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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2017

A genetically encoded adhesin toolbox for programming multicellular morphologies and patterns

Synthetic multicellular systems hold promise for understanding natural development of biofilms and higher organisms1,2, as well as for engineering complex multi-component metabolic pathways2,3 and materials4. However, such efforts will require tools to adhere cells into defined morphologies and patterns, and these tools are currently lacking1,5,6. Here we report the first 100% genetically encoded synthetic platform for modular cell-cell adhesion in Escherichia coli, which provides control over multicellular self-assembly. Adhesive selectivity is provided by a library of outer membrane-displayed nanobody7,8 and antigen peptides with orthogonal intra-library specificities, while affinity is controlled by intrinsic adhesin affinity, competitive inhibition, and inducible expression. We demonstrate the resulting capabilities for rational design of well-defined morphologies and patterns through homophilic and heterophilic interactions, lattice-like self-assembly, phase separation, differential adhesion, and sequential layering. This adhesion toolbox, compatible with synthetic biology standards9, will enable construction of high-level multicellular designs and shed light on the evolutionary transition to multicellularity6,10.

synthetic biology↗

bioRxiv2018

Time-scale separation and stochasticity conspire to impact phenotypic dynamics in the canonical and inverted Bacillus subtilis core genetic regulation circuits.

In this work, we study two seemingly unrelated aspects of core genetic nonlinear dynamical control of the competence phenotype in Bacillus subtilis, a common Gram-positive bacterium living in the soil. We focus on hitherto unchartered aspects of the dynamics by exploring the effect of time scale separation between transcription and translation and, as well, the effect of intrinsic molecular stochasticity. We consider these aspects of regulatory control as two possible evolutionary handles. Hence, using theory and computations, we study how the onset of oscillations breaks the excitability-based competence phenotype in two topologically close evolutionary competing circuits: the canonical \"wild-type\" regulation circuit selected by Evolution and, the indirect-feedback inverted circuit that failed to be selected by Evolution, as was shown elsewhere, due to dynamical reasons.

systems biology↗

bioRxiv2018

A systematic review and meta-analysis of Drosophila short-term-memory genetics: robust reproducibility, but little independent replication

Geneticists use olfactory conditioning in Drosophila to identify learning genes; however, little is known about how these genes are integrated into short-term memory (STM) pathways. Here, we investigated the hypothesis that the STM evidence base is weak. We performed systematic review and meta-analysis of the field. Using metrics to quantify variation between discovery articles and follow-up studies, we found that seven genes were both highly replicated, and highly reproducible. However, ~80% of STM genes have never been replicated. While only a few studies investigated interactions, the reviewed genes could account for >1000% memory. This large summed effect size could indicate irreproducibility, many shared pathways, or that current assay protocols lack the specificity needed to identify core plasticity genes. Mechanistic theories of memory will require the convergence of evidence from system, circuit, cellular, molecular, and genetic experiments; systematic data synthesis is an essential tool for integrated neuroscience.

animal behavior and cognition↗

bioRxiv2018

The effect of education on adult mortality, health, and income: triangulating across genetic and policy reforms

1On average, educated people are healthier, wealthier and have higher life expectancy than those with less education. Numerous studies have attempted to determine whether these differences are caused by education, or are merely correlated with it and are ultimately caused by another factor. Previous studies have used a range of natural experiments to provide causal evidence. Here we exploit two natural experiments, perturbation of germline genetic variation associated with education which occurs at conception, known as Mendelian randomization, and a policy reform, the raising of the school leaving age in the UK in 1972. Previous studies have suggested that the differences in outcomes associated with education may be due to confounding. However, the two independent sources of variation we exploit largely imply consistent causal effects of education on outcomes much later in life.

epidemiology↗

bioRxiv2018

Developmental genetics of corolla tube formation: role of the tasiRNA-ARF pathway

More than 80,000 angiosperm species produce flowers with petals fused into a corolla tube. As an important element of the tremendous diversity of flower morphology, the corolla tube plays a critical role in many specialized interactions between plants and animal pollinators (e.g., beeflies, hawkmoths, hummingbirds, nectar bats), which in turn drives rapid plant speciation. Despite its clear significance in plant reproduction and evolution, the corolla tube remains one of the least understood plant structures from a developmental genetics perspective. Through mutant analyses and transgenic experiments, here we show that the tasiRNA-ARF pathway is required for corolla tube formation in the monkeyflower species Mimulus lewisii. Loss-of-function mutations in the M. lewisii orthologs of ARGONAUTE7 and SUPPRESSOR OF GENE SILENCING 3 cause a dramatic decrease in abundance of TAS3-derived small RNAs and a moderate up-regulation of AUXIN RESPONSE FACTOR 3 (ARF3) and ARF4, which lead to inhibition of lateral expansion of the bases of petal primordia and complete arrest of the upward growth of the inter-primordial regions, resulting in unfused corollas. By using an auxin reporter construct, we discovered that auxin distribution is continuous along the petal primordium base and the inter-primordial region during the critical stage of corolla tube formation in the wild-type, and that this auxin distribution is much weaker and more restricted in the mutant. Together, these results suggest a new conceptual model highlighting the central role of auxin directed synchronized growth of the petal primordium base and the inter-primordial region in corolla tube formation.

plant biology↗

bioRxiv2018

Reliability of Whole-Exome Sequencing for Assessing Intratumor Genetic Heterogeneity

Multi-region sequencing is used to detect intratumor genetic heterogeneity (ITGH) in tumors. To assess whether genuine ITGH can be distinguished from sequencing artifacts, we whole-exome sequenced (WES) three anatomically distinct regions of the same tumor with technical replicates to estimate technical noise. Somatic variants were detected with three different WES pipelines and subsequently validated by high-depth amplicon sequencing. The cancer-only pipeline was unreliable, with about 69% of the identified somatic variants being false positive. Even with matched normal DNA where 82% of the somatic variants were detected reliably, only 36%-78% were found consistently in technical replicate pairs. Overall 34%-80% of the discordant somatic variants, which could be interpreted as ITGH, were found to constitute technical noise. Excluding mutations affecting low mappability regions or occurring in certain mutational contexts was found to reduce artifacts, yet detection of subclonal mutations by WES in the absence of orthogonal validation remains unreliable.

genomics↗

bioRxiv2018

Genetics behind the biosynthesis of nonulosonic acid containing lipooligosaccharides in Campylobacter coli

Campylobacter jejuni and Campylobacter coli are the most common cause of bacterial gastroenteritis in the world. Ganglioside mimicry by C. jejuni lipooligosaccharide (LOS) is the triggering factor of Guillain-Barre syndrome (GBS), an acute polyneuropathy. Sialyltransferases from the glycosyltransferase (GT) family 42 are essential for the expression of ganglioside mimics in C. jejuni. Recently, two novel GT-42 genes, cstIV and cstV, have been identified in C. coli. Despite being present in [~]11% of currently available C. coli genomes, the biological role of cstIV and cstV is unknown. In the present study, mutation studies in two strains expressing either cstIV or cstV were performed and mass spectrometry was used to investigate differences in the chemical composition of LOS. Attempts were made to identify donor and acceptor molecules using in vitro activity tests with recombinant GT-42 enzymes. Here, we show that CstIV and CstV are involved in C. coli LOS biosynthesis. In particular, cstV is associated with LOS sialylation, while cstIV is linked to the addition of a diacetylated nonulosonic acid residue.\n\nIMPORTANCEDespite being a major foodborne pathogen, Campylobacter coli glycobiology has been largely neglected. The genetic makeup of the C. coli lipooligosaccharide biosynthesis locus was largely unknown until recently. C. coli harbour a large set of genes associated to lipooligosaccharide biosynthesis, including several putative glycosyltransferases involved in the synthesis of sialylated lipooligosaccharide in Campylobacter jejuni. In the present study, C. coli was found to express lipooligosaccharide structures containing sialic acid and other nonulosonate acids. These findings have a strong impact in understanding C. coli ecology, host-pathogen interaction, and pathogenesis.

microbiology↗

bioRxiv2018

Genetically encoded ratiometric indicators for potassium ion

Potassium ion (K+) homeostasis and dynamics play critical roles in regulating various biological activities, and the ability to monitor K+ spatial-temporal dynamics is critical to understanding these biological functions. Here we report the design and characterization of a Forster resonance energy transfer (FRET)-based genetically encoded K+ indicator, KIRIN1, constructed by inserting a bacterial cytosolic K+ binding protein (Kbp) between a fluorescent protein (FP) FRET pair, mCerulean3 and cp173Venus. Binding of K+ induces a conformational change in Kbp, resulting in an increase in FRET efficiency. KIRIN1 was able to detect K+ at physiologically relevant concentrations in vitro and is highly selective toward K+ over Na+. We further demonstrated that KIRIN1 allowed real-time imaging of pharmacologically induced depletion of cytosolic K+ in live cells, and KIRIN1 also enabled optical tracing of K+ efflux and reuptake in neurons upon glutamate stimulation in cultured primary neurons. These results demonstrate that KIRIN1 is a valuable tool to detect K+ in vitro and in live cells.

bioengineering↗

bioRxiv2018

A genetically encoded biosensor reveals location bias of opioid drug action

Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. One way that drugs can differ is by imposing selective effects on the conformational equilibrium of individual ORs. We wondered if drugs can also impose selective effects on the location of OR activation in individual OR-expressing neurons. Here we develop a genetically encoded biosensor that directly localizes ligand-induced activation and deactivation of ORs in living cells, and use it to generate the first real-time map of the spatiotemporal organization of - and {delta}-OR activation in neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by uniquely driving OR activation in the somatic Golgi apparatus and extending throughout the dendritic arbor in Golgi outposts. These results demonstrate a new approach to probe the cellular basis of neuromodulation and reveal that drugs profoundly distort the spatiotemporal landscape of neuronal OR activation.

neuroscience↗

bioRxiv2018

A complete nanonpore-only assembly of an XDR Mycobacterium tuberculosis Beijing lineage strain identifies novel genetic variation in repetitive PE/PPE gene regions

A better understanding of the genomic changes that facilitate the emergence and spread of drug resistant M. tuberculosis strains is required. Short-read sequencing methods have limited capacity to identify long, repetitive genomic regions and gene duplications. We sequenced an extensively drug resistant (XDR) Beijing sub-lineage 2.2.1.1 \"epidemic strain\" from the Western Province of Papua New Guinea using long-read sequencing (Oxford Nanopore MinION(R)). With up to 274 fold coverage from a single flow-cell, we assembled a 4404947bp circular genome containing 3670 coding sequences that include the highly repetitive PE/PPE genes. Comparison with Illumina reads indicated a base-level accuracy of 99.95%. Mutations known to confer drug resistance to first and second line drugs were identified and concurred with phenotypic resistance assays. We identified mutations in efflux pump genes (Rv0194), transporters (secA1, glnQ, uspA), cell wall biosynthesis genes (pdk, mmpL, fadD) and virulence genes (mce-gene family, mycp1) that may contribute to the drug resistance phenotype and successful transmission of this strain. Using the newly assembled genome as reference to map raw Illumina reads from representative M. tuberculosis lineages, we detect large insertions relative to the reference genome. We provide a fully annotated genome of a transmissible XDR M. tuberculosis strain from Papua New Guinea using Oxford Nanopore MinION sequencing and provide insight into genomic mechanisms of resistance and virulence.\n\nData SummaryO_LISample Illumina and MinION sequencing reads generated and analyzed are available in NCBI under project accession number PRJNA386696 (https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA386696)\nC_LIO_LIThe assembled complete genome and its annotations are available in NCBI under accession number CP022704.1 (https://www.ncbi.nlm.nih.gov/sra/?term=CP022704.1)\nC_LI\n\nImpact statementWe recently characterized a Modern Beijing lineage strain responsible for the drug resistance outbreaks in the Western province, Papua New Guinea. With some of the genomic markers responsible for its drug resistance and transmissibility are known, there is need to elucidate all molecular mechanisms that account for the resistance phenotype, virulence and transmission. Whole genome sequencing using short reads has widely been utilized to study MTB genome but it does not generally capture long repetitive regions as variants in these regions are eliminated using analysis. Illumina instruments are known to have a GC bias so that regions with high GC or AT rich are under sampled and this effect is exacerbated in MTB, which has approximately 65% GC content. In this study, we utilized Oxford Nanopore Technologies (ONT) MinION sequencing to assemble a high-quality complete genome of an extensively drug resistant strain of a modern Beijing lineage. We were able to able to assemble all PE/PPE (proline-glutamate/proline-proline-glutamate) gene families that have high GC content and repetitive in nature. We show the genomic utility of ONT in offering a more comprehensive understanding of genetic mechanisms that contribute to resistance, virulence and transmission. This is important for settings up predictive analytics platforms and services to support diagnostics and treatment.

genomics↗

bioRxiv2018

Biological and clinical insights from genetics of insomnia symptoms

Insomnia is a common disorder linked with adverse long-term medical and psychiatric outcomes, but underlying pathophysiological processes and causal relationships with disease are poorly understood. Here we identify 57 loci for self-reported insomnia symptoms in the UK Biobank (n=453,379) and confirm their impact on self-reported insomnia symptoms in the HUNT study (n=14,923 cases, 47,610 controls), physician diagnosed insomnia in Partners Biobank (n=2,217 cases, 14,240 controls), and accelerometer-derived measures of sleep efficiency and sleep duration in the UK Biobank (n=83,726). Our results suggest enrichment of genes involved in ubiquitin-mediated proteolysis, phototransduction and muscle development pathways and of genes expressed in multiple brain regions, skeletal muscle and adrenal gland. Evidence of shared genetic factors is found between frequent insomnia symptoms and restless legs syndrome, aging, cardio-metabolic, behavioral, psychiatric and reproductive traits. Evidence is found for a possible causal link between insomnia symptoms and coronary heart disease, depressive symptoms and subjective well-being.\n\nOne Sentence SummaryWe identify 57 genomic regions associated with insomnia pointing to the involvement of phototransduction and ubiquitination and potential causal links to CAD and depression.

genomics↗

bioRxiv2018

Lentiviral co-packaging mitigates the effects of intermolecular recombination and multiple integrations in pooled genetic screens

Lentiviral vectors are widely used for functional genomic screens, enabling efficient and stable transduction of target cells with libraries of genetic elements. Unfortunately, designs that rely on integrating multiple variable sequences, such as combinatorial perturbations or perturbations linked to barcodes, may be compromised by unintended consequences of lentiviral packaging. Intermolecular recombination between library elements and integration of multiple perturbations (even at limiting virus dilution) can negatively impact the sensitivity of pooled screens. Here, we describe a simple approach to prevent recombination between lentiviral vectors containing multiple linked variable elements, such as the recently reported CRISP-seq, Perturb-seq, and Mosaic-seq designs. We show that modifying the packaging protocol by diluting the perturbation library with a carrier plasmid increases the fraction of correct, single integrations from <60% to >90%, at the cost of reducing titer by 100-fold.

genomics↗

bioRxiv2018

Triggering the expression of a silent gene cluster from genetically intractable bacteria results in scleric acid discovery

Herein we report a strategy for the rapid and rational characterisation of novel microbial natural products from silent gene clusters. A conserved set of five regulatory genes was used as a query to search genomic databases and identify atypical biosynthetic gene clusters (BGCs). A 20-kb BGC from the genetically intractable Streptomyces sclerotialus bacterial strain was captured using yeast-based homologous recombination and introduced into validated heterologous hosts. CRISPR/Cas9-mediated genome editing was then employed to rationally inactivate the key transcriptional repressor and trigger production of an unprecedented class of hybrid natural products exemplified by (2-(benzoyloxy)acetyl)-L-proline, named scleric acid. Subsequent rounds of CRISPR/Cas9-mediated gene deletions afforded a selection of biosynthetic gene mutant strains which led to a plausible biosynthetic pathway for scleric acid assembly. Scleric acid and a key biosynthetic intermediate were also synthesised and used as authentic standards. The assembly of scleric acid involves two unique enzymatic condensation reactions that respectively link a proline and a benzoyl residue to each end of a rare hydroxyethyl-ACP intermediate. Scleric acid was then shown to exhibit moderate activity against Mycobacterium tuberculosis, as well as modest inhibition of the cancer-associated metabolic enzyme Nicotinamide N-methyltransferase (NNMT).

synthetic biology↗

bioRxiv2018

A Likelihood-Free Inference Framework for Population Genetic Data using Exchangeable Neural Networks

An explosion of high-throughput DNA sequencing in the past decade has led to a surge of interest in population-scale inference with whole-genome data. Recent work in population genetics has centered on designing inference methods for relatively simple model classes, and few scalable general-purpose inference techniques exist for more realistic, complex models. To achieve this, two inferential challenges need to be addressed: (1) population data are exchangeable, calling for methods that efficiently exploit the symmetries of the data, and (2) computing likelihoods is intractable as it requires integrating over a set of correlated, extremely high-dimensional latent variables. These challenges are traditionally tackled by likelihood-free methods that use scientific simulators to generate datasets and reduce them to hand-designed, permutation-invariant summary statistics, often leading to inaccurate inference. In this work, we develop an exchangeable neural network that performs summary statistic-free, likelihood-free inference. Our frame-work can be applied in a black-box fashion across a variety of simulation-based tasks, both within and outside biology. We demonstrate the power of our approach on the recombination hotspot testing problem, outperforming the state-of-the-art.

evolutionary biology↗

bioRxiv2018

Non-antibiotic antimicrobial triclosan induces multiple antibiotic resistance through genetic mutation

Antibiotic resistance poses a major threat to public health. Overuse and misuse of antibiotics are generally recognised as the key factors contributing to antibiotic resistance. However, whether non-antibiotic, anti-microbial (NAAM) chemicals can directly induce antibiotic resistance is unclear. We aim to investigate whether the exposure to a NAAM chemical triclosan (TCS) has an impact on inducing antibiotic resistance on Escherichia coli. Here, we report that at a concentration of 0.2 mg/L TCS induces multi-drug resistance in wild-type Escherichia coli after 30-day TCS exposure. The oxidative stress induced by TCS caused genetic mutations in genes such as fabI, frdD, marR, acrR and soxR, and subsequent up-regulation of the transcription of genes encoding beta-lactamase and multi-drug efflux pump, together with down-regulation of genes related to membrane permeability. The findings advance our understanding of the potential role of NAAM chemicals in the dissemination of antibiotic resistance in microbes, and highlights the need for controlling biocide applications.

microbiology↗

bioRxiv2018

Quantifying genetic, environmental and individual stochastic variability in Plantago lanceolata

Predicting ecological and evolutionary population dynamics requires understanding how genetic and environmental parameters influence variation in survival and reproduction among individuals. However such a focus often neglects the stochastic events that individuals experience throughout their lives that also influence survival and reproduction. With an illustrative example, we quantify and illustrate the influence of such non-selective demographic variability on population dynamics using size-structured matrix models of an experimental population of Plantago lanceolata. Our analysis shows that variation in survival and reproduction among individuals explained by environment, genes, and their interaction was modest compared to the stochastic variation in lifespan and reproduction. We illustrate how expectations on population growth, based on expected lifetime reproduction and generation time, can be misleading when variance in reproduction among individuals of the same genotype (full sibs) was large. Such large within genotype variance can lower population growth, fitness. Our results accompany recent investigations that call for more focus on stochastic variation in survival and reproduction, rather than dismissal of this variation as uninformative noise.

evolutionary biology↗

bioRxiv2018

Enterococcus faecalis AHG0090 is a genetically tractable bacterium and produces a secreted peptidic bioactive that suppresses NF-kB activation in human gut epithelial cells

Enterococcus faecalis is an early coloniser of the human infant gut and contributes to the development of intestinal immunity. To better understand the functional capacity of E. faecalis we constructed a broad host range RP4 mobilisable vector, pEHR513112, that confers chloramphenicol resistance and used a metaparental mating approach to isolate E. faecalis AHG0090 from a faecal sample collected from a healthy human infant. We demonstrated that E. faecalis AHG0090 is genetically tractable and could be manipulated using traditional molecular microbiology approaches. E. faecalis AHG0090 was comparable to the gold-standard anti-inflammatory bacterium Faecalibacterium prausnitzii A2-165 in its ability to suppress cytokine mediated NF-[Kcy]B activation in human gut derived LS174T goblet cell-like and Caco-2 enterocyte-like cell lines. E. faecalis AHG0090 and F. prausnitzii A2-165 produced secreted low molecular weight NF-[Kcy]B suppressive peptidic bioactives. Both bioactives were sensitive to heat and proteinase K treatments although the E. faecalis AHG0090 bioactive was more resilient to both forms of treatment. As expected, E. faecalis AHG0090 suppressed IL-1{beta} induced NF-[Kcy]B-p65 subunit nuclear translocation and expression of the NF-[Kcy]B regulated genes IL-6, IL-8 and CXCL-10. Finally, we determined that E. faecalis AHG0090 is distantly related to other commensal strains and likely encodes niche factors that support effective colonisation of the infant gut.

microbiology↗

bioRxiv2018

From proteins to polysaccharides: lifestyle and genetic evolution of Coprothermobacter proteolyticus.

Microbial communities that degrade lignocellulosic biomass are typified by high levels of species- and strain-level complexity as well as synergistic interactions between both cellulolytic and non-cellulolytic microorganisms. Coprothermobacter proteolyticus frequently dominates thermophilic, lignocellulose-degrading communities with wide geographical distribution, which is in contrast to reports that it ferments proteinaceous substrates and is incapable of polysaccharide hydrolysis. Here we deconvolute a highly efficient cellulose-degrading consortium (SEM1b) that is co-dominated by Clostridium (Ruminiclostridium) thermocellum- and multiple heterogenic strains affiliated to C. proteolyticus. Metagenomic analysis of SEM1b recovered metagenome-assembled genomes (MAGs) for each constituent population, whilst in parallel two novel strains of C. proteolyticus were successfully isolated and sequenced. Annotation of all C. proteolyticus genotypes (two strains and one MAG) revealed their genetic acquisition of various carbohydrate-active enzymes (CAZymes), presumably derived from horizontal gene transfer (HGT) events involving C. thermocellum- or Thermotogae-affiliated populations that are historically co-located. HGT material included whole saccharolytic operons and dockerin-encoding enzymatic subunits that are synonymous with cellulosomes. Finally, temporal genome-resolved metatranscriptomic analysis of SEM1b revealed expression of C. proteolyticus CAZymes at different SEM1b life-stages as well as co-expression of CAZymes from multiple SEM1b populations, inferring deeper microbial interactions that are dedicated towards co-degradation of cellulose and hemicellulose. We show that C. proteolyticus, a ubiquitous keystone population, consists of closely related strains that have adapted via HGT to degrade both oligo- and longer polysaccharides present in decaying plants and microbial cell walls, thus explaining its dominance in thermophilic anaerobic digesters on a global scale.

microbiology↗