title: Lonely in the dark: trauma memory and sex-specific dysregulation of amygdala reactivity to fear signals

abstract: Loneliness exacerbates psychological distress and increases the risk of psychopathology after trauma exposure. The prevalence of trauma-associated disorders varies substantially between sexes, and accumulating evidence indicates sex-specific effects of loneliness. However, it is still unclear whether a lack of social connectedness affects trauma-induced intrusions and the neural processing of fear signals. Moreover, it is uncertain, whether loneliness plays a different role in women and men. We used a prestratification strategy and recruited n=47 (n=20 women) healthy individuals with high loneliness and n=35 controls (n=18 women). Participants were exposed to an experimental trauma and evoked intrusive thoughts in daily life were monitored for three consecutive days. Functional magnetic resonance imaging was used to assess neural habituation to fearful faces and fear learning (conditioning and extinction) prior to trauma exposure. The total number of intrusions and amygdala reactivity in neural fear processing served as the primary study outcomes. Our results revealed a significant interaction between loneliness and sex such that loneliness was associated with more intrusions in men, but not in women. A similar pattern emerged at the neural level, with both reduced amygdala habituation to repeated fearful faces and amygdala hyperreactivity during the conditioning of fear signals in lonely men, but not in women. Our findings indicate that loneliness may confer vulnerability to intrusive memories after trauma exposure in healthy men and that this phenotype relates to altered limbic processing of fear signals. Collectively, interventions targeting social connectedness may mitigate the sequelae of traumatic experiences.


title: Asymmetrical choice-related ensemble activity in direct and indirect-pathway striatal neurons drives perceptual decisions

abstract: Action selection during decision-making depends on the basal ganglia circuits that comprise the direct and indirect pathways known to oppositely control movement. However, the mechanism for coordinating these opponent pathways during decision-making remains unclear. We address this by employing deep-brain two-photon imaging and optogenetic manipulations of the direct- and indirect-pathway spiny projection neurons (dSPNs and iSPNs) in the posterior striatum during an auditory decision-making behavior. We show that while dSPNs and iSPNs play opposite causal roles during decision-making, each subtype contains divergent ensembles preferring different choices. The ensembles in dSPNs show stronger contralateral dominance than those in iSPNs manifested by higher-level activation and synchronization. Consistent with this asymmetrical contralateral dominance, optogenetic disinhibition of both pathways promoted contralateral choices. A computational model incorporating the striatal ensemble asymmetry recapitulated the causal behavioral effects. Our results uncover the asymmetry between opponent SPN ensembles as a circuit mechanism for action selection during decision-making.

category: neuroscience


title: Mapping morphological malformation to genetic dysfunction in blood vessel organoids with 22q11.2 Deletion Syndrome

abstract: DiGeorge Syndrome, or 22q11.2 deletion syndrome (22q11.2 DS), is a genetic disorder caused by microdeletions in chromosome 22, impairing the function of endothelial cells (EC) and/or mural cells and leading to deficits in blood vessel development such as abnormal aortic arch morphology, tortuous retinal vessels, and tetralogy of Fallot. The mechanism by which dysfunctional endothelial cells and pericytes contribute to the vasculopathy, however, remains unknown. In this study, we used human blood vessel organoids (VOs) generated from iPSC of 22q11.2 DS patients to model the vascular malformations and genetic dysfunctions. We combined high-resolution lightsheet imaging and single-cell transcriptome analysis to link the genetic profile and vascular phenotype at the single-cell level. We developed a comprehensive analytical methodology by integrating deep learning-mediated blood vessel segmentation, network graph construction, and tessellation analysis for automated morphology characterization. We report that 22q11.2DS VOs demonstrate a smaller size with increased angiogenesis/sprouting, suggesting a less stable vascular network. Overall, clinical presentations of smaller vascular diameter, less connected vasculature, and increased branch points were recapitulated in 22q11.2DS VOs. Single-cell transcriptome profiling showed heterogeneity in both 22q11.2DS and control VOs, but the former demonstrated alterations in endothelial characteristics that are organ-specific and suggest a perturbation in the vascular developmental process. Intercellular communication analysis indicated that the vascular dysfunctions in 22q11.2 deletion were due to a lower cell-cell contact and upregulated extracellular matrix organization involving collagen and fibronectin. Voronoi diagram-based tessellation analysis also indicated that the colocalization of endothelial tubes and mural cells was different between control and 22q11.2 VOs, indicating that alterations in EC and mural interactions might contribute to the deficits in vascular network formation. This study illustrates the utility of VO in revealing the pathogenesis of 22q11.2DS vasculopathy.

category: bioengineering


title: 2-photon-fabricated nano-fluidic traps for extended detection of single macromolecules and colloids in solution

abstract: The analysis of nanoscopic species, such as proteins and colloidal assemblies, at the single-molecule level has become vital in many areas of fundamental and applied research. Approaches to increase the detection timescales for single molecules in solution without immobilising them onto a substrate surface and applying external fields are much sought after. Here we present an easy-to-implement and versatile nanofluidics-based approach that enables increased observational-timescale analysis of single biomacromolecules and nanoscale colloids in solution. We use two-photon-based hybrid lithography in conjunction with soft lithography to fabricate nanofluidic devices with nano-trapping geometries down to 100 nm in height. We provide a rigorous description and characterisation of the fabrication route that enables the writing of nanoscopic 3D structures directly in photoresist and allows for the integration of nano-trapping and nano-channel geometries within micro-channel devices. Using confocal fluorescence burst detection, we validated the functionality of particle confinement in our nano-trap geometries through measurement of particle residence times. All species under study, including nanoscale colloids, -synuclein oligomers, and double-stranded DNA, showed a three to five-fold increase in average residence time in the detection volume of nano-traps, due to the additional local steric confinement, in comparison to free space diffusion in a nearby micro-channel. Our approach thus opens-up the possibility for single-molecule studies at prolonged observational timescales to analyse and detect nanoparticles and protein assemblies in solution without the need for surface immobilisation.

category: biophysics


title: Effects of crude oil on juvenile threespine stickleback somatic and immune development

abstract: Aquatic oil spills have resounding effects on surrounding ecosystems, and thus significant resources are committed to oil spill responses to remove the oil from the environment as quickly as possible. Oil has immunotoxic effects and may be particularly harmful to larval and juvenile fish as it can cause a number of developmental defects and stunt growth. In spite of significant efforts to clean oil, it is unclear whether larval and juvenile fish can recover from the effects of oil and no work has been done on the effect crude oil has on developing threespine stickleback (Gasterosteus aculeatus) fish. Threespine stickleback are a ubiquitous sentinel species in the northern hemisphere and are an important food source for many larger, economically valuable fish. As fish with fully marine, anadromous, and freshwater populations, stickleback are exposed to oil in a variety of aquatic environments. We hypothesized that oil exposure would suppress both growth and immunity of developing stickleback, but that fish health could be recovered by removal of the crude oil. Fish were exposed to Alaska North Slope crude oil and then were moved to water without crude oil for two weeks (depuration). Measurements of growth and immunity were taken before and after the depuration. We found that crude oil effected different developmental pathways independently, significantly impacting some but not others. This is the first study to examine the effect crude oil has on early stages of stickleback development, and that stickleback fish are unable to recover from exposure after being transferred to clean water for two-weeks, suggesting larval/juvenile stickleback exposed to crude oil need longer than two-weeks to recover if they are able to recover at all.

category: pharmacology and toxicology


title: Effects of food availability cycles on phase and period of activity/rest rhythm in Drosophila melanogaster

abstract: Foraging and feeding are indispensable for survival and their timing depends not only on the metabolic state of the animal but also on the availability of food resources in their environment. Since both these aspects are subject to change over time, these behaviours exhibit rhythmicity in occurrence. As the locomotor activity of an organism is related to its disposition to acquire food, and peak feeding in fruit flies has been shown to occur at a particular time of the day, we asked if cyclic food availability can entrain their rhythmic activity. By subjecting flies to cyclic food availability i.e., feeding/starvation (FS) cycles, we provided food cues contrasting to the preferred activity times and observed if this imposed cycling in food availability could entrain the activity/rest rhythm. We found that phase control, which is a property integral to entrainment, was not achieved despite increasing starvation duration of FS cycles (FS12:12, FS10:14 and FS8:16). We also found that flies subjected to T21 and T26 FS cycles were unable to match period of the activity rhythm to short or long T-cycles. Taken together these results show that external food availability cycles do not entrain the activity/rest rhythm of fruit flies. However, we find that starvation induced hyperactivity causes masking which results in phase changes. Additionally, T-cycle experiments resulted in minor period changes during FS treatment. These findings highlight that food cyclicity by itself may not be a potent zeitgeber but may act in unison with other abiotic factors like light and temperature to help flies time their activity appropriately.

category: animal behavior and cognition


title: Trimethylamine N-Oxide (TMAO) links peripheral insulin resistance and cognitive deficiencies in a senescence accelerated mouse model

abstract:
It has been established that ageing is the major risk factor for cognitive deficiency or neurodegenerative diseases such as Alzheimers disease (AD) and it is becoming increasingly evident that insulin resistance is another factor. Biological plausibility for a link between insulin resistance and dementia is relevant for understanding disease etiology, and to form bases for prevention efforts to decrease disease burden.

The dysfunction of the insulin signaling system and glucose metabolism has been proposed to be responsible for brain aging. Normal insulin signaling in the brain is required to mediate growth, metabolic functions, and the survival of neurons and glia. Insulin receptors are densely expressed in the olfactory bulb, the cerebral cortex and the hippocampus and regulate neurotransmitter release and receptor recruitment. In normal elderly individuals, reduced glucose tolerance and decreased insulin levels in the aged brain are typically observed. Furthermore, insulin signaling is aberrantly activated in the AD brain, leading to non-responsive insulin receptor signaling.

The senescence accelerated mouse (SAMP8) mouse was one of the accelerated senescence strains that spontaneously developed from breeding pairs of the AKR/J series. The SAMP8 mouse develops early learning and memory deficits (between 6 and 8 months) together with other characteristics similar to those seen in Alzheimers disease. The present project proposes the investigation of the missing link between aging, insulin resistance and dementia.

Peripheral but not central insulin resistance was found in SAMP8 mice accompanied by cognitive deficiencies. Furthermore, a marked peripheral inflammatory state (i.e. significantly higher adipose tissue TNF- and IL6 levels) were observed in SAMP8 mice, followed by neuroinflammation that could be due to a higher cytokine leaking into the brain across a aging-disrupted BBB. Moreover, aging-induced gut dysbiosis produces higher TMAO that could also contribute to the peripheral and central inflammatory tone as well as to the cognitive deficiencies observed in SAMP8 mice. All those alterations were reversed by DMB, a treatment inhibits the transformation of choline, carnitine and crotonobetaine, decreaseing TMAO levels.

The ever-increasing incidence of neurodegenerative diseases not only limits the life quality of the affected individuals and their families but also poses an enormous demand on the societies. Thus, it is instrumental to pursue novel promising approaches to prevent and treat it at the highest possible speed to rapidly translate them to clinical practice. From this point of view, data obtained from this project will be instrumental to validate the principle approach of microbial dysbiosis and increased TMAO secretion as a key link between aging, insulin resistance and dementia. Collectively, the proposed experiments ideally integrate the aim to promote a novel approach to improve the lives of those suffering from cognitive disturbances.


title: Hippocampal Neuropeptide Y2 receptor blockade improves spatial memory retrieval and modulates limbic brain metabolism

abstract:
IntroductionThe neuropeptide Y (NPY) is broadly distributed in the central nervous system (CNS), and it has been related to neuroprotective functions. NPY seems to be an important component to counteract brain damage and cognitive impairment mediated by drugs of abuse and neurodegenerative diseases, and both NPY and its Y2 receptor (Y2R) are highly expressed in the hippocampus, critical for learning and memory. We have recently demonstrated its influence on cognitive functions; however, the specific mechanism and involved brain regions where NPY modulates spatial memory by acting on Y2R remain unclear.

MethodsHere, we examined the involvement of the hippocampal NPY Y2R in spatial memory and associated changes in brain metabolism by bilateral administration of the selective antagonist BIIE0246 into the rat dorsal hippocampus. To further evaluate the relationship between memory functions and neuronal activity, we analysed the regional expression of the mitochondrial enzyme cytochrome c oxidase (CCO) as an index of oxidative metabolic capacity in limbic and non-limbic brain regions.

ResultsThe acute blockade of NPY Y2R significantly improved spatial memory recall in rats trained in the Morris water maze that matched metabolic activity changes in spatial memory processing regions. Specifically, CCO activity changes were found in the dentate gyrus of the dorsal hippocampus and CA1 subfield of the ventral hippocampus, the infralimbic region of the PFC and the mammillary bodies.

ConclusionsThese findings suggest that the NPY hippocampal system, through its Y2R receptor, influences spatial memory recall (retrieval) and exerts control over patterns of brain activation that are relevant for associative learning, probably mediated by Y2R modulation of long-term potentiation and long-term depression.

HighlightsO_LIUnder hippocampal Y2R antagonism, place preference memory retrieval is enhanced
C_LIO_LISpatial retrieval enhancement under Y2R blockade is correlated with changes in regional brain energy metabolism
C_LIO_LIEnhanced retrieval associated CCO activity increases in the dorsal DG, while decreasing in the ventral CA1, IL cortex and mammillary bodies
C_LIO_LIY2R exert control over patterns of brain activation that are relevant for spatial memory expression
C_LI


title: Virtual reality validation of naturalistic modulation strategies to counteract fading in retinal stimulation

abstract:
ObjectiveTemporal resolution is a key challenge in artificial vision. Several prosthetic approaches are limited by the perceptual fading of evoked phosphenes upon repeated stimulation from the same electrode. Therefore, implanted patients are forced to perform active scanning, via head movements, to refresh the visual field viewed by the camera. However, active scanning is a draining task, and it is crucial to find compensatory strategies to reduce it.

ApproachTo address this question, we implemented perceptual fading in simulated prosthetic vision using virtual reality. Then, we quantified the effect of fading on two indicators: the time to complete a reading task and the head rotation during the task. We also tested if stimulation strategies previously proposed to increase the persistence of responses in retinal ganglion cells to electrical stimulation could improve these indicators.

Main resultsThis study shows that stimulation strategies based on interrupted pulse trains and randomisation of the pulse duration allows significant reduction of both the time to complete the task and the head rotation during the task.

SignificanceThe stimulation strategy used in retinal implants is crucial to counteract perceptual fading and to reduce active head scanning during prosthetic vision. In turn, less active scanning might improve the patients comfort in artificial vision.


title: A multiplexed cell-free assay to screen for antimicrobial peptides in double emulsion droplets

abstract: The global surge in bacterial resistance against traditional antibiotics triggered intensive research for novel compounds, with antimicrobial peptides (AMPs) identified as a promising candidate. Automated methods to systematically generate and screen AMPs according to their membrane preference, however, are still lacking. We introduce a novel microfluidic system for the simultaneous cell-free production and screening of AMPs for their membrane specificity. On our device, AMPs are cell-free produced within water-in-oil-in-water double emulsion droplets, generated at high frequency. Within each droplet, the peptides can interact with different classes of co-encapsulated liposomes, generating a membrane-specific fluorescent signal. The double emulsions can be incubated and observed in a hydrodynamic trapping array or analysed via flow cytometry. Our approach provides a valuable tool for the discovery and development of membrane-active antimicrobials.


title: FDJD: RNA-Seq Based Fusion Transcript Detection Using Jaccard Distance

abstract: Gene fusions events, which are the result of two genes fused together to create a hybrid gene, were first described in cancer cells in the early 1980s. These events are relatively common in many cancers including prostate, lymphoid, soft tissue, and breast. Recent advances in next-generation sequencing (NGS) provide a high volume of genomic data, including cancer genomes. The detection of possible gene fusions requires fast and accurate methods. However, current methods suffer from inefficiency, lack of sufficient accuracy, and a high false-positive rate. We present an RNA-Seq fusion detection method that uses dimensionality reduction and parallel computing to speed up the computation. We convert the RNA categorical space into a compact binary array called binary fingerprints, which enables us to reduce the memory usage and increase efficiency. The search and detection of fusion candidates are done using the Jaccard distance. The detection of candidates is followed by refinement. We benchmarked our fusion prediction accuracy using both simulated and genuine RNA-Seq datasets. Paired-end Illumina RNA-Seq genuine data were obtained from 60 publicly available cancer cell line data sets. The results are compared against the state-of-the-art-methods such as STAR-Fusion, InFusion, and TopHat-Fusion. Our results show that FDJD exhibits superior accuracy compared to popular alternative fusion detection methods. We achieved 90% accuracy on simulated fusion transcript inputs, which is the highest among the compared methods while maintaining comparable run time.

category: bioinformatics


title: Missense variants reveal functional insights into the human ARID family of gene regulators

abstract: Missense variants are alterations to protein coding sequences that result in amino acid substitutions. They can be deleterious if the amino acid is required for maintaining structure or/and function, but are likely to be tolerated at other sites. Consequently, missense variation within a healthy population can mirror the effects of negative selection on protein structure and function, such that functional sites on proteins are often depleted of missense variants. Advances in high-throughput sequencing have dramatically increased the sample size of available human variation data, allowing for population-wide analysis of selective pressures. In this study, we developed a convenient set of tools, called 1D-to-3D, for visualizing the positions of missense variants on protein sequences and structures. We used these tools to characterize human homologues of the ARID family of gene regulators. ARID family members are implicated in multiple cancer types, developmental disorders, and immunological diseases but current understanding of their mechanistic roles is incomplete. Combined with phylogenetic and structural analyses, our approach allowed us to characterise sites important for protein-protein interactions, histone modification recognition, and DNA binding by the ARID proteins. We find that comparing missense depletion patterns among paralogs can reveal sub-functionalization at the level of domains. We propose that visualizing missense variants and their depletion on structures can serve as a valuable tool for complementing evolutionary and experimental findings.


title: Meta-Transcriptome Detector (MTD): a novel pipeline for metatranscriptome analysis of bulk and single-cell RNAseq data

abstract: RNA-seq data contains not only host transcriptomes but also non-host information that comprises transcripts from active microbiota in the host cells. Therefore, joint and integrative analyses of both host and meta-transcriptome can reveal gene expression of microbial community in a given sample as well as the correlative and interactive dynamics of host response to the microbiome. However, there are no convenient tools that can systemically analyze host-microbiota interactions by simultaneously quantifying host and meta-transcriptome in the same sample at the tissue and the single-cell level, which poses a challenge for interested researchers with a limited expertise in bioinformatics. Here, we developed a software pipeline that can comprehensively and synergistically analyze and correlate the host and meta-transcriptome in a single sample using bulk and single-cell RNA-seq data. This pipeline, named MTD, can extensively identify and quantify microbiome, including viruses, bacteria, protozoa, fungi, plasmids, and vectors in the host cells and correlate the microbiome with the host transcriptome. MTD is easy to install and run, involving only a few lines of simple commands. It empowers researchers with unique genomics insights into host immune responses to microorganisms.


title: MTD: a unique pipeline for host and meta-transcriptome joint and integrative analyses of RNA-seq data


title: Structural comparative modeling of multi-domain ΔF508 CFTR

abstract:
Cystic Fibrosis (CF) is a common genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), an epithelial anion channel expressed in several vital organs. Absence of functional CFTR results in imbalanced osmotic equilibrium and subsequent mucus build up in the lungs - which increases the risk of infection and eventually causes death. CFTR is an ATP binding cassette (ABC) transporter composed of two transmembrane domains (TMDs), two nucleotide binding domains (NBDs), and an unstructured regulatory domain. The most prevalent patient mutation is the deletion of F508 ({Delta}F508), making {Delta}F508 CFTR the primary target for current FDA approved CF therapies. However, no experimental multi-domain {Delta}F508 CFTR structure has been determined and few studies have modeled {Delta}F508 using multi-domain WT CFTR structures. Here, we used cryo-EM density data and Rosetta comparative modeling (RosettaCM) to compare a {Delta}F508 model with published experimental data on CFTR NBD1 thermodynamics. We then apply this modeling method to generate multi-domain WT and {Delta}F508 CFTR structural models. These models demonstrate the destabilizing effects of {Delta}F508 on NBD1 and the NBD1/TMD interface in both the closed and open conformation of CFTR. Furthermore, we modeled {Delta}F508/R1070W and {Delta}F508 bound to the CFTR corrector VX-809. Our models reveal the stabilizing effects of R1070W and VX-809 on multi-domain models of {Delta}F508 CFTR and pave the way for rational design of additional drugs that target {Delta}F508 CFTR for treatment of CF.

Author SummaryProteins three-dimension shape determines their function, so when genetic mutation compromises the shape of vital proteins, it may cause disease. Such is the case in Cystic Fibrosis, a chronic genetic disease caused by mutations in the protein Cystic Fibrosis Transmembrane Conductance Regulator. Here, we work backwards from the shape of the wild-type protein - found in healthy people, to computationally model the shape of the most common Cystic Fibrosis mutant. Our computer models reveal distinct defects in the shape of the mutant Cystic Fibrosis Transmembrane Conductance Regulator protein in the area surrounding the mutation. We also model an important FDA approved Cystic Fibrosis drug, VX-809, into the mutant protein structure and show how VX-809 stabilizes the protein around the location of the mutation. The method we developed will pave the way for computational drug design for Cystic Fibrosis.


title: CRAFT: a bioinformatics software for custom prediction of circular RNA functions

abstract:
Circular RNAs (circRNAs), transcripts generated by backsplicing, are particularly stable and pleiotropic molecules, whose dysregulation drives human diseases and cancer by modulating gene expression and signaling pathways. CircRNAs can regulate cellular processes by different mechanisms, including interaction with microRNAs (miRNAs) and RNA-binding proteins (RBP), and encoding specific peptides. The prediction of circRNA functions is instrumental to interpret their impact in diseases, and to prioritize circRNAs for functional investigation. Currently, circRNA functional predictions are provided by web databases that do not allow custom analyses, while self-standing circRNA prediction tools are mostly limited to predict only one type of function, mainly focusing on the miRNA sponge activity of circRNAs. To solve these issues, we developed CRAFT (CircRNA Function prediction Tool), a freely available computational pipeline that predicts circRNA sequence and molecular interactions with miRNAs and RBP, along with their coding potential. Analysis of a set of circRNAs with known functions has been used to appraise CRAFT predictions and to optimize its setting. CRAFT provides a comprehensive graphical visualization of the results, links to several knowledge databases, and extensive functional enrichment analysis. Moreover, it originally combines the predictions for different circRNAs. CRAFT is a useful tool to help the user explore the potential regulatory networks involving the circRNAs of interest and generate hypotheses about the cooperation of circRNAs into the modulation of biological processes.

Key pointsO_LICRAFT is a self standing tool for comprehensive circRNA function prediction.
C_LIO_LICRAFT functions include circRNA sequence reconstruction, microRNA and RNA-binding protein response elements and coding potential prediction.
C_LIO_LIPredictions for multiple circRNAs are connected to infer possible cooperation networks and illustrate the potential impact of circRNAs on biological and disease processes.
C_LI


title: Adversarial domain translation networks enable fast and accurate large-scale atlas-level single-cell data integration

abstract: The rapid emergence of large-scale atlas-level single-cell RNA-seq datasets presents remarkable opportunities for broad and deep biological investigations through integrative analyses. However, harmonizing such datasets requires integration approaches to be not only computationally scalable, but also capable of preserving a wide range of fine-grained cell populations. We created Portal, a unified framework of adversarial domain translation to learn harmonized representations of datasets. With innovation in model and algorithm designs, Portal achieves superior performance in preserving biological variation during integration, while achieving integration of millions of cells in minutes with low memory consumption. We show that Portal is widely applicable to integrating datasets across samples, platforms and data types (including scRNA-seq, snRNA-seq and scATAC-seq). Finally, we demonstrate the power of Portal by applying it to the integration of cross-species datasets with limited shared information among them, elucidating biological insights into the similarities and divergences in the spermatogenesis process among mouse, macaque and human.


title: Adversarial domain translation networks for fast and accurate integration of large-scale atlas-level single-cell datasets


title: A Thin-Film Lubrication Model for Biofilm Expansion Under Strong Adhesion

abstract: Understanding microbial biofilm growth is important to public health, because biofilms are a leading cause of persistent clinical infections. In this paper, we develop a thin-film model for microbial biofilm growth on a solid substratum to which it adheres strongly. We model biofilms as two-phase viscous fluid mixtures of living cells and extracellular fluid. The model tracks the movement, depletion, and uptake of nutrients explicitly, and incorporates cell proliferation via a nutrient-dependent source term. Notably, our thin-film reduction is two-dimensional and includes the vertical dependence of cell volume fraction. Numerical solutions show that this vertical dependence is weak for biologically-feasible parameters, reinforcing results from previous models in which this dependence was neglected. We exploit this weak dependence by writing and solving a simplified one-dimensional model that is computationally more efficient than the full model. We use both the one and two-dimensional models to predict how model parameters affect expansion speed and biofilm thickness. This analysis reveals that expansion speed depends on cell proliferation, nutrient availability, cell-cell adhesion on the upper surface, and slip on the biofilm-substratum interface. Our numerical solutions provide a means to qualitatively distinguish between the extensional flow and lubrication regimes, and quantitative predictions that can be tested in future experiments.


title: Cooperative protein allosteric transition mediated by a fluctuating transmission network

abstract: Allosteric communication between distant protein sites represents a key mechanism of biomolecular regulation and signal transduction. Compared to other processes such as protein folding, however, the dynamical evolution of allosteric transitions is still not well understood. As example of allosteric coupling between distant protein regions, we consider the global open-closed motion of the two domains of T4 lysozyme, which is triggered by local motion in the hinge region. Combining extensive molecular dynamics simulations with a correlation analysis of interresidue contacts, we identify a network of interresidue distances that move in a concerted manner. The cooperative process originates from a cogwheel-like motion of the hydrophobic core in the hinge region, which constitutes a flexible transmission network. Through rigid contacts and the protein backbone, the small local changes of the hydrophobic core are passed on to the distant terminal domains and lead to the emergence of a rare global conformational transition. As in an Ising-type model, the cooperativity of the allosteric transition can be explained via the interaction of local fluctuations.


title: Systems medicine dissection of chromosome 1q amplification reveals oncogenic regulatory circuits and informs targeted therapy in cancer

abstract:
Understanding the biological and clinical impact of copy number aberrations (CNA) in cancer remains an unmet challenge. Genetic amplification of chromosome 1q (chr1q-amp) is a major CNA conferring adverse prognosis in several cancers, including the blood cancer, multiple myeloma (MM). Although several chr1q genes portend high-risk MM disease, the underpinning molecular aetiology remains elusive. Here we integrate patient multi-omics datasets with genetic variables to identify 103 adverse prognosis genes in chr1q-amp MM. Amongst these, the transcription factor PBX1 is ectopically expressed by genetic amplification and epigenetic activation of its own preserved 3D regulatory domain. By binding to reprogrammed super-enhancers, PBX1 directly regulates critical oncogenic pathways, whilst in co-operation with FOXM1, activates a proliferative gene signature which predicts adverse prognosis across multiple cancers. Notably, pharmacological disruption of the PBX1-FOXM1 axis, including with a novel PBX1 inhibitor is selectively toxic against chr1q-amp cancer cells. Overall, our systems medicine approach successfully identifies CNA-driven oncogenic circuitries, links them to clinical phenotypes and proposes novel CNA-targeted therapy strategies in cancer.

SignificanceWe provide a comprehensive systems medicine strategy to unveil oncogenic circuitries and inform novel precision therapy decisions against CNA in cancer. This first clinical multi-omic analysis of chr1q-amp in MM identifies a central PBX1-FOXM1 regulatory axis driving high-risk prognosis, as a novel therapeutic target against chr1q-amp in cancer.

category: cancer biology


title: Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression

abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus which causes significant economic losses to the swine industry worldwide. Here, we use ribosome profiling (RiboSeq) and parallel RNA sequencing (RNASeq) to characterise the transcriptome and translatome of both species of PRRSV and analyse the host response to infection. We quantified viral gene expression over a timecourse of infection, and calculated the efficiency of programmed ribosomal frameshifting (PRF) at both sites on the viral genome. At the nsp2 frameshift site (a rare example of protein-stimulated frameshifting), -2 PRF efficiency increases over time, likely facilitated by accumulation of the PRF- stimulatory viral protein (nsp1{beta}) during infection. This marks arteriviruses as the second example of temporally regulated PRF. Surprisingly, we also found PRF efficiency at the canonical ORF1ab frameshift site increases over time, in apparent contradiction of the common assumption that RNA structure-directed frameshift sites operate at a fixed efficiency. This has potential implications for the numerous other viruses with canonical PRF sites. Furthermore, we discovered several highly translated additional viral ORFs, the translation of which may be facilitated by multiple novel viral transcripts. For example, we found a 125-codon ORF overlapping nsp12, which is expressed as highly as nsp12 itself at late stages of replication, and is likely translated from novel subgenomic (sg) RNA transcripts that overlap the 3' end of ORF1b. Similar transcripts were discovered for both PRRSV-1 and PRRSV- 2, suggesting a potential conserved mechanism for temporal regulation of expression of the 3'-proximal region of ORF1b. In addition, we identified a highly translated, short upstream ORF (uORF) in the 5' UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. This is the first application of RiboSeq to arterivirus-infected cells, and reveals new features which add to the complexity of gene expression programmes in this important family of nidoviruses.

category: pathology


title: Remodeling of dermal adipose tissue alleviates cutaneous toxicity induced by anti-EGFR therapy

abstract:
Anti-epidermal growth factor receptor (EGFR) therapy-associated cutaneous toxicity is a syndrome characterized by papulopustular rash, local inflammation, folliculitis and microbial infection, resulting in a decrease in quality of life and dose interruption. However, no effective clinical intervention is available for this adverse effect. Here, we report the atrophy of dermal white adipose tissue (dWAT), a highly plastic adipose tissue with various skin-specific functions, correlates with rash occurrence and exacerbation in a murine model of EGFR inhibitor-induced rash. The reduction in dWAT is due to the inhibition of adipogenic differentiation by defects in peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) signaling, and increased lipolysis by the induced expression of the lipolytic cytokine IL6. The activation of PPAR{gamma} by rosiglitazone maintains adipogenic differentiation and represses the transcription of IL6, eventually improving skin functions and ameliorating the severity of rash without altering the antitumor effects. Thus, activation of PPAR{gamma} represents a promising approach to ameliorate cutaneous toxicity in patients with cancer who receive anti-EGFR therapy.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: Cortical microtubule pulling forces contribute to the union of the parental genomes in the C. elegans zygote

abstract: Previously, we reported that the Polo-like kinase PLK-1 phosphorylates the single C. elegans lamin (LMN-1) to trigger lamina depolymerization during mitosis. We showed that this event is required for the formation of a pronuclear envelopes scission event that removes membranes on the juxtaposed oocyte and sperm pronuclear envelopes in the zygote, allowing the parental chromosomes to merge in a single nucleus after segregation (Velez-Aguilera, 2020). Here we show that cortical microtubule pulling forces contribute to pronuclear envelopes scission by promoting mitotic spindle elongation. We also demonstrate that weakening of the pronuclear envelopes, via PLK-1-mediated lamina depolymerization, is a prerequisite for the astral microtubule pulling forces to trigger pronuclear membranes scission. Finally, we provide evidence that PLK-1 mainly acts via lamina depolymerization in this process. These observations thus indicate that temporal coordination between lamina depolymerization and mitotic spindle elongation facilitates pronuclear envelopes scission and parental genomes unification.

category: cell biology


title: When less is more - Endogenous tagging with TurboID increases the sensitivity of proximity labelling-based experiments

abstract: In recent years, proximity labelling has established itself as an unbiased and powerful approach to map the interactome of specific proteins. Generally, protein fusions with labelling enzymes are transiently overexpressed to perform these experiments. Using a pipeline for the rapid generation CRISPR-Cas9 knock-ins (KIs) based on antibiotic selection, we were able to compare the performance of commonly used labelling enzymes when endogenously expressed. We found TurboID and its shorter variant miniTurboID to be superior above other labelling enzymes at physiological expression levels. Endogenous tagging of the  subunit of the AP-1 complex increased the sensitivity for detection of interactors in a proximity labelling experiment and resulted in a more comprehensive mass spectrometry data set. We were able to identify several known interactors of the complex and cargo proteins that simple overexpression of a labelling enzyme fusion protein could not reveal. Our approach greatly simplifies the execution of proximity labelling experiments for proteins in their native cellular environment and allows going from CRISPR transfection to mass spectrometry data in just over a month.


title: Revisiting the effect of pharmaceuticals on transmission stage formation in the malaria parasite Plasmodium falciparum

abstract: Malaria parasites rely on specialized stages, called gametocytes, to ensure human-to-human transmission. The formation of these sexual precursor cells is initiated by commitment of blood stage parasites to the sexual differentiation pathway. Plasmodium falciparum, the most virulent of six parasite species infecting humans, employs nutrient sensing to control the rate at which sexual commitment is initiated, and the presence of stress-inducing factors, including antimalarial drugs, has been linked to increased gametocyte production in vitro and in vivo. These observations suggest that therapeutic interventions may promote gametocytogenesis and malaria transmission. Here, we engineered a P. falciparum reporter line to quantify sexual commitment rates after exposure to antimalarials and other pharmaceuticals commonly prescribed in malaria-endemic regions. Our data reveal that some of the tested drugs indeed have the capacity to elevate sexual commitment rates in vitro. Importantly, however, these effects are only observed at drug concentrations that inhibit parasite survival and only rarely result in a net increase of gametocyte production. Using a drug-resistant parasite reporter line, we further show that the gametocytogenesis-promoting effect of drugs is linked to general stress responses rather than to compound-specific activities. Altogether, we provide conclusive evidence for the absence of mechanistic links between the regulation of sexual commitment and the activity of commonly used pharmaceuticals in vitro. Our data hence contradict scenarios in which therapeutic interventions would promote the spread of drug-resistant parasites or malaria transmission in general.


title: AMPK promotes Arf6 activation in a kinase-independent manner upon energy deprivation

abstract: AMP-activated protein kinase (AMPK) is a crucial cellular nutrient and energy sensor that maintains energy homeostasis. AMPK also governs cancer cell invasion and migration by regulating gene expression and activating multiple cellular signaling pathways. ADP-ribosylation factor 6 (Arf6) can be activated via nucleotide exchange by guanine nucleotide exchange factors (GEFs), and its activation also regulates tumor invasion and migration. By studying GEF-mediated Arf6 activation, we elucidated that AMPK functions as a noncanonical GEF for Arf6 in a kinase-independent manner. Moreover, by examining the physiological role of the AMPK-Arf6 axis, we determined that AMPK activates Arf6 upon glucose starvation and 5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside (AICAR) treatment. We further identified the binding motif in the C-terminal regulatory domain of AMPK that is responsible for promoting Arf6 activation and thus inducing cell migration and invasion. These findings reveal a noncanonical role of AMPK in which its C-terminal regulatory domain serves as a GEF for Arf6 during energy deprivation.


title: Vitronectin acts as a key regulator of adhesion and migration in umbilical cord-derived MSCs under different stress conditions

abstract: Mesenchymal stem cell (MSC)-based therapy gets compromised as adverse microenvironmental conditions like nutrient deprivation, ischemia, hypoxia at the target site affect migration, engraftment and viability, of MSCs post transplantation. To improve the treatment efficacy, it is critical to identify factors involved in regulating migration and adhesion of MSCs under such microenvironmental stress conditions. In our study, we observed that human Whartons jelly-derived MSCs (WJ-MSCs) exhibited increase in cell spread area and adhesion, with reduction in cellular migration under serum starvation stress. The changes in adhesion and migration characteristics were accompanied by formation of large number of super mature focal adhesions along with extensive stress fibres and altered ECM gene expression with notable induction in vitronectin (VTN) expression. NF-{kappa}{beta} was found to be a positive regulator of VTN expression while ERK pathway regulated it negatively. Molecular and phenotypic comparison studies with inhibition of these signalling pathways or knocking down of VTN under serum starvation established the correlation between increase in VTN expression and increased cellular adhesion with corresponding reduction in cell migration. VTN knockdown also resulted in reduction of super mature focal adhesions and extensive stress fibres, which were formed under serum starvation. Additionally, VTN induction was not detected in hypoxia-treated WJ-MSCs, and the MSCs showed no significant change in the adhesion or migration properties under hypoxia. However, when VTN expression was induced under hypoxia by ERK pathway inhibition, similar increase in cell spread area and adhesion were observed. Our study thus highlights VTN as a key player which is induced under serum starvation stress and possibly regulates the adhesion and migration properties of WJ-MSCs via focal adhesion signalling.


title: Vitronectin acts as a key regulator of adhesion and migration in human umbilical cord-derived MSCs under different stress conditions


title: Human Cytomegalovirus modifies placental small extracellular vesicle secretion and composition towards a proviral phenotype to enhance infection of fetal recipient cells

abstract:
Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during human cytomegalovirus (hCMV) congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of hCMV infection on sEVs production, composition and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from either an ex vivo model of infected histocultures from early placenta or from the amniotic fluid of patients naturally infected by hCMV at the beginning of pregnancy. Based on these findings, we propose that placental sEVs could be key actors favoring viral dissemination to the fetal brain during hCMV congenital infection.

Significance StatementHuman cytomegalovirus (hCMV) infection is a major issue during pregnancy, affecting 1% of births in western countries. Despite extensive research, the pathophysiology of this congenital infection remains unclear. Recently, increasing evidence point to the key role of placental small extracellular vesicles (sEVs) in materno-fetal communication during pregnancy. Here, we examined the impact of hCMV infection on the protein composition and function of placental sEVs. We observe that hCMV infection leads to major changes in placental sEV protein content. Functional studies show the ability of sEVs produced by placental infected cells to facilitate further infection of naive recipient fetal cells, notably human neural stem cells. Our study demonstrates that placental sEVs are key players of hCMV pathophysiology during congenital infection.


title: Common variation in EDN1 regulatory regions highlights the role of PPARγ as a key regulator of Endothelin in vitro

abstract:
Pulmonary Arterial Hypertension (PAH) is a rare disease caused by the obliteration of the pulmonary arterioles, increasing pulmonary vascular resistance and eventually causing right heart failure. Endothelin-1 is a vasoconstrictor peptide whose levels are indicators of disease progression and its pathway is one of the commonest targeted by current treatments.

We sequenced the EDN1 untranslated regions of a small subset of PAH patients, predicted the effect in silico, and used a luciferase assay with the different genotypes to analyze its influence on gene expression. Finally, we used siRNAs against the major transcription factors predicted for these regions (PPAR{gamma}, KLF4, and VDR) to assess Endothelin-1 expression in cell culture and validate the binding sites.

First, we detected a SNP in the 5UTR (rs397751713) and another in the 3regulatory region (rs2859338) that increased luciferase activity in vitro depending on their genotype. We determined in silico that KLF4/PPAR{gamma} could be binding in the rs397751713 and VDR in rs2859338. By using siRNAs and luciferase, we determined that PPAR{gamma} binds differentially in rs397751713. PPAR{gamma} and VDR Knock-Down, increased EDN1 mRNA levels and Endothelin-1 production in PAECs, while PPAR{gamma} and KLF4 Knock-Down increased the Endothelin-1 production in HeLa.

In conclusion, common variants in EDN1 regulatory regions could alter Endothelin-1 levels. We were able to validate that PPAR{gamma} binds in rs397751713 and is key to regulate Endothelin-1. Also, KLF4 and VDR regulate Endothelin-1 production in a cell-dependent manner, but for VDR this interaction does not happen by binding directly to the regions we studied.


title: LiveCellMiner: A New Tool to Analyze Mitotic Progression

abstract: Live-cell imaging has become state of the art to accurately identify the nature of mitotic and cell cycle defects. Low- and high-throughput microscopy setups have yield huge data amounts of cells recorded in different experimental and pathological conditions. Tailored semi-automated and automated image analysis approaches allow the analysis of high-content screening data sets, saving time and avoiding bias. However, they were mostly designed for very specific experimental setups, which restricts their flexibility and usability. The general need for dedicated experiment-specific user-annotated training sets and experiment-specific user-defined segmentation parameters remains a major bottleneck for fully automating the analysis process. In this work we present LiveCellMiner, a highly flexible open-source software tool to automatically extract, analyze and visualize both aggregated and time-resolved image features with potential biological relevance. The software tool allows analysis across high-content data sets obtained in different platforms, in a quantitative and unbiased manner. As proof of principle application, we analyze here the dynamic chromatin and tubulin cytoskeleton features in human cells passing through mitosis highlighting the versatile and flexible potential of this tool set.


title: Methylglyoxal Induces Endothelial Dysfunction via Stunning

abstract: Elevated levels of methylglyoxal (MG) and its associated post-translational modifications have been reported to be associated with progression and development of numerous pathological conditions. Despite such extensive evidence, it still remains unclear what the specific effects of MG are other than that induction of cytotoxicity. Here we evaluated the effects of MG in cardiac endothelial cells in vitro. We found that MG leads to a non-proliferative state and endothelial dysfunction, which is reversible as MG-H1, a major post-translational modification induced by MG, is turned over by lysosomal degradation. MG-induced cellular stunning/paralysis describes a new hallmark for cellular dysfunction which could lead to alterations in tissue homeostasis as well as cell-to-cell interactions, thereby contributing to the pathogenesis of diseases.


title: Robust temporal map of human in vitro myelopoiesis using single-cell genomics

abstract: Myeloid cells have a central role in homeostasis and tissue defence. Characterising the current in vitro protocols of myelopoiesis is imperative for their use in research and immunotherapy as well as for understanding the early stages of myeloid differentiation in humans. Here, we profiled the transcriptome of more than 400k cells and generated a robust molecular map of the differentiation of human induced pluripotent stem cells (iPSC) into macrophages. By integrating our in vitro datasets with in vivo single-cell developmental atlases, we found that in vitro macrophage differentiation recapitulates features of in vivo yolk sac hematopoiesis, which happens prior to the appearance of definitive hematopoietic stem cells (HSC). During in vitro myelopoiesis, a wide range of myeloid cells are generated, including erythrocytes, mast cells and monocytes, suggesting that, during early human development, the HSC-independent immune wave gives rise to multiple myeloid cell lineages. We leveraged this model to characterize the transition of hemogenic endothelium into myeloid cells, uncovering poorly described myeloid progenitors and regulatory programs. Taking advantage of the variety of myeloid cells produced, we developed a new protocol to produce type 2 conventional dendritic cells (cDC2) in vitro. We found that the underlying regulatory networks coding for myeloid identity are conserved in vivo and in vitro. Using genetic engineering techniques, we validated the effects of key transcription factors important for cDC2 and macrophage identity and ontogeny. This roadmap of early myeloid differentiation will serve as an important resource for investigating the initial stages of hematopoiesis, which are largely unexplored in humans, and will open up new therapeutic opportunities.

category: developmental biology


title: Esrrγa regulates nephron development and ciliogenesis by controlling prostaglandin synthesis and cooperation with Ppargc1a

abstract: Cilia are essential for the ontogeny and function of many tissues, including the kidney. In mammals, Esrr{gamma} has been previously established as a significant determinant of renal health, with decreased expression linked to age related dysfunction, cyst formation, and kidney disease. Here, we report that the Esrr{gamma} vertebrate ortholog estrogen related receptor gamma a (esrr{gamma}a) is essential for proper cell fate choice within kidney functional units (nephrons) as well as ciliogenesis. Deficiency of esrr{gamma}a resulted in nephrons with alterations in proximodistal segmentation and a decreased multiciliated epithelial cell populace. Surprisingly, esrr{gamma}a deficiency disrupted renal ciliogenesis and caused a similar abrogation within the developing node and otic vesicle--all defects that occurred independently of changes in cell polarity or basal body organization. These phenotypes were consistent with interruptions in prostaglandin signaling, and we found that ciliogenesis was rescued in esrr{gamma}a deficient embryos with exogenous PGE2 or through overexpression of the cyclooxygenase enzyme Ptgs1. Through genetic interaction studies, we found that peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (ppargc1a), which acts upstream of Ptgs1-mediated prostaglandin synthesis, has a synergistic relationship with esrr{gamma}a in the ciliogenic pathway. These data position esrr{gamma}a as a novel link between ciliogenesis and nephrogenesis through regulation of prostaglandin signaling and cooperation with ppargc1a, and highlight esrr{gamma}a as a potential new target for future ciliopathic treatments.


title: How do avian embryos resume development following diapause? A new role for TGF-β in regulating pluripotency-related genes

abstract: Avian embryos can halt their development for long periods at low temperature in a process called diapause and successfully resume development when reincubated at maternal body temperature. Successful resumption of development depends on different factors, including temperature. We have recently shown that embryos that enter diapause at 18 {degrees}C present a significant reduction in their ability to develop normally when put back into incubation, compared to embryos entering diapause at 12 {degrees}C. However, the mechanisms underlying these differences are unknown. To address this question, transcriptome analysis was performed to compare the effect of diapause temperature on gene expression, and to identify pathways involved in the process. Genetic comparison and pathway-enrichment analysis revealed that TGF-{beta} and pluripotency-related pathways are differentially regulated at the two temperatures, with higher expression at 12 {degrees}C compared to 18 {degrees}C. Investigating the involvement of the TGF-{beta} pathway revealed an essential role for BMP4 in regulating the expression of the transcription factors Nanog and Id2, which are known to regulate pluripotency and self-renewal in embryonic stem cells. BMP4 gain- and loss-of-function experiments in embryos in diapause at the different temperatures revealed the main role of BMP4 in enabling resumption of normal development following diapause. Collectively, these findings identify molecular regulators that facilitate embryos ability to undergo diapause at different temperatures and resume a normal developmental program.


title: Landscape structure and dispersal rate drive large scale catastrophic shifts in spatially explicit metapopulations

abstract: Even when environments deteriorate gradually, ecosystems may shift abruptly from one state to another. Such catastrophic shifts are difficult to predict and reverse (hysteresis). While well studied in simplified contexts, we lack a general understanding of how catastrophic shifts spread in realistic spatial contexts. For different types of landscape structure, including typical terrestrial modular and riverine dendritic networks, we here investigate landscape-scale stability in metapopulations made of bistable patches. We find that such metapopulations usually exhibit large scale catastrophic shifts and hysteresis, and that the properties of these shifts depend strongly on metapopulation spatial structure and dispersal rate: intermediate dispersal rates and a riverine spatial structure can largely reduce hysteresis size. Interestingly, our study suggests that large-scale restoration is easier with spatially clustered restoration efforts and in populations characterized by an intermediate dispersal rate.

category: ecology


title: Edaphic controls on genome size and GC content of bacteria in soil microbial communities

abstract: Genomic traits, such as genome size, GC content, codon usage, and amino acid content, shed insight into the evolutionary processes of bacteria and selective forces behind microbial community composition. Nutrient limitation has been shown to reduce bacterial genome size and influence nucleotide composition, yet little research has been conducted in the soil environment, and the factors which shape soil bacterial genomic traits remain largely unknown. Here we determined average genome size, GC content, codon usage, and amino acid content from 398 soil metagenomes across the United States from the National Ecological Observation Network (NEON) and observed the distribution of these traits across numerous environmental gradients. We found that genomic trait averages were most strongly related to pH, which we suggest results in both physiological constraints on growth as well as affects availability of nutrients in soil. Low pH soils had higher carbon to nitrogen ratios (C:N) and tended to have communities with larger genomes and lower GC-content, potentially a result of increased physiological stress and increased metabolic diversity. Conversely, smaller genomes with high GC content were associated with high pH and low soil carbon to nitrogen ratios, indicating potential resource driven selection against carbon-rich AT base pairs. We found that this relationship of nutrient conservation also applied to amino acid stoichiometry, where bacteria in soils with C:N ratios tended to code for amino acids with lower C:N. Together, these relationships point towards fundamental mechanisms which underpin nucleotide and amino acid selection in soil bacterial communities.


title: The relationship between land cover and microbial community composition in European lakes

abstract: Microbes such as bacteria, archaea, and protists are essential for element cycling and ecosystem functioning, but many questions central to the understanding of the role of microbes in ecology are still open. Here, we analyze the relationship between lake microbiomes and the land cover surrounding the lakes. By applying machine learning methods, we quantify the covariance between land cover categories and the microbial community composition recorded in the largest amplicon sequencing dataset of European lakes available to date. We identify microbial bioindicators for these land cover categories. Combining land cover and physico-chemical bioindicators identified from the same amplicon sequencing dataset, we develop two novel similarity metrics that facilitate insights into the ecology of the lake microbiome. We show that the bioindicator network, i.e., the graph linking OTUs indicative of the same environmental parameters, corresponds to microbial co-occurrence patterns. Taken together, we demonstrate the strength of machine learning approaches to identify correlations between microbial diversity and environmental factors, potentially opening new approaches to integrate environmental molecular diversity into monitoring and water quality assessments.


title: Quantifying asymmetry in non-symmetrical morphologies, with an example from Cetacea

abstract:
1. Three-dimensional measurements of morphology are key to gaining an understanding of a species biology and to answering subsequent questions regarding the processes of ecology (or palaeoecology), function, and evolution. However, the collection of morphometric data is often focused on methods designed to produce data on bilaterally symmetric morphologies which may mischaracterise asymmetric structures.

2. Using 3D landmark and curve data on 3D surface meshes of specimens, we present a method for first quantifying the level of asymmetry in a specimen and second, accurately capturing the morphology of asymmetric specimens for further geometric analyses.

3. We provide an example of the process from initial landmark placement, including details on how to place landmarks to quantify the level of asymmetry, and then on how to use this information to accurately capture the morphology of asymmetric morphologies or structures. We use toothed whales (odontocetes) as a case study and include examples of the consequences of mirroring landmarks and curves, a method commonly used in bilaterally symmetrical specimens, on asymmetric specimens.

4. We conclude by presenting a step-by-step method to collecting 3D landmark data on asymmetric specimens. Additionally, we provide code for placing landmarks and curves on asymmetric specimens in a manner designed to both save time and ultimately accurately quantify morphology. This method can be used as a first crucial step in morphometric analyses of any biological specimens by assessing levels of asymmetry and then if required, accurately quantifying this asymmetry. The latter not only saves the researcher time, but also accurately represents the morphology of asymmetric structures.


title: Impacts of Taxon-Sampling Schemes on Bayesian Molecular Dating under the Unresolved Fossilized Birth-Death Process

abstract: Evolutionary timescales can be estimated using a combination of genetic data and fossil evidence based on the molecular clock. Bayesian phylogenetic methods such as tip dating and total-evidence dating provide a powerful framework for inferring evolutionary timescales, but the most widely used priors for tree topologies and node times often assume that present-day taxa have been sampled randomly or exhaustively. In practice, taxon sampling is often carried out so as to include representatives of major lineages, such as orders or families. We examined the impacts of these diversified sampling schemes on Bayesian molecular dating under the unresolved fossilized birth-death (FBD) process, in which fossil taxa are topologically constrained but their exact placements are not inferred. We used synthetic data generated by simulation of nucleotide sequence evolution, fossil occurrences, and diversified taxon sampling. Our analyses show that increasing sampling density does not substantially improve divergence-time estimates under benign conditions. However, when the tree topologies were fixed to those used for simulation or when evolutionary rates varied among lineages, the performance of Bayesian tip dating improves with sampling density. By exploring three situations of model mismatches, we find that including all relevant fossils without pruning off those inappropriate for the FBD process can lead to underestimation of divergence times. Our reanalysis of a eutherian mammal data set confirms some of the findings from our simulation study, and reveals the complexity of diversified taxon sampling in phylogenomic data sets. In highlighting the interplay of taxon-sampling density and other factors, the results of our study have useful implications for Bayesian molecular dating in the era of phylogenomics.

category: evolutionary biology


title: Phylogenomic analyses using genomes and transcriptomes do not resolve relationships among major clades in Phrymaceae

abstract:
Premise of the studyPhylogenomic datasets using genomes and transcriptomes provide rich opportunities beyond resolving bifurcating phylogenetic relationships. Monkeyflower (Phrymaceae) is a model system for evolutionary ecology. However, it lacks a well-supported phylogeny for a stable taxonomy and for macroevolutionary comparisons.

MethodsWe sampled 24 genomes and transcriptomes in Phrymaceae and closely related families, including eight newly sequenced transcriptomes. We reconstructed the phylogeny using IQ-TREE and ASTRAL, evaluated gene tree discordance using PhyParts, Quartet Sampling, and cloudogram, and carried out phylogenetic network analyses using PhyloNet and HyDe. We searched for whole genome duplication (WGD) events using chromosome numbers, synonymous distance, and gene duplication events.

Key resultsMost gene trees support the monophyly of Phrymaceae and each of its tribes. Most gene trees also support the tribe Mimuleae being sister to Phrymeae + Diplaceae + Leucocarpeae, with extensive gene tree discordance among the latter three. Despite the discordance, polyphyly of Mimulus s.l. is strongly supported, and no particular reticulation event among the Phrymaceae tribes is well supported. Reticulation likely occurred among Erythranthe bicolor and close relatives. No ancient WGD event was detected in Phrymaceae. Instead, small-scale duplications are among potential drivers of macroevolutionary diversification of Phrymaceae.

ConclusionsWe show that analysis of reticulate evolution is sensitive to taxon sampling and methods used. We also demonstrate that genome-scale data do not always fully "resolve" phylogenetic relationships. They present rich opportunities to investigate reticulate evolution, and gene and genome evolution involved in lineage diversification and adaptation.


title: Genetic and antigenic characterization of an expanding H3 influenza A virus clade in US swine visualized by Nextstrain

abstract:
Defining factors that influence spatial and temporal patterns of influenza A virus (IAV) is essential to inform vaccine strain selection and strategies to reduce the spread of potentially zoonotic swine-origin IAV. The relative frequency of detection of the H3 phylogenetic clade 1990.4.a (colloquially known as C-IVA) in US swine declined to 7% in 2017, but increased to 32% in 2019. We conducted phylogenetic and phenotypic analyses to determine putative mechanisms associated with increased detection. We created an implementation of Nextstrain to visualize the emergence, spatial spread, and genetic evolution of H3 IAV-S, identifying two C-IVA clades that emerged in 2017 and cocirculated in multiple US states. Phylodynamic analysis of the HA gene documented low relative genetic diversity from 2017 to 2019, suggesting clonal expansion. The major H3 C-IVA clade contained an N156H amino acid substitution, but HI assays demonstrated no significant antigenic drift. The minor HA clade was paired with the NA clade N2-2002B prior to 2016, but acquired and maintained N2-2002A in 2016, resulting in a loss in antigenic cross-reactivity between N2-2002B and -2002A containing H3N2 strains. The major C-IVA clade viruses acquired a nucleoprotein (NP) of the H1N1pdm09 lineage through reassortment in replacement of the North American swine lineage NP. Instead of genetic or antigenic diversity within the C-IVA HA, our data suggest that population immunity to H3 2010.1, along with antigenic diversity of the NA and acquisition of the H1N1pdm09 NP gene likely explain the re-emergence and transmission of C-IVA H3N2 in swine.

ImportanceGenetically distinct clades of influenza A virus (IAV) in swine undermines efforts to control the disease. Swine producers commonly use vaccines, and vaccine strains are selected by identifying the most common hemagglutinin (HA) gene from viruses detected in a farm or a region. In 2019, we identified an increase in detection frequency of an H3 phylogenetic clade, C-IVA, which was previously circulating at much lower levels in U.S. swine. Our study identified genetic and antigenic factors contributing to its resurgence by linking comprehensive phylodynamic analyses with empirical wet-lab experiments and visualized these evolutionary analyses in a Nextstrain implementation. The contemporary C-IVA HA genes did not demonstrate an increase in genetic diversity nor significant antigenic changes. N2 genes did demonstrate antigenic diversity, and the expanding C-IVA clade acquired a nucleoprotein (NP) gene segment via reassortment. Virus phenotype and vaccination targeting prior dominant HA clades likely contributed to the clades success.


title: Integrating transcriptomes to investigate genes associated with adaptation to aquatic environments, and assess phylogenetic conflict and whole-genome duplications in Alismatales

abstract: Land plants first evolved from freshwater algae, and flowering plants returned to water as early as the Cretaceous and multiple times subsequently. Alismatales is the largest clade of aquatic angiosperms including all marine angiosperms, as well as terrestrial plants. We used Alismatales to explore plant adaptation to aquatic environments by analyzing a data set that included 95 samples (89 Alismatales species) covering four genomes and 91 transcriptomes (59 generated in this study). To provide a basis for investigating adaptation, we assessed phylogenetic conflict and whole-genome duplication (WGD) events in Alismatales. We recovered a relationship for the three main clades in Alismatales as (Tofieldiaceae, Araceae) + core Alismatids. We also found phylogenetic conflict among the backbone of the three main clades that could be explained by incomplete lineage sorting and introgression. Overall, we identified 18 putative WGD events across Alismatales. One of them occurred at the most recent common ancestor of core Alismatids, and three occurred at seagrass lineages. We also found that lineage and life-form were both important for different evolutionary patterns for the genes related to freshwater and marine adaptation. For example, several light or ethylene-related genes were lost in the seagrass Zosteraceae, but are present in other seagrasses and freshwater species. Stomata-related genes were lost in both submersed freshwater species and seagrasses. Nicotianamine synthase genes, which are important in iron intake, expanded in both submersed freshwater species and seagrasses. Our results advance the understanding of the adaptation to aquatic environments and whole-genome duplications using phylogenomics.


title: Phylogenomic analyses of Alismatales shed light into adaptations to aquatic environments


title: Effect of RNA preservation methods on RNA quantity and quality of field collected avian whole blood

abstract:
A limitation of comparative transcriptomic studies of wild avian populations continues to be sample acquisition and preservation to achieve resulting high-quality RNA (i.e., ribonucleic acids that transfers, translates, and regulates the genetic code from DNA into proteins). Field sampling of wild bird samples provides challenges as RNA degradation progresses quickly and because cryopreservation is often not feasible at remote locations. We collected blood samples from songbirds, as avian blood is nucleated and provides sufficient transcriptionally active material in a small and non-lethal sample, to compare the efficacy of widely available RNA stabilizing buffers, RNAlater (Ambion) and DNA/RNA Shield (Zymo) at differing concentrations along with a dry ice-based flash freezing method (Isopropanol 99% and dry ice mixture, -109{degrees}C). Each blood sample was divided among five different preservation treatments (dry ice-based flash freezing, RNAlater with 1:5 or 1:10 dilution, or DNA/RNA Shield with 1:2 or 1:3 dilution). A new protocol was optimized for total RNA extraction from avian blood samples with small starting volumes enabling sampling of small passerines. We quantified quality measures, RNA integrity numbers (RINe), rRNA ratios, and total RNA concentrations. We found that RNA preservation buffers, RNAlater and DNA/RNA Shield at all concentrations, provide sample protection from RNA degradation. We suggest caution against using dry ice-based flash-freezing alone for samples preservation as these samples resulted in lower quality measures then samples in preservation buffer. Total RNA concentration was generally not affected by preservation treatment and may vary due to differences in initial samples volumes and carryover across processing steps.

LAY SUMMARYO_LIPreserving RNA samples collected in field conditions, under extreme or variable conditions, remains a challenge and continues to limit the study of gene expression in wild birds.
C_LIO_LIWe optimized RNA extractions for small-volume whole avian blood samples.
C_LIO_LIWe compared commonly used RNA preservations methods, commercial RNA buffers at varying concentrations along with a dry ice-based flash freezing method, for field collected blood bird samples.
C_LIO_LIAll RNA buffers preserved samples resulted in high quality RNA extractions suitable for downstream RNA sequencing, while dry ice-based flash freezing resulted in less reliable sample preservation.
C_LIO_LIOur results support the feasibility of blood sampling as a method of non-lethal sampling that will enable the increased usage RNA sequencing in wild bird studies.
C_LI

KEY TERMSO_ST_ABSRNA-SeqC_ST_ABShigh-throughput sequencing which profiles RNA molecules in a sample at the collection timepoint. Provides content and abundance information and may identify novel genes and isoforms. Prior to sequencing, library preparation may either target mRNA or total RNA with the use of enrichment or depletion steps.

Transcriptomeall RNA transcripts, both coding and non-coding, in an individual.

rRNA ratio (28S/18S)commonly used ratio of 28S and 18S ribosomal RNA (rRNA) (band sizes or peak area ratio) that informs the integrity of RNA. Appropriate ratios vary by taxonomic groups and associated rRNA sizes. Acceptable ratios of 28S to 18S are approximately 2.0 for eukaryotes.

Electropherograma quantitative distribution of fragment size.

Qubit 4 Fluorometerfluorescence-based quantification of RNA molecule concentration using fluorescent intercalating dyes which bind to target molecules.

Agilent 4200 TapeStationhigh-throughput analyzer which uses microfluidics, microcapillary electrophoresis, and fluorescence detection that allows for size determination of the isolated molecules. Used for quality control and quantification of DNA and RNA samples for downstream methods. Reports metrics to assess sample (DNA, RNA, protein) integrity and quantification, which includes: electropherogram, gel-like image, concentration values, 28S/18S rRNA ratios, and an algorithmically determined integrity score (RINe). Provides a more complete characterization of the measured samples then previous analyzers for downstream analysis.

Fast zonearea between small RNAs and the 18S rRNA fragment (~200 bp to 1.8 kb). The fast zone is where the degradation of the 18S and 28S peaks accumulates.

RINeRNA integrity number (RIN equivalent) which is determined by quantitative measurements of total RNA degradation. Measurements are based on features of the electropherogram which assesses degradation or lack of degradation by measuring the ratio of area in the fast zone to the 18S peak signal. RIN of 10 signifies the highest quality intact RNA and zero is the worst score indicates a low-quality degraded sample.


title: Monophyly of Diverse Bigyromonadea and their Impact on Phylogenomic Relationships Within Stramenopiles

abstract: Stramenopiles are a diverse but relatively well-studied eukaryotic supergroup with considerable genomic information available (Sibbald and Archibald, 2017). Nevertheless, the relationships between major stramenopile subgroups remain unresolved, in part due to a lack of data from small nanoflagellates that make up a lot of the genetic diversity of the group. This is most obvious in Bigyromonadea, which is one of four major stramenopile subgroups but represented by a single transcriptome. To examine the diversity of Bigyromonadea and how the lack of data affects the tree, we generated transcriptomes from seven novel bigyromonada species described in this study: Develocauda condao, Develocanicus komovi, Develocanicus vyazemskyi, Cubaremonas variflagellatum, Pirsonia chemainus, Feodosia pseudopoda, and Koktebelia satura. Both maximum likelihood and Bayesian phylogenomic trees based on a 247 gene-matrix recovered a monophyletic Bigyromonadea that includes two diverse subgroups, Developea and Pirsoniales, that were not previously related based on single gene trees. Maximum likelihood analyses show Bigyromonadea related to oomycetes, whereas Bayesian analyses and topology testing were inconclusive. We observed similarities between the novel bigyromonad species and motile zoospores of oomycetes in morphology and the ability to self- aggregate. Rare formation of pseudopods and fused cells were also observed, traits that are also found in members of labyrinthulomycetes, another osmotrophic stramenopiles. Furthermore, we report the first case of eukaryovory in the flagellated stages of Pirsoniales. These analyses reveal new diversity of Bigyromonadea, and altogether suggest their monophyly with oomycetes, collectively known as Pseudofungi, is the most likely topology of the stramenopile tree.


title: A new lineage of non-photosynthetic green algae with extreme organellar genomes

abstract:
BackgroundThe plastid genomes of the green algal order Chlamydomonadales tend to expand their non-coding regions, but this phenomenon is poorly understood. Here we shed new light on organellar genome evolution in Chlamydomonadales by studying a previously unknown non-photosynthetic lineage. We established cultures of two new Polytoma-like flagellates, defined their basic characteristics and phylogenetic position, and obtained complete organellar genome sequences and a transcriptome assembly for one of them.

ResultsWe discovered a novel deeply diverged chlamydomonadalean lineage that has no close photosynthetic relatives and represents an independent case of photosynthesis loss. To accommodate these organisms we establish the new genus Leontynka, with two species (L. pallida and L. elongata) distinguishable through both their morphological and molecular characteristics. Notable features of the colourless plastid of L. pallida deduced from the plastid genome (plastome) sequence and transcriptome assembly include the retention of ATP synthase, thylakoid-associated proteins, the carotenoid biosynthesis pathway, and a plastoquinone-based electron transport chain, the latter two modules having an obvious functional link to the eyespot present in Leontynka. Most strikingly, the ~362 kbp plastome of L. pallida is by far the largest among the non-photosynthetic eukaryotes investigated to date due to an extreme proliferation of sequence repeats. These repeats are also present in coding sequences, with one repeat type found in the exons of 11 out of 34 protein-coding genes, with up to 36 copies per gene, thus affecting the encoded proteins. The mitochondrial genome of L. pallida is likewise exceptionally large, with its >104 kbp surpassed only by the mitogenome of Haematococcus lacustris among all members of Chlamydomonadales hitherto studied. It is also bloated with repeats, though entirely different from those in the L. pallida plastome, which contrasts with the situation in H. lacustris where both the organellar genomes have accumulated related repeats. Furthermore, the L. pallida mitogenome exhibits an extremely high GC content in both coding and non-coding regions and, strikingly, a high number of predicted G-quadruplexes.

ConclusionsWith its unprecedented combination of plastid and mitochondrial genome characteristics, Leontynka pushes the frontiers of organellar genome diversity and is an interesting model for studying organellar genome evolution.


title: Trait Association and Prediction Through Integrative K-mer Analysis

abstract: Genome-wide association study (GWAS) with single nucleotide polymorphisms (SNPs) has been widely used to explore genetic controls of phenotypic traits. Here we employed an GWAS approach using k-mers, short substrings from sequencing reads. Using maize cob and kernel color traits, we demonstrated that k-mer GWAS can effectively identify associated k-mers. Co-expression analysis of kernel color k-mers and pathway genes directly found k-mers from causal genes. Analyzing complex traits of kernel oil and leaf angle resulted in k-mers from both known and candidate genes. Evolution analysis revealed most k-mers positively correlated with kernel oil were strongly selected against in maize populations, while most k-mers for upright leaf angle were positively selected. In addition, phenotypic prediction of kernel oil, leaf angle, and flowering time using k-mer data showed at least a similarly high prediction accuracy to the standard SNP-based method. Collectively, our results demonstrated the bridging role of k-mers for data integration and functional gene discovery.

category: genetics


title: Kinetic networks identify key regulatory nodes and transcription factor functions in early adipogenesis

abstract: Sequence-specific transcription factors (TFs) bind DNA, modulate chromatin structure, regulate gene expression, and or- chestrate transcription cascades. Activation and repression of TFs drive tightly controlled regulatory programs that lead to cellular processes such as differentiation. We measured chromatin accessibility and nascent transcription at seven time points over the first four hours of induced adipogenesis of 3T3-L1 mouse preadipocytes to construct dynamic gene regulatory networks. Regulatory networks describe successive waves of TF binding and dissociation followed by direct regulation of proximal genes. We identified 14 families of TFs that coordinate with and antagonize each other to regulate early adipogenesis. We developed a compartment model to quantify individual TF contributions to RNA polymerase initiation and pause release rates. Network analysis showed that the glucocorticoid receptor and AP1 drive immediate gene activation, including induction of Twist2. Twist2 is a highly interconnected node within the network and its expression leads to repression of target genes. Although Twist2s role in adipogenesis has not been previously appreciated, both Twist2 knockout mice and Setleis syndrome (Twist2-/-) patients lack subcutaneous and brown adipose tissue. We found that kinetic networks integrating chromatin structure and nascent transcription dynamics identify key genes, TF functions, and coordinate interactions within regulatory cascades.

category: genomics


title: Net charges of the ribosomal proteins of the S10 and spc clusters of halophiles are inversely related to the degree of halotolerance

abstract:
Net positive charge(s) on ribosomal proteins (r-proteins) have been reported to influence the assembly and folding of ribosomes. A high percentage of r-proteins from extremely halophilic archaea are known to be acidic or even negatively charged. Those proteins that remain positively charged are typically far less so. Herein the analysis is extended to the non-archaeal halophilic bacteria, eukaryotes and halotolerant archaea. The net charges (pH 7.4) of r-proteins that comprise the S10-spc operon/cluster from individual microbial and eukaryotic genomes were estimated and intercompared. It was observed that as a general rule, as the salt tolerance of the bacterial strains increased from 5 to 15%, the net charges of the individual proteins remained mostly basic. The most striking exceptions were the extremely halophilic bacterial strains, Salinibacter ruber SD01, Acetohalobium arabaticum DSM 5501 and Selenihalanaerobacter shriftii ATCC BAA-73, which are reported to require a minimum of 18%-21% of salt for their growth. All three strains have a higher number of acidic S10-spc cluster r-proteins than what is seen in the moderate halophiles or the halotolerant strains. Of the individual proteins, only uL2 never became acidic. uS14 and uL16 also seldom became acidic. The net negative charges on several of the S10-spc cluster r-proteins is a feature generally shared by all extremely halophilic archaea and bacteria. The S10-spc cluster r-proteins of halophilic fungi and algae (eukaryotes) were exceptions. They were positively charged despite the halophilicity of the organisms.

ImportanceThe net charges (at pH 7.4) of the ribosomal proteins (r-proteins) that comprise the S10-spc cluster show an inverse relationship with the halophilicity/halotolerance levels in both bacteria and archaea. In non-halophilic bacteria, the S10-spc cluster r-proteins are generally basic (positively charged), while the rest of the proteomes in these strains are generally acidic. On the other hand, the whole proteomes of the extremely halophilic strains are overall negatively charged including the S10-spc cluster r-proteins. Given that the distribution of charged residues in the ribosome exit tunnel influences co-translational folding, the contrasting charges observed in the S10-spc cluster r-proteins has potential implications for the rate of passage of these proteins through the ribosomal exit tunnel. Furthermore, the universal protein uL2 which lies in the oldest part of the ribosome is always positively charged irrespective of the strain/organism it belongs to. This has implications for its role in the prebiotic context.


title: The natural adaptation and human selection history of African sheep genomes

abstract: African sheep manifest diverse but distinct physio-anatomical traits which are the outcomes of natural- and human-driven selection. Here, we generated 34.8 million variants from 150 indigenous African sheep genomes sequenced at an average depth of [~]54x for 130 samples (Ethiopia, Libya) and [~]10x for 20 samples (Sudan), representing sheep from diverse environments, tail morphology and post-Neolithic introductions to Africa. Phylogenetic and model-based admixture analysis provided evidence of four genetic groups that correspond to altitudinal geographic origins and tail morphotypes. Comparative genomic analysis identified targets of selection spanning conserved haplotype structures overlapping genes and gene families relating to hypoxia responses, caudal vertebrae and tail skeleton length, ear morphology, and tail fat-depot structure. Our findings provide novel insights underpinning variation and response to human selection and environmental adaptation, and possible pleiotropic gene interactions in indigenous African sheep genomes, which guaranteed the successful establishment of the species on the continent.


title: Morpholino-mediated knockdown of the brain mineralocorticoid receptor affects glucocorticoid signaling and neuroplasticity in wild ocellated wrasse (Symphodus ocellatus)

abstract: Uncovering the genetic, physiological, and developmental mechanisms underlying phenotypic variation is necessary for understanding how genetic and genomic variation shape phenotypic variation and for discovering possible targets of selection. Although the neural and endocrine mechanisms underlying social behavior are evolutionarily ancient, we lack an understanding of the proximate causes and evolutionary consequences of variation in these mechanisms. Here, we examine in the natural environment the behavioral, neuromolecular, and fitness consequences of a morpholino-mediated knockdown of the mineralocorticoid receptor (MR) in the brain of nesting males of the ocellated wrasse, Symphodus ocellatus, a species with male alternative reproductive tactics. Even though MR knockdown did not significantly change male behavior directly, this experimental manipulation strongly altered glucocorticoid signaling and neuroplasticity in the preoptic area, the putative hippocampus homolog, and the putative basolateral amygdala homolog. We also found that individual variation in stress axis gene expression and neuroplasticity is strongly associated with variation in male behavior and fitness-related traits. The brain region-specific effects of MR knockdown on phenotypic integration in the wild reported here suggest specific neuroendocrine and neuroplasticity pathways that may be targets of selection.


title: A3D Database: Structure-based Protein Aggregation Predictions for the Human Proteome

abstract:
MotivationProtein aggregation is associated with highly debilitating human disorders and constitutes a major bottleneck for producing therapeutic proteins. Our knowledge of the human protein structures repertoire has dramatically increased with the recent development of the AlphaFold (AF) deep-learning method. This structural information can be used to understand better protein aggregation properties and the rational design of protein solubility. This article uses the Aggrescan3D (A3D) tool to compute the structure-based aggregation predictions for the human proteome and make the predictions available in a database form.

ResultsHere, we present the A3D Database, in which we analyze the AF-predicted human protein structures (for over 17 thousand non-membrane proteins) in terms of their aggregation properties using the A3D tool. Each entry of the A3D Database provides a detailed analysis of the structure-based aggregation propensity computed with A3D. The A3D Database implements simple but useful graphical tools for visualizing and interpreting protein structure datasets. We discuss case studies illustrating how the database could be used to analyze physiologically relevant proteins. Furthermore, the database enables testing the influence of user-selected mutations on protein solubility and stability, all integrated into a user-friendly interface.

Availability and implementationA3D Database is freely available at: http://biocomp.chem.uw.edu.pl/A3D2/hproteome


title: Characterizing disease-associated human proteins without available protein structures or homologues

abstract: Mutations in human proteins lead to diseases. The structure of these proteins can help understand the mechanism of such diseases and develop therapeutics against them. With improved deep learning techniques such as RoseTTAFold and AlphaFold, we can predict the structure of proteins even in the absence of structural homologues. We modeled and extracted the domains from 553 disease-associated human proteins without known protein structures or close homologues in the Protein Databank (PDB). We noticed that the model quality was higher and the RMSD lower between AlphaFold and RoseTTAFold models for domains that could be assigned to CATH families as compared to those which could only be assigned to Pfam families of unknown structure or could not be assigned to either. We predicted ligand-binding sites, protein-protein interfaces, conserved residues in these predicted structures. We then explored whether the disease-associated missense mutations were in the proximity of these predicted functional sites, if they destabilized the protein structure based on ddG calculations or if they were predicted to be pathogenic. We could explain 80% of these disease-associated mutations based on proximity to functional sites, structural destabilization or pathogenicity. When compared to polymorphisms a larger percentage of disease associated missense mutations were buried, closer to predicted functional sites, predicted as destabilising and/or pathogenic. Usage of models from the two state-of-the-art techniques provide better confidence in our predictions, and we explain 93 additional mutations based on RoseTTAFold models which could not be explained based solely on AlphaFold models.


title: Characterizing and explaining impact of disease-associated mutations in proteins without known structures or structural homologues


title: The evolution of genomic, transcriptomic, and single-cell protein markers of metastatic upper tract urothelial carcinoma

abstract: The molecular characteristics of metastatic upper tract urothelial carcinoma (UTUC) are unknown. The genomic and transcriptomic differences between primary and metastatic UTUC is not well described either. We combined whole-exome sequencing, RNA-sequencing, and Imaging Mass Cytometry (IMC) of 44 tumor samples from 28 patients with high-grade primary and metastatic UTUC. IMC enables spatially resolved single-cell analyses to examine the evolution of cancer cell, immune cell, and stromal cell markers using mass cytometry with lanthanide metal-conjugated antibodies. We discovered that actionable genomic alterations are frequently discordant between primary and metastatic UTUC tumors in the same patient. In contrast, molecular subtype membership and immune depletion signature were stable across primary and matched metastatic UTUC. Molecular and immune subtypes were consistent between bulk RNA-sequencing and mass cytometry of protein markers from 340,798 single-cells. Molecular subtyping at the single cell level was highly conserved between primary and metastatic UTUC tumors within the same patient.


title: Gene expression based inference of drug resistance in cancer

abstract: Inter and intra-tumoral heterogeneity are major stumbling blocks in the treatment of cancer and are responsible for imparting differential drug responses in cancer patients. Recently, the availability of large-scale drug screening datasets has provided an opportunity for predicting appropriate patient-tailored therapies by employing machine learning approaches. In this study, we report a predictive modeling approach to infer treatment response in cancers using gene expression data. In particular, we demonstrate the benefits of considering integrated chemogenomics approach, utilizing the molecular drug descriptors and pathway activity information as opposed to gene expression levels. We performed extensive validation of our approach on tissue-derived single-cell and bulk expression data. Further, we constructed several prostate cancer cell lines and xenografts, exposed to differential treatment conditions to assess the predictability of the outcomes. Our approach was further assessed on pan-cancer RNA-sequencing data from The Cancer Genome Atlas (TCGA) archives, as well as an independent clinical trial study describing the treatment journey of three melanoma patients. To summarise, we benchmarked the proposed approach on cancer RNA-seq data, obtained from cell lines, xenografts, as well as humans. We concluded that pathway-activity patterns in cancer cells are reasonably indicative of drug resistance, and therefore can be leveraged in personalized treatment recommendations.


title: Automated assembly scaffolding elevates a new tomato system for high-throughput genome editing

abstract: Advancing crop genomics requires efficient genetic systems enabled by high-quality personalized genome assemblies. Here, we introduce RagTag, a toolset for automating assembly scaffolding and patching, and we establish chromosome-scale reference genomes for the widely used tomato genotype M82 along with Sweet-100, a rapid-cycling genotype that we developed to accelerate functional genomics and genome editing. This work outlines strategies to rapidly expand genetic systems and genomic resources in other plant species.

category: plant biology