulation. In addition, we observed strong and persistent increases in neuronal excitability after stimulation, with increases in membrane resistance and reduction in spike threshold. A pharmacological agent that blocks changes in membrane resistance can revert this effect. These significant increases in excitability may partly explain the observed biphasic synaptic plasticity. We propose that cell-intrinsic changes in excitability are involved in the formation of neuronal ensembles. We propose an "iceberg" model, by which increased neuronal excitability makes subthreshold connections suprathreshold, enhancing the effect of already existing synapses, and generating a new neuronal ensemble.

Significance StatementWe investigated the synaptic and cellular mechanisms underlying the formation of neuronal ensembles, i.e., spontaneously coactive groups of neurons. Using in vitro electrophysiology and optogenetic in slices of mouse neocortex we replicated a protocol that generates ensembles in vivo. After optogenetic and electrical stimulation, we observed biphasic synaptic plasticity and, unexpectedly, major increases in excitability, input resistance, and reductions in firing threshold. The increased excitability can explain the observed synaptic plasticity. Our results reveal a major role for intrinsic excitability in the establishment of neuronal ensembles.


title: MafF is an antiviral host factor that suppresses transcription from Hepatitis B Virus core promoter

abstract:
Hepatitis B Virus (HBV) is a stealth virus that exhibits only minimal induction of the interferon system that is required for both innate and adaptive immune responses. However, 90% of acutely infected adults can clear the virus, suggesting the presence of additional mechanisms that facilitate viral clearance. Herein, we report that Maf bZIP transcription factor F (MafF) promotes host defense against infection with HBV. Using siRNA library and an HBV/NL reporter virus, we screened to identify anti-HBV host factors. Our data showed that silencing of MafF led to a 6-fold increase in luciferase activity after HBV/NL infection. Overexpression of MafF reduced HBV core promoter transcriptional activity, which was relieved upon mutating the putative MafF binding region. Loss of MafF expression by CRISPR/CAS9 (in HepG2-hNTCP-C4 cells) or siRNA silencing (in primary hepatocytes [PXB]), induced HBV core and HBV pregenomic RNA (pgRNA) levels, respectively, after HBV infection. MafF physically binds to HBV core promoter and competitively inhibits HNF-4 binding to an overlapping sequence in HBV enhancer II sequence (EnhII) as seen by ChIP analysis. MafF expression was induced by IL-1{beta}/TNF- treatment in both HepG2 and PXB cells, in an NF-{kappa}B-dependent manner. Consistently, MafF expression levels were significantly enhanced and positively correlated with the levels of these cytokines in patients with chronic HBV infection, especially in the immune clearance phase.

ImportanceHBV is a leading cause of chronic liver diseases, infecting about 250 million people worldwide. HBV has developed strategies to escape interferon-dependent innate immune responses. Hence, the identification of other anti-HBV mechanisms is important for understanding HBV pathogenesis, and developing anti-HBV strategies. MafF was shown to suppress transcription from HBV core promoter, leading to a significant suppression of HBV life cycle. Furthermore, MafF expression was induced in chronic HBV patients and in primary human hepatocytes (PXB). This induction correlated with the levels of inflammatory cytokines (IL-1{beta} and TNF-). These data suggest that the induction of MafF contributes to the hosts antiviral defense by suppressing transcription from selected viral promoters. Our data shed light on a novel role for MafF as anti-HBV host restriction factor.


title: Single-nuclei transcriptomics of schizophrenia prefrontal cortex primarily implicates neuronal subtypes

abstract: Transcriptomic studies of bulk neural tissue homogenates from persons with schizophrenia and controls have identified differentially expressed genes in multiple brain regions. However, the brains heterogeneous nature prevents identification of relevant cell types. This study analyzed single-nuclei transcriptomics of ~275,000 nuclei from frozen human postmortem dorsolateral prefrontal cortex samples from males with schizophrenia (n = 12) and controls (n = 14). 4,766 differential expression events were identified in 2,994 unique genes in 16 of 20 transcriptomically-distinct cell populations. ~96% of differentially expressed genes occurred in five neuronal cell types, and differentially expressed genes were enriched for genes associated with schizophrenia and bipolar GWAS loci. Downstream analyses identified cluster-specific enriched gene ontologies, KEGG pathways, and canonical pathways. Additionally, microRNAs and transcription factors with overrepresented neuronal cell type-specific targets were identified. These results expand our knowledge of disrupted gene expression in specific cell types and permit new insight into the pathophysiology of schizophrenia.


title: The influence of autotoxicity on the dynamics of vegetation spots

abstract: Plant autotoxicity has proved to play an essential role in the behaviour of local vegetation. We analyse a reaction-diffusion-ODE model describing the interactions between vegetation, water, and autotoxicity. The presence of autotoxicity is seen to induce movement and deformation of spot patterns in some parameter regimes, a phe-nomenon which does not occur in classical biomass-water models. We aim to analytically quantify this novel feature, by studying travelling wave solutions in one spatial dimension. We use geometric singular perturbation theory to prove the existence of symmetric, stationary and non-symmetric, travelling pulse solutions, by constructing appropriate homoclinic orbits in the associated 5-dimensional dynamical system. In the singularly perturbed context, we perform an extensive scaling analysis of the dynamical system, identifying multiple asymptotic scaling regimes where (travelling) pulses may or may not be constructed. We show that, while the analytically constructed stationary pulse corresponds to its numerical counterpart, there is a discrepancy between the numerically observed travelling pulse and its analytical counterpart. Our findings indicate how the inclusion of an additional ODE may significantly influence the properties of classical biomass-water models of Klausmeier/Gray-Scott type.


title: Non-parametric modelling of temporal and spatial counts data from RNA-seq experiments

abstract:
AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSMotivationC_ST_ABSThe negative binomial distribution has been shown to be a good model for counts data from both bulk and single-cell RNA-sequencing (RNA-seq). Gaussian process (GP) regression provides a useful non-parametric approach for modeling temporal or spatial changes in gene expression. However, currently available GP regression methods that implement negative binomial likelihood models do not scale to the increasingly large datasets being produced by single-cell and spatial transcriptomics.

ResultsThe GPcounts package implements GP regression methods for modelling counts data using a negative binomial likelihood function. Computational efficiency is achieved through the use of variational Bayesian inference. The GP function models changes in the mean of the negative binomial likelihood through a logarithmic link function and the dispersion parameter is fitted by maximum likelihood. We validate the method on simulated time course data, showing that it is better able to identify changes in over-dispersed counts data than methods based on Gaussian or Poisson likelihoods. To demonstrate temporal inference, we apply GPcounts to single-cell RNA-seq datasets after pseudotime and branching inference. To demonstrate spatial inference, we apply GPcounts to data from the mouse olfactory bulb to identify spatially variable genes and compare to two published GP methods. We also provide the option of modelling additional dropout using a zero-inflated negative binomial. Our results show that GPcounts can be used to model temporal and spatial counts data in cases where simpler Gaussian and Poisson likelihoods are unrealistic.

AvailabilityGPcounts is implemented using the GPflow library in Python and is available at https://github.com/ManchesterBioinference/GPcounts along with the data, code and notebooks required to reproduce the results presented here.

Contactnuha.bintayyash@manchester.ac.uk or magnus.rattray@manchester.ac.uk


title: Pulsations and flows in tissues: two collective dynamics with simple cellular rules

abstract: Collective motions of epithelial cells in vivo are essential for morphogenesis in developmental biology. Tissues elongate, contract, flow, and oscillate, sculpting embryos. These tissue level dynamics are known, but their physical mechanisms at the cellular level are unclear, with various behaviors that depend on the tissues and species. Moreover, investigations on in vitro tissue behavior usually focus on only one type of cell dynamics and use diverse theoretical approaches, making systematic comparisons between studies challenging. Here, we show that the very same epithelial monolayer of Madine Darby Canine Kidney (MDCK) cells can exhibit two types of tissue kinematics, pulsations, and coherent flows. We also report that these distinct motions can be controlled with internal and external cues by using quantitative live imaging of the acto-myosin cytoskeleton, specific inhibitors, microfabrication, and friction modulation of the substrate. We further demonstrate with a unified vertex model that both behaviors depend on the interplay between velocity alignment and random diffusion of cell polarization. When alignment and diffusion are comparable, a pulsatile flow emerges, whereas the tissue undergoes long-range flows when velocity alignment dominates. We propose that environmental friction, acto-myosin distributions, and cell polarization kinetics serve as the essential factors regulating the dynamics of tissue morphogenesis.


title: Functional and genetic determinants of mutation rate variability in regulatory elements of cancer genomes

abstract:
BackgroundCancer genomes are shaped by mutational processes with complex spatial variation at multiple scales. Entire classes of regulatory elements are affected by local variations in mutation frequency. However, the underlying mutational mechanisms with functional and genetic determinants remain poorly understood.

ResultsWe characterised the mutational landscape of 1.3 million gene regulatory and chromatin architectural elements in 2,419 whole cancer genomes with transcriptional and pathway activity, functional conservation and recurrent driver events. We developed RM2, a statistical model that quantifies mutational enrichment or depletion in classes of genomic elements through genetic, trinucleotide and megabase-scale effects. We report a map of localised mutational processes affecting CTCF binding sites, transcription start sites (TSS) and tissue-specific open-chromatin regions. We show that increased mutational frequency in TSSs correlates with mRNA abundance in most cancer types, while open-chromatin regions are generally enriched in mutations. We identified [~]10,000 CTCF binding sites with core DNA motifs and constitutive binding in 66 cell types that represent focal points of local mutagenesis. We detected site-specific mutational signatures, such as SBS40 in open-chromatin regions in prostate cancer and SBS17b in CTCF binding sites in gastrointestinal cancers. We also proposed candidate drivers of localised mutagenesis: BRAF mutations associate with mutational enrichments at CTCF binding sites in melanoma, and ARID1A mutations with TSS-specific mutations in pancreatic cancer.

ConclusionsOur method and catalogue of localised mutational processes provide novel perspectives to cancer genome evolution, mutagenesis, DNA repair and driver discovery. Functional and genetic correlates of localised mutagenesis provide mechanistic hypotheses for future studies.


title: How does an ectodomain of membrane-associated proteins stand upright and exert robust signal

abstract: Even after decades of research, a comprehensive mechanism that elucidates the underpinnings of signaling through the cell membrane is still elusive. Here, we address a simple question- "how does the ectodomain of a membrane-associated protein consisting of multiple domains and connected by flexible linkers stand  upright on the membrane?". Our analysis based on large amount of available structural and functional data, looking for a pattern of association of these molecules in the crystal structures and with the concept that  random things seldom repeat lead to a surprisingly interesting and consistent observation that (1) the weak cis-interaction mediated symmetric oligomerization of signaling molecules not only support their  upright orientation but often bury their ligand-binding surface to avoid spurious signaling (2) the linkers connecting the domains are probably not flexible as presumed. This analysis provides a model for pre-liganded receptor supramolecular organization that resolves some of the mysteries unanswered by hypothesis such as  lipid-rafts and  fence and pickets. With CD4, pMHCII, CD2 and TNFR1 as examples, we show that the observed cis-association of molecules also correlate well with their functional role. Further, our analysis reconciles the long-standing controversies related to these molecules and appear to be generic enough to be applied to other signaling molecules.


title: Per1/Per2-Igf2 axis-mediated circadian regulation of myogenic differentiation

abstract:
Circadian rhythms regulate cell proliferation and differentiation but circadian control of tissue regeneration remains elusive at the molecular level. Here, we show that proper myoblast differentiation and muscle regeneration are regulated by the circadian master regulators Per1 and Per2. Depletion of Per1 or Per2 suppressed myoblast differentiation in vitro and muscle regeneration in vivo, demonstrating their non-redundant functions. Both Per1 and Per2 were required for the activation of Igf2, an autocrine promoter of myoblast differentiation, accompanied by Per-dependent recruitment of RNA polymerase II, dynamic histone modifications at the Igf2 promoter and enhancer, and the promoter-enhancer interaction. This circadian epigenetic priming created a preferred time window for initiating myoblast differentiation. Consistently, muscle regeneration was faster if initiated at night when Per1, Per2, and Igf2 were highly expressed compared with morning. This study reveals the circadian timing as a significant factor for effective muscle cell differentiation and regeneration.

eTOC SummaryKatoku-Kikyo et al. show that the circadian master regulators Per1 and Per2 control the efficiency of myoblast differentiation via Igf2 activation. This pathway creates a preferred circadian time window for myoblast differentiation in vitro and muscle regeneration in vivo.

category: developmental biology