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Results for “biochemistry”

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bioRxiv2017

The essential genome of Escherichia coli K-12

Transposon-Directed Insertion-site Sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries and therefore it remains unclear whether the two methodologies are comparable. To address this, a high density transposon library was constructed in Escherichia coli K-12. Essential genes predicted from sequencing of this library were compared to existing essential gene databases. To decrease false positive identification of essential gene candidates, statistical data analysis included corrections for both gene length and genome length. Through this analysis new essential genes and genes previously incorrectly designated as essential were identified. We show that manual analysis of TraDIS data reveals novel features that would not have been detected by statistical analysis alone. Examples include short essential regions within genes, orientation-dependent effects and fine resolution identification of genome and protein features. Recognition of these insertion profiles in transposon mutagenesis datasets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry.\n\nIMPORTANCEIncentives to define lists of genes that are essential for bacterial survival include the identification of potential targets for antibacterial drug development, genes required for rapid growth for exploitation in biotechnology, and discovery of new biochemical pathways. To identify essential genes in E. coli, we constructed a very high density transposon mutant library. Initial automated analysis of the resulting data revealed many discrepancies when compared to the literature. We now report more extensive statistical analysis supported by both literature searches and detailed inspection of high density TraDIS sequencing data for each putative essential gene for the model laboratory organism, Escherichia coli. This paper is important because it provides a better understanding of the essential genes of E. coli, reveals the limitations of relying on automated analysis alone and a provides new standard for the analysis of TraDIS data.

microbiology↗

bioRxiv2018

Pyrimidine starvation activates a bistable phenotypic switch leading to ribosome provisioning and rapid exit from stationary phase

Observations of bacteria at the single-cell level have revealed many instances of phenotypic heterogeneity within otherwise clonal populations, but the selective causes, molecular bases and broader ecological relevance remain poorly understood. In an earlier experiment in which the bacterium Pseudomonas fluorescens SBW25 was propagated under a selective regime that mimicked the host immune response, a genotype evolved that stochastically switched between capsulation states. The genetic cause was a mutation in carB that decreased the pyrimidine pool (and growth rate), lowering the activation threshold of a pre-existing but hitherto unrecognised phenotypic switch. Genetic components surrounding bifurcation of UTP flux towards DNA/RNA or UDP-glucose (a precursor of colanic acid forming the capsules) were implicated as key components. Extending these molecular analyses - and based on a combination of genetics, transcriptomics, biochemistry and mathematical modelling - we show that pyrimidine limitation triggers an increase in ribosome biosynthesis and that switching is caused by competition between ribosomes and CsrA/RsmA proteins for the mRNA transcript of a feed-forward regulator of colanic acid biosynthesis. We additionally show that in the ancestral bacterium the switch is part of a programme that determines stochastic entry into the semi-quiescent capsulated state, ensures that such cells are provisioned with excess ribosomes, and enables provisioned cells to exit rapidly from stationary phase under permissive conditions.

microbiology↗

bioRxiv2018

Adult Plant Resistance in Maize to Northern Leaf Spot Is a Feature of Partial Loss-of-function Alleles of Hm1

Adult plant resistance (APR) is an enigmatic phenomenon in which resistance genes are ineffective in protecting seedlings from disease but confer robust resistance at maturity. Maize has multiple cases in which genes confer APR to northern leaf spot, a lethal disease caused by Cochliobolus carbonum race 1 (CCR1). The first identified case of APR in maize is encoded by a hypomorphic allele, Hm1A, at the hm1 locus. In contrast, wild type alleles of hm1 provide complete protection at all developmental stages and in every part of the maize plant. Hm1 encodes an NADPH-dependent reductase, which inactivates HC-toxin, a key virulence effector of CCR1. Cloning and characterization of Hm1A ruled out differential transcription or translation for its APR phenotype and identified an amino acid substitution that reduced HC-toxin reductase (HCTR) activity. The possibility of a causal relationship between the weak nature of Hm1A and its APR phenotype was confirmed by the generation of two new APR alleles of Hm1 by mutagenesis. The HCTRs encoded by these new APR alleles had undergone relatively conservative missense changes that partially reduced their enzymatic activity similar to HM1A. No difference in accumulation of HCTR was observed between adult and juvenile plants, suggesting that the susceptibility of seedlings derives from a greater need for HCTR activity, not reduced accumulation of the gene product. Conditions and treatments that altered the photosynthetic output of the host had a dramatic effect on resistance imparted by the APR alleles, demonstrating a link between the energetic or metabolic status of the host and disease resistance affected by HC-toxin catabolism by the APR alleles of HCTR.\n\nAUTHOR SUMMARYAdult plant resistance (APR) is a phenomenon in which disease resistance genes are able to confer resistance at the adult stages of the plant but somehow fail to do so at the seedling stages. Despite the widespread occurrence of APR in various plant diseases, the mechanism underlying this trait remains obscure. It is not due to the differential transcription of these genes, and here we show that it is also not due to the differential translation or activity of the APR alleles of the maize hm1 gene at different stages of development. Using a combination of molecular genetics, biochemistry and physiology, we present multiple lines of evidence that demonstrate that APR is a feature or symptom of weak forms of resistance. While the mature parts of the plant are metabolically robust enough to manifest resistance, seedling tissues are not, leaving them vulnerable to disease. Growth conditions that compromise the photosynthetic output of the plant further deteriorate the ability of the seedlings to protect themselves from pathogens.\n\nOne sentence summaryCharacterization of adult plant resistance in the maize-CCR1 pathosystem reveals a causal link between weak resistance and APR.

pathology↗

bioRxiv2018

Quantitative variation within a species for traits underpinning C4 photosynthesis

Engineering C4 photosynthesis into C3 crops such as rice or wheat could substantially increase their yield by alleviating photorespiratory losses1,2. This objective is challenging because the C4 pathway involves complex modifications to the biochemistry, cell biology and anatomy of leaves3. Forward genetics has provided limited insight into the mechanistic basis of these characteristics and there have been no reports of significant quantitative intra-specific variation of C4 attributes that would allow trait-mapping4,5. Here we show that accessions of C4 Gynandropsis gynandra collected from locations across Africa and Asia exhibit natural variation in key characteristics of C4 photosynthesis. Variable traits include bundle sheath size and vein density, gas exchange parameters and carbon-isotope discrimination associated with the C4 state, but also abundance of transcripts encoding core enzymes of the C4 cycle. Traits relating to water use showed more quantitative variation than those associated with carbon assimilation. We propose variation in these traits likely adapted the hydraulic system for increased water use efficiency rather than improving carbon fixation, indicating that selection pressure may drive C4 diversity in G. gynandra by acting to modify water use rather than photosynthesis. As these accessions can be easily crossed and produce fertile offspring, our findings indicate that natural variation within a C4 species is sufficiently large to allow genetic-mapping of key anatomical C4 traits and regulators.

plant biology↗

bioRxiv2018

A saturation mutagenesis approach to understanding PTEN lipid phosphatase activity and genotype-phenotypes relationships

Phosphatase and tensin homolog (PTEN) is a tumor suppressor frequently mutated in diverse cancers. Germline PTEN mutations are also associated with a range of clinical outcomes, including PTEN hamartoma tumor syndrome (PHTS) and autism spectrum disorder (ASD). To empower new insights into PTEN function and clinically relevant genotype-phenotype relationships, we systematically evaluated the effect of PTEN mutations on lipid phosphatase activity in vivo. Using a massively parallel approach that leverages an artificial humanized yeast model, we derived high-confidence estimates of functional impact for 7,244 single amino acid PTEN variants (86% of possible). These data uncovered novel insights into PTEN protein structure, biochemistry, and mutation tolerance. Variant functional scores can reliably discriminate likely pathogenic from benign alleles. Further, 32% of ClinVar unclassified missense variants are phosphatase deficient in our assay, supporting their reclassification. ASD associated mutations generally had less severe fitness scores relative to PHTS associated mutations (p = 7.16x10-5) and a higher fraction of hypomorphic mutations, arguing for continued genotype-phenotype studies in larger clinical datasets that can further leverage these rich functional data.

genomics↗

bioRxiv2018

End-to-end differentiable learning of protein structure

Predicting protein structure from sequence is a central challenge of biochemistry. Co-evolution methods show promise, but an explicit sequence-to-structure map remains elusive. Advances in deep learning that replace complex, human-designed pipelines with differentiable models optimized end-to-end suggest the potential benefits of similarly reformulating structure prediction. Here we report the first end-to-end differentiable model of protein structure. The model couples local and global protein structure via geometric units that optimize global geometry without violating local covalent chemistry. We test our model using two challenging tasks: predicting novel folds without co-evolutionary data and predicting known folds without structural templates. In the first task the model achieves state-of-the-art accuracy and in the second it comes within 1-2[A]; competing methods using co-evolution and experimental templates have been refined over many years and it is likely that the differentiable approach has substantial room for further improvement, with applications ranging from drug discovery to protein design.

bioinformatics↗

bioRxiv2018

Label-free imaging of cholesterol and lipid distributions in model membranes

Over recent decades, lipid membranes have become standard models for examining the biophysics and biochemistry of cell membranes. Interrogation of lipid domains within biomembranes is generally done with fluorescence microscopy via exogenous chemical probes. However, most fluorophores have limited partitioning tunability, with the majority segregating in the least biologically relevant domains (i.e., low-density liquid domains). Therefore, a molecular-level picture of the majority of non-labeled lipids forming the membrane is still elusive. Here, we present simple, label-free imaging of domain formation in lipid monolayers, with chemical selectivity in unraveling lipid and cholesterol composition in all domain types. Exploiting conventional vibrational contrast in spontaneous Raman imaging, combined with chemometrics analysis, allows for examination of ternary systems containing saturated lipids, unsaturated lipids, and cholesterol. We confirm features commonly observed by fluorescence microscopy, and provide an unprecedented analysis of cholesterol distribution at the single-membrane level.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC=\"FIGDIR/small/279794_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (76K):\norg.highwire.dtl.DTLVardef@1395718org.highwire.dtl.DTLVardef@1851c93org.highwire.dtl.DTLVardef@1702f5org.highwire.dtl.DTLVardef@c33555_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2018

The biosynthetic origin of psychoactive kavalactones in kava

For millennia, humans have used plants for medicinal purposes. However, our limited understanding of plant biochemistry hinders the translation of such ancient wisdom into modern pharmaceuticals1. Kava (Piper methysticum) is a medicinal plant native to the Polynesian islands with anxiolytic and analgesic properties supported by over 3,000 years of traditional use as well as numerous recent clinical trials2-5. The main psychoactive principles of kava, kavalactones, are a unique class of polyketide natural products known to interact with central nervous system through mechanisms distinct from those of the prescription psychiatric drugs benzodiazepines and opioids6,7. Here we report de novo elucidation of the biosynthetic pathway of kavalactones, consisting of seven specialized metabolic enzymes. Based on phylogenetic and crystallographic analyses, we highlight the emergence of two paralogous styrylpyrone synthases, both of which have neofunctionalized from an ancestral chalcone synthase to catalyze the formation of the kavalactone scaffold. Structurally diverse kavalactones are then biosynthesized by subsequent regio- and stereo-specific tailoring enzymes. We demonstrate the feasibility of engineering heterologous production of kavalactones and their derivatives in bacterial, yeast, and plant hosts, thus opening an avenue towards the development of new psychiatric therapeutics for anxiety disorders, which affect over 260 million people globally8.

plant biology↗

bioRxiv2018

Discovery of biomarkers for glycaemic deterioration before and after the onset of type 2 diabetes: an overview of the data from the epidemiological studies within the IMI DIRECT Consortium

Abstract/SummaryO_ST_ABSBackground and aimsC_ST_ABSUnderstanding the aetiology, clinical presentation and prognosis of type 2 diabetes (T2D) and optimizing its treatment might be facilitated by biomarkers that help predict a persons susceptibility to the risk factors that cause diabetes or its complications, or response to treatment. The IMI DIRECT (Diabetes Research on Patient Stratification) Study is a European Union (EU) Innovative Medicines Initiative (IMI) project that seeks to test these hypotheses in two recently established epidemiological cohorts. Here, we describe the characteristics of these cohorts at baseline and at the first main follow-up examination (18-months).\n\nMaterials and methodsFrom a sampling-frame of 24,682 European-ancestry adults in whom detailed health information was available, participants at varying risk of glycaemic deterioration were identified using a risk prediction algorithm and enrolled into a prospective cohort study (n=2127) undertaken at four study centres across Europe (Cohort 1: prediabetes). We also recruited people from clinical registries with recently diagnosed T2D (n=789) into a second cohort study (Cohort 2: diabetes). The two cohorts were studied in parallel with matched protocols. Endogenous insulin secretion and insulin sensitivity were modelled from frequently sampled 75g oral glucose tolerance (OGTT) in Cohort 1 and with mixed-meal tolerance tests (MMTT) in Cohort 2. Additional metabolic biochemistry was determined using blood samples taken when fasted and during the tolerance tests. Body composition was assessed using MRI and lifestyle measures through self-report and objective methods.\n\nResultsUsing ADA-2011 glycaemic categories, 33% (n=693) of Cohort 1 (prediabetes) had normal glucose regulation (NGR), and 67% (n=1419) had impaired glucose regulation (IGR). 76% of the cohort was male, age=62(6.2) years; BMI=27.9(4.0) kg/m2; fasting glucose=5.7(0.6) mmol/l; 2-hr glucose=5.9(1.6) mmol/l [mean(SD)]. At follow-up, 18.6(1.4) months after baseline, fasting glucose=5.8(0.6) mmol/l; 2-hr OGTT glucose=6.1(1.7) mmol/l [mean(SD)]. In Cohort 2 (diabetes): 65% (n=508) were lifestyle treated (LS) and 35% (n=271) were lifestyle + metformin treated (LS+MET). 58% of the cohort was male, age=62(8.1) years; BMI=30.5(5.0) kg/m2; fasting glucose=7.2(1.4)mmol/l; 2-hr glucose=8.6(2.8) mmol/l [mean(SD)]. At follow-up, 18.2(0.6) months after baseline, fasting glucose=7.8(1.8) mmol/l; 2-hr MMTT glucose=9.5(3.3) mmol/l [mean(SD)].\n\nConclusionThe epidemiological IMI DIRECT cohorts are the most intensely characterised prospective studies of glycaemic deterioration to date. Data from these cohorts help illustrate the heterogeneous characteristics of people at risk of or with T2D, highlighting the rationale for biomarker stratification of the disease - the primary objective of the IMI DIRECT consortium.\n\nAbbreviations

epidemiology↗

bioRxiv2018

The RhoGAP myosin 9/HUM-7 integrates membrane signals to modulate Rho/RHO-1 during embryonic morphogenesis in C. elegans

During embryonic morphogenesis, cells and tissues undergo dramatic movements under the control of F-actin regulators. Our studies of epidermal cell migrations in developing C. elegans embryos have identified multiple plasma membrane signals that regulate the Rac GTPase, thus regulating WAVE and Arp2/3 complexes, to promote branched F-actin formation and polarized enrichment. We describe here a pathway that acts in parallel to Rac to transduce membrane signals to control epidermal F-actin through the GTPase Rho. Rho contributes to epidermal migrations through effects on underlying neuroblasts. Here we identify signals to regulate Rho in the epidermis. HUM-7, the C. elegans homolog of human Myo9A and Myo9B, regulates F-actin dynamics during epidermal migrations, by controlling Rho. Genetics and biochemistry support that HUM-7 behaves as GAP for the Rho GTPase, so that loss of HUM-7 enhances Rho-dependent epidermal cell behaviors. We identify SAX-3/ROBO as an upstream signal that contributes to attenuated Rho activation through its regulation of HUM-7/Myo9. These studies identify a new role for Rho during epidermal cell migrations, and suggest that Rho activity is regulated by SAX-3/ROBO acting on the RhoGAP HUM-7.

developmental biology↗

bioRxiv2018

Investigation of lanthanide-dependent methylotrophy uncovers complementary roles for alcohol dehydrogenase enzymes

The discovery that methylotrophic bacteria can utilize lanthanides as catalysts for methanol metabolism has opened new areas of biology and biochemistry. Recent studies of lanthanide-dependent enzymes have focused on biochemical and kinetic properties or the regulation of encoding genes. Kinetic analysis of a pyrroloquinoline quinone methanol dehydrogenase, XoxF1 (MexAM1_1746), from the model methylotroph Methylobacterium extorquens AM1 confirms the use of different lanthanides as cofactors and formaldehyde as a product of methanol oxidation, showing that not all XoxF MDH produce formate as the only end product in vivo. The dephosphotetrahydromethanopterin pathway for formaldehyde oxidation is still required for lanthanide-methylotrophic growth, as a fae mutant does not grow with methanol in the presence of exogenous lanthanides. Increases of 15-22% in growth rate and 10-12.5% in growth yield are observed when M. extorquens AM1 is grown in the presence of lanthanides with methanol. RNA-sequencing transcriptomics indicates remodeling of methanol, formaldehyde and formate oxidation gene expression, and targeted metabolomics shows increased accumulation of intracellular formate and decreased pools of several assimilatory intermediates. Methanol sensitivity growth assays show that the lanthanide-dependent pyrroloquinoline quinone alcohol dehydrogenase ExaF (MexAM1_1139), but not XoxF1, can reduce formaldehyde toxicity when lanthanides are present, providing evidence of a role for ExaF during lanthanide-dependent methylotrophy. We conclude from these results that lanthanide-dependent methylotrophy is more efficient than calcium-dependent methylotrophy in M. extorquens AM1, and that this change is due, at least in part, to the lanthanide-dependent enzymes XoxF1 and ExaF.\n\nIMPORTANCELanthanides serve as cofactors for pyrroloquinoline quinone containing alcohol dehydrogenase enzymes in methylotrophic bacteria. The present study addresses a fundamental gap in our understanding of how lanthanides impact metabolism, including a detailed assessment of the metabolic modifications to accommodate enhanced efficiency during methylotrophy. Kinetic characterization of XoxF1 provides a detailed description of the impact of diverse lanthanides on catalytic function for a lanthanide-dependent methanol dehydrogenase. We further show that the lanthanide-dependent ethanol dehydrogenase ExaF can oxidize formaldehyde in vivo, revealing complementary roles for these enzymes. This study provides novel insight into the effects of lanthanides on bacterial metabolism, highlighting the implementation of multiple, redundant and complementary oxidation systems.

microbiology↗

bioRxiv2018

Long-term acclimation to different stress types: revealing tradeoffs between mesophyll and stomatal conductance

Ziziphus spina-christi, a thermophilic tree, became more abundant in the Mediterranean, presumably due to increased winter temperatures. In order to predict the plant acclimation to future climate changes, we attempted to understand which factors underlie photosynthetic stress acclimation.\n\nStress acclimation to three major long-term abiotic stresses (drought, salinity and temperature) was investigated by measuring growth, gas exchange, chlorophyll fluorescence and leaf structure. We developed a stress index that allowed to precisely define stress level, enabling a comparison between stress types. For each stress, photosynthesis-limiting factors were compared: stomatal conductance (gs), mesophyll conductance (gm) and maximum capacity for Rubisco carboxylation (Vcmax).\n\nPhotosynthesis under all stresses was limited mostly by gs and gm (80-90%); whereas biochemistry (Vcmax) made a minor contribution (10-20%). The relative contribution of gs and gm on photosynthetic limitation was influenced by stress type. During acclimation to drought or salinity, photosynthesis was limited by a decline in gs, while intolerance to low temperatures was driven by decline in gm. Low mesophyll-imposed limitation was the best predictor of abiotic stress tolerance.\n\nThe results demonstrate how warming climate benefits thermophilic species. Moreover, current work gives methodology for stress studies, and defines the main factors underlying the plant response to climate change.\n\nHighlightWe have compared the photosynthesis limitation resulting from long-term acclimation to three major a-biotic stresses (drought, salinity and temperature) to understand which limiting-factor are dominant under each type of stress.

plant biology↗

bioRxiv2018

Ligand Modulates Cross-Coupling between Riboswitch Folding and Transcriptional Pausing

HighlightsO_LIsmFRET, biochemistry and simulations probe co-transcriptional riboswitch folding\nC_LIO_LINascent RNA folding is disfavored by the DNA template and aided by RNAP\nC_LIO_LITranscriptional pausing is stabilized by RNA pseudoknot and destabilized by ligand\nC_LI\n\nSUMMARYNumerous classes of riboswitches have been found to regulate bacterial gene expression in response to physiological cues, offering new paths to anti-bacterial drugs. As common studies of isolated riboswitches lack the functional context of the transcription machinery, we here combine single-molecule, biochemical and simulation approaches to investigate the coupling between co-transcriptional folding of the pseudoknot-structured preQ1 riboswitch and RNA polymerase (RNAP) pausing. We show that pausing at a site immediately downstream of the riboswitch requires a ligand-free pseudoknot in the nascent RNA, a precisely spaced sequence resembling the pause consensus, and electrostatic and steric interactions with the RNAP exit channel. While interactions with RNAP stabilize the native fold of the riboswitch, binding of the ligand signals RNAP release from the pause. Our results demonstrate that the nascent riboswitch and its ligand actively modulate the function of RNAP and vice versa, a paradigm likely to apply to other cellular RNA transcripts.

biophysics↗

bioRxiv2018

Phosphoregulation of Cdt1 in G2 and M phases prevents re-replication independently of Geminin

Achieving complete and precise genome duplication requires that each genomic segment be replicated only once per cell division cycle. Protecting large eukaryotic genomes from re-replication requires an overlapping set of molecular mechanisms that prevent the first DNA replication step, the DNA loading of MCM helicase complexes to license replication origins. Previous reports have defined many such origin licensing inhibition mechanisms, but the temporal relationships among them are not clear, particularly with respect to preventing re-replication in G2 and M phases. Using a combination of mutagenesis, biochemistry, and single cell analyses in human cells, we define a new mechanism that prevents re-replication through hyperphosphorylation of the essential MCM loading protein, Cdt1. We demonstrate that Cyclin A/CDK1 hyperphosphorylates Cdt1 to inhibit MCM re-loading in G2 phase. The mechanism of inhibition is to block Cdt1 binding to MCM independently of other known Cdt1 inactivation mechanisms such as Cdt1 degradation during S phase or Geminin binding. Moreover, we provide evidence that protein phosphatase 1-dependent Cdt1 dephosphorylation at the mitosis-to-G1 phase transition re-activates Cdt1. We propose that multiple distinct, non-redundant licensing inhibition mechanisms act in a series of sequential relays through each cell cycle phase to ensure precise genome duplication. Author SummaryThe initial step of DNA replication is loading the DNA helicase, MCM, onto DNA during the first phase of the cell division cycle. If MCM loading occurs inappropriately onto DNA that has already been replicated, then cells risk DNA re-replication, a source of endogenous DNA damage and genome instability. How mammalian cells prevent any sections of their very large genomes from re-replicating is still not fully understood. We found that the Cdt1 protein, one of the critical MCM loading factors, is inhibited specifically in late cell cycle stages through a mechanism involving protein phosphorylation. This phosphorylation prevents Cdt1 from binding MCM; when Cdt1 cant be phosphorylated MCM is inappropriately re-loaded onto DNA and cells are prone to re-replication. When cells divide and transition into G1 phase, Cdt1 is then dephosphorylated to re-activate it for MCM loading. Based on these findings we assert that the different mechanisms that cooperate to avoid re-replication are not redundant, but rather distinct mechanisms are dominant in different cell cycle phases. These findings have implications for understanding how genomes are duplicated precisely once per cell cycle and shed light on how that process is perturbed by changes in Cdt1 levels or phosphorylation activity.

molecular biology↗

bioRxiv2018

Antibiotic resistance evasion is explained by rare mutation frequency and not by lack of compensatory mechanisms

Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for decades without appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We investigated why Mycobacterium tuberculosis fails to become resistant to D-cycloserine. To address this question we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low mutation frequency associated with D-cycloserine resistance is the dominant factor delaying the appearance of clinical resistance to this antibiotic in bacteria infecting humans, and not lack of potential compensatory mechanisms.\n\nOne Sentence SummaryWe show that the lack of D-cycloserine resistance in Mycobacterium tuberculosis is due its ultra-low mutation frequency rather than lack of compensatory mechanisms.

microbiology↗

bioRxiv2018

Toward deciphering developmental patterning with deep neural network

Complex biological functions are carried out by the interaction of genes and proteins. Uncovering the gene regulation network behind a function is one of the central themes in biology. Typically, it involves extensive experiments of genetics, biochemistry and molecular biology. In this paper, we show that much of the inference task can be accomplished by a deep neural network (DNN), a form of machine learning or artificial intelligence. Specifically, the DNN learns from the dynamics of the gene expression. The learnt DNN behaves like an accurate simulator of the system, on which one can perform in-silico experiments to reveal the underlying gene network. We demonstrate the method with two examples: biochemical adaptation and the gap-gene patterning in fruit fly embryogenesis. In the first example, the DNN can successfully find the two basic network motifs for adaptation - the negative feedback and the incoherent feed-forward. In the second and much more complex example, the DNN can accurately predict behaviors of essentially all the mutants. Furthermore, the regulation network it uncovers is strikingly similar to the one inferred from experiments. In doing so, we develop methods for deciphering the gene regulation network hidden in the DNN "black box". Our interpretable DNN approach should have broad applications in genotype-phenotype mapping. SignificanceComplex biological functions are carried out by gene regulation networks. The mapping between gene network and function is a central theme in biology. The task usually involves extensive experiments with perturbations to the system (e.g. gene deletion). Here, we demonstrate that machine learning, or deep neural network (DNN), can help reveal the underlying gene regulation for a given function or phenotype with minimal perturbation data. Specifically, after training with wild-type gene expression dynamics data and a few mutant snapshots, the DNN learns to behave like an accurate simulator for the genetic system, which can be used to predict other mutants behaviors. Furthermore, our DNN approach is biochemically interpretable, which helps uncover possible gene regulatory mechanisms underlying the observed phenotypic behaviors.

developmental biology↗

bioRxiv2018

A standardised framework to identify optimal animal models for efficacy assessment in drug development

IntroductionPoor translation of efficacy data derived from animal models is a potential contributor to costly and unnecessary attrition in clinical trials.\n\nObjectivesTo develop a tool to assess, validate and compare the clinical translatability of animal models used for the preliminary assessment of efficacy.\n\nDesign and ResultsWe conducted an exploratory literature search to identify the key aspects to validate animal models. Eight aspects (Epidemiology, Pathophysiology, Genetic, Biochemistry, Aetiology, Histology, Pharmacology and Endpoints) were identified for which questions were drafted to evaluate the different faces of the human disease simulation. Features of the framework include standardised instructions, a weighting and scoring system to compare models as well as contextualising factors regarding model similarity and evidence uncertainty. We included a quality assessment of the internal validity of drug intervention studies included in the Pharmacological validation section for both effective and ineffective drugs in humans. A web-based survey was conducted with experts from different stakeholders to gather input on the framework. Finally, we present a case study of a preliminary validation and comparison of two animal models for Duchenne Muscular Dystrophy (mdx mouse and GRMD dog) and Diabetes Type 2 (ZDF rat and db/db mouse). We show that there are significant differences between the mdx mouse and the GRMD dog, the latter mimicking the human condition to a greater extent than the mouse despite the considerable lack of published data. In DT2, both the ZDF rat and the db/db mouse are comparable with minor differences in pathophysiology.\n\nConclusionsFIMD facilitates drug development by serving as the basis to select the most relevant model that can provide meaningful and translatable results to progress drug candidates to the clinic.

pharmacology and toxicology↗

bioRxiv2018

EGFR confers exquisite specificity of Wnt9a-Fzd9b signaling in hematopoietic stem cell development

The mechanisms of Wnt-Frizzled (Fzd) signaling selectivity and their biological implications remain unclear. We demonstrate for the first time that the epidermal growth factor receptor (EGFR) is required as a co-factor for Wnt signaling. Using genetic studies in zebrafish, paired with in vitro cell biology and biochemistry, we have determined that Fzd9b signals specifically with Wnt9a in vivo and in vitro to elicit {beta}-catenin dependent Wnt signals that regulate hematopoietic stem and progenitor cell (HSPC) development in the dorsal aorta. This requirement is conserved in the derivation of HSPCs from human embryonic stem cells. Wnt9a-Fzd9b specificity requires two intracellular domains in Fzd9b, which interact with EGFR as a required co-factor to promote signal transduction. EGFR phosphorylates one tyrosine residue on Fzd9b, a requirement for the Wnt signal. These findings indicate that Wnt signaling interactions can be exquisitely specific and inform protocols for derivation of HSPCs in vitro.\n\nHighlightsO_LIAn in vitro signaling screen identifies Fzd9b as a Wnt9a-specific receptor.\nC_LIO_LIFzd9b and Wnt9a regulate hematopoietic stem cell development as a cognate pair.\nC_LIO_LIWNT9A and FZD9 are required for HSPC derivation from human pluripotent cells in vitro.\nC_LIO_LIEGFR confers specificity to Wnt9a-Fzd9b signaling in zebrafish and human cells.\nC_LI

developmental biology↗