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bioRxiv2017

Intracellular Passage Of Na+ In An Active State G Protein Coupled Receptor

Playing a central role in cell signalling, GPCRs have evolved into the largest superfamily of membrane proteins and form the majority of drug targets in humans. How extracellular agonist binding triggers the activation of GPCRs and associated intracellular effector proteins remains, however, poorly understood. High resolution structural studies have recently revealed that inactive class-A GPCRs harbour a conserved binding site for Na+ ions in the centre of their transmembrane domain, accessible from the extracellular space. Here, we show that the opening of a conserved hydrated channel in the activated state receptors allows the Na+ ion to egress from its binding site into the cytosol. Coupled with protonation changes, this ion movement occurs without significant energy barriers, and can be driven by physiological transmembrane ion and voltage gradients. We propose that Na+ ion exchange with the cytosol is a key step in GPCR activation, which locks receptors in long-lived active-state conformations.

biophysics↗

bioRxiv2017

Evolutionary compromises to environmental toxins: ammonia and urea tolerance in Drosophila suzukii and Drosophila melanogaster.

The invasive species Drosophila suzukii has evolved morphological and behavioral adaptations to lay eggs under the skin of fresh fruits. This results in severe damage of a wide range of small and stone fruits, thus making this species a serious agricultural and economical threat.\n\nDrosophila suzukii females typically lay few eggs per fruit, preferring not infested fruits. Hence larvae are exposed to a reduced amount of nitrogenous waste products. On the contrary, the innocuous Drosophila melanogaster lays eggs on fermented fruits already infested by conspecifics, with larvae developing in a crowded environment characterized by accumulation of nitrogenous waste such as ammonia and urea. Given these differences in oviposition and larval ecological niche, we expected different behavioral and physiological mechanisms in the two species to cope with nitrogenous waste. We investigated the impact of different concentrations of ammonia and urea on fecundity and larval development in both species. Females and larvae of D. suzukii showed a greater sensitivity to high concentration of both compounds, with a dramatic decrease in fecundity and egg viability.\n\nTo better understand the pathways underlying these differences, we evaluated the effect on ornithine aminotransferase and glutathione-S-transferase, two enzymes involved in nitrogen metabolism and stress response that are expressed during larval development. Under ammonia and urea exposure, the expression of these enzymes was significantly reduced in D. suzukii.\n\nThe fact that D. suzukiis shift from rotten to fresh fruit as oviposition and larval substrate resulted in less efficient detoxifying and excretory mechanisms represents a potential approach for its control. Fecundity and larval development are in fact dramatically impaired by nitrogen waste products. These findings can help in planning effective strategies of sustainable pest management that targets both females and larvae.

evolutionary biology↗

bioRxiv2017

Household triclosan and triclocarban exposure impacts the adult intestinal microbiome but not the infant intestinal microbiome

In 2016, the US Food and Drug Administration banned the use of specific microbicides in some household and personal wash products. This decision was due to concerns that these chemicals might induce antibiotic resistance or disrupt human microbial communities. Triclosan and triclocarban (referred to as TCs) are the most common antimicrobials in household and personal care products, but the extent to which TC exposure perturbs microbial communities in humans, particularly during infant development, was unknown. We conducted a randomized intervention of TC-containing household and personal care products during the first year following birth to characterize whether TC exposure from wash products perturbs microbial communities in mothers and their infants. Longitudinal survey of the intestinal microbiota using 16S ribosomal RNA amplicon sequencing showed that TC exposure from wash products did not induce global reconstruction of either infant or maternal intestinal microbiotas following 10 months of exposure after birth. However, broadly antibiotic-resistant species from the phylum Proteobacteria were enriched in stool samples from mothers in TC households only after the introduction of triclosan-containing toothpaste. Despite the minimal effects of TC exposure from wash products on the gut microbial community of infants and adults, these results suggest detected taxonomic differences are associated with potential harmful effects on host physiology, highlighting the need for consumer safety testing of self-care products not subject to the ban on the human microbiome and health outcomes.

microbiology↗

bioRxiv2017

Histopathological image QTL discovery of thyroid autoimmune disease variants

Genotype-to-phenotype association studies typically use macroscopic physiological measurements or molecular readouts as quantitative traits. There are comparatively few suitable quantitative traits available between cell and tissue length scales, a limitation that hinders our ability to identify variants affecting phenotype at many clinically informative levels. Here we show that quantitative image features, automatically extracted from histopathological imaging data, can be used for image Quantitative Trait Loci (iQTL) mapping and variant discovery. Using thyroid pathology images, clinical metadata, and genomics data from the Genotype and Tissue Expression (GTEx) project, we establish and validate a quantitative imaging biomarker for immune cell infiltration. A total of 100,215 variants were selected for iQTL profiling, and tested for genotype-phenotype associations with our quantitative imaging biomarker. Significant associations were found in HDAC9 and TXNDC5. We validated the TXNDC5 association using GTEx cis-expression QTL data, and an independent hypothyroidism dataset from the Electronic Medical Records and Genomics network.\n\nOne Sentence SummaryWe use a histopathological image QTL analysis to identify genomic variants associated with immune cell infiltration.

genomics↗

bioRxiv2017

Experience-dependent plasticity in accessory olfactory bulb interneurons following male-male social interaction

Conflict of interestThe authors declare no competing financial interests.\n\nAcknowledgementsWe thank Jennifer Torres, Salma Ferdous, and Natasha Browder for technical support. We thank Kimberly Huber and Todd Roberts for critical feedback on the manuscript. This work was funded by the National Institute on Deafness and Other Communication Disorders and the National Institute on Drug Abuse of the National Institutes of Health under award numbers R00DC011780, R01DC015784 (JPM), and T32DA007290 (HLC). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Partial support was also provided by the National Science Foundation (Grant IOS-1451034; JPM, co-PI).\n\nChemosensory information processing in the mouse accessory olfactory system (AOS) guides the expression of social behavior. After salient chemosensory encounters, the accessory olfactory bulb (AOB) experiences changes in the balance of excitation and inhibition at reciprocal synapses between mitral cells (MCs) and local interneurons. The mechanisms underlying these changes remain controversial. Moreover, it remains unclear whether MC-interneuron plasticity is unique to specific behaviors, such as mating, or whether it is a more general feature of the AOB circuit. Here, we describe targeted electrophysiological studies of AOB inhibitory internal granule cells (IGCs), many of which upregulate the immediate-early gene Arc after male-male social experience. Following the resident-intruder paradigm, Arc-expressing IGCs in acute AOB slices from resident males displayed stronger excitation than non-expressing neighbors when sensory inputs are stimulated. The increased excitability of Arc-expressing IGCs was not correlated with changes in the strength or number of excitatory synapses with MCs, but is instead associated with increased intrinsic excitability and decreased HCN channel-mediated IH currents. Consistent with increased inhibition by IGCs, MCs responded to sensory input stimulation with decreased depolarization and spiking following resident-intruder encounters. These results reveal that non-mating behaviors drive AOB inhibitory plasticity, and indicate that increased MC inhibition involves intrinsic excitability changes in Arc-expressing interneurons.\n\nSignificance StatementThe accessory olfactory bulb (AOB) is a site of experience-dependent plasticity between excitatory mitral cells (MCs) and inhibitory internal granule cells (IGCs), but the physiological mechanisms and behavioral conditions driving this plasticity remain unclear. Here, we report studies of AOB neuronal plasticity following male-male social chemosensory encounters. We show that the plasticity-associated immediate-early gene Arc is selectively expressed in IGCs from resident males following the resident-intruder assay. Following behavior, Arc-expressing IGCs are more strongly excited by sensory input stimulation and MC activation is suppressed. Arc-expressing IGCs do not show increased excitatory synaptic drive, but instead show increased intrinsic excitability. These data indicate that MC-IGC plasticity is induced after male-male social chemosensory encounters, resulting in MC suppression by Arc-expressing IGCs.

neuroscience↗

bioRxiv2017

Enzymes And Substrates Are Balanced At Minimal Combined Mass Concentration In Vivo

A fundamental problem in biology is how cells organize their resource investment. Cellular metabolism, for example, typically involves hundreds of enzymes and metabolites, but it is unclear according to which principles their concentrations are set. Reasoning that natural selection will drive cells towards achieving a given physiological state at minimal cost, we derive a general equation that predicts the concentration of a metabolite from the concentration of the most abundant and costly enzyme consuming it. Simulations of cellular growth as well as experimental data demonstrate that costs are approximately proportional to molecular masses. For effectively irreversible reactions, the cell maximizes its metabolic efficiency by investing equally into substrate and unbound enzyme molecules. Without fitting any free parameters, the resulting model predicts in vivo substrate concentrations from enzyme concentrations and substrate affinities with high accuracy across data from E. coli and diverse eukaryotes (R2=0.79, geometric mean fold-error 1.74). The corresponding organizing principle - the minimization of the summed mass concentrations of solutes - may facilitate reducing the complexity of kinetic models and will contribute to the design of more efficient synthetic cellular systems.

systems biology↗

bioRxiv2017

Regulatory RNPs: A novel class of ribonucleoproteins that contribute to ribosome heterogeneity

Ribonucleoproteins (RNPs), which are comprised of non-coding RNA and associated proteins, are involved in essential cellular processes such as translation and pre-mRNA splicing. One class of RNP is the small Cajal body-specific RNP (scaRNP), which contributes to the biogenesis of small nuclear RNPs (snRNPs) that are central components of the spliceosome. Interestingly, three scaRNAs are internally processed, generating stable nucleolus-enriched RNAs of unknown function. Here we provide evidence that these RNAs become part of novel RNPs we term regulatory RNPs (regRNPs). We postulate that regRNPs can impact rRNA modifications via interactions with the guide RNA component of small nucleolar RNPs (snoRNPs). Most modifications within rRNA (predominantly pseudouridylation and ribose 2-O-methylation) are conducted by snoRNPs, and we hypothesize that the activity of at least some of these snoRNPs is under the control of regRNPs. Ribosome heterogeneity leading to specialized ribosomes is an exciting emerging concept. Because modifications within rRNA can vary in different physiological or pathological situations, rRNA modifications are thought to be the major source of ribosome heterogeneity. Our identification of regRNPs thus provides important and timely insight into how ribosome heterogeneity may be accomplished. This work also provides additional functional connections between the Cajal body and the nucleolus.\n\nSummary StatementProcessed scaRNAs give rise to a novel regulatory RNP which regulates the modification of ribosomal RNA. These findings provide insight into the mechanisms governing ribosome heterogeneity.

molecular biology↗

bioRxiv2017

Defects of myelination are common pathophysiology in syndromic and idiopathic autism spectrum disorders

Autism spectrum disorder (ASD) affects approximately 1:68 individuals and has incalculable burdens on affected individuals, their families, and health care systems. While the genetic contributions to idiopathic ASD are heterogeneous and largely unknown, the causal mutations for syndromic forms of ASD - including truncations and copy number variants - provide a genetic toehold with which to gain mechanistic insights1-3. Models of these syndromic disorders have been used to better characterize the molecular and physiological processes disrupted by these mutations4. Two fundamental questions remain - how biologically similar are the mouse models of syndromic forms of ASD, and how relevant are these mouse models to their human analogs? To address these questions, we performed integrative transcriptomic analyses of seven independent mouse models of three syndromic forms of ASD generated across five laboratories, and assessed dysregulated genes and their pathways in human postmortem brain from patients with ASD and unaffected controls. These cross-species analyses converged on shared disruptions in myelination and axon development across both syndromic and idiopathic ASD, highlighting both the face validity of mouse models for these disorders and identifying novel convergent molecular phenotypes amendable to rescue with therapeutics.

neuroscience↗

bioRxiv2017

Profiling Of Lysine-Acetylated Proteins In Human Urine

Biomarker is the measurable change associated with a physiological or pathophysiological process, its nature is change. Contrast to the blood which is under homeostatic controls, urine reflects changes in the body earlier and more sensitive therefore is a better biomarker source. Lysine acetylation is an abundant and highly regulated post-translational modification. It plays a pivotal role in modulating diverse biological processes and is associated with various important diseases. Enrichment or visualization of proteins with specific post-translational modifications provides a method for sampling the urinary proteome and reducing sample complexity. In this study, we used anti-acetyllysine antibody-based immunoaffinity enrichment combined with high-resolution mass spectrometry to profile lysine-acetylated proteins in normal human urine. A total of 629 acetylation sites on 315 proteins were identified, including some very low-abundance proteins. This is the first proteome-wide characterization of lysine acetylation proteins in normal human urine. Our dataset provides a useful resource for the further discovery of the lysine acetylated proteins as biomarker in urine.

molecular biology↗

bioRxiv2017

Aging, Mortality And The Fast Growth Trade-Off Of Schizosaccharomyces pombe

Replicative aging has been demonstrated in asymmetrically dividing unicellular organisms, seemingly caused by unequal damage partitioning. Although asymmetric segregation and inheritance of potential aging factors also occurs in symmetrically dividing species, it nevertheless remains controversial whether this results in aging. Based on large-scale single-cell lineage data obtained by time-lapse microscopy with a microfluidic device, in this report, we demonstrate the absence of replicative aging in old-pole cell lineages of Schizosaccharomyces pombe cultured under constant favorable conditions. By monitoring more than 1,500 cell lineages in seven different culture conditions, we showed that both cell division and death rates are remarkably constant for at least 50-80 generations. Our measurements revealed that the death rate per cellular generation increases with division rate, pointing to a physiological trade-off with fast growth under balanced growth conditions. We also observed the formation and inheritance of Hsp104-associated protein aggregates, which are a potential aging factor in old-pole cell lineages, and found that these aggregates exhibited a tendency to preferentially remain at the old-poles for several generations. However, the aggregates were eventually segregated from old-pole cells upon cell division and probabilistically allocated to new-pole cells. The quantity and inheritance of protein aggregates increased neither cellular generation time nor cell death initiation rates. Furthermore, our results revealed that unusually large amounts of protein aggregates induced by oxidative stress exposure did not result in aging; old-pole cells resumed normal growth upon stress removal, despite the fact that most of them inherited significant quantities of aggregates. These results collectively indicate that protein aggregates are not a major determinant of cell fate in S. pombe, and thus cannot be an appropriate molecular marker or index for replicative aging under both favorable and stressful environmental conditions.

microbiology↗

bioRxiv2017

Beyond The Linear Genome: Comprehensive Determination Of The Endogenous Circular Elements In C. elegans And Human Genomes Via An Unbiased Genomic-Biophysical Method

Investigations aimed at defining the 3-D configuration of eukaryotic chromosomes have consistently encountered an endogenous population of chromosome-derived circular genomic DNA, referred to as extrachromosomal circular DNA (eccDNA). While the production, distribution, and activities of eccDNAs remain understudied, eccDNA formation from specific regions of the linear genome has profound consequences on the regulatory and coding capabilities for these regions. High-throughput sequencing has only recently made extensive genomic mapping of eccDNA sequences possible and had yet to be applied using a rigorous approach that distinguishes ascertainment bias from true enrichment. Here, we define eccDNA distribution, utilizing a set of unbiased topology-dependent approaches for enrichment and characterization. We use parallel biophysical, enzymatic, and informatic approaches to obtain a comprehensive profiling of eccDNA in C. elegans and in three human cell types, where eccDNAs were previously uncharacterized. We also provide quantitative analysis of the eccDNA loci at both unique and repetitive regions. Our studies converge on and support a consistent picture in which endogenous genomic DNA circles are present in normal physiological DNA metabolism, and in which the circles come from both coding and noncoding genomic regions. Prominent among the coding regions generating DNA circles are several genes known to produce a diversity of protein isoforms, with mucin proteins and titin as specific examples.

genomics↗

bioRxiv2017

High-resolution analysis of spatiotemporal virulence gene regulation during food-borne infection with Escherichia coli O157:H7 within a live host

Food-borne infection with enterohemorrhagic Escherichia coli (EHEC) is a major cause of diarrheal illness in humans, and can lead to severe complications such as hemolytic uremic syndrome. Cattle and other ruminants are the main reservoir of EHEC, which enters the food-chain through contaminated meat, dairy, or vegetables. However, how EHEC transitions from the transmission vector to colonizing the intestinal tract, and how virulence-specific genes are regulated during this transition, is not well understood. Here, we describe the establishment of a vertebrate model for food-borne EHEC infection, using the protozoan Paramecium caudatum as a vector and the zebrafish (Danio rerio) as a host. At 4 days post fertilization, zebrafish have a fully developed intestinal tract, yet are fully transparent. This allows us to follow intestinal colonization, microbe-host cell interactions, and microbial gene induction within the live host and in real time throughout the infection. Additionally, this model can be adapted to compare food- and water-borne infections, under gnotobiotic conditions or against the backdrop of an endogenous (and variable) host microbiota. Finally, the zebrafish allows for investigation of factors affecting shedding and transmission of bacteria to naive hosts. High-resolution analysis of EHEC gene expression within the zebrafish host emphasizes the need for tight transcriptional regulation of virulence factors for within-host fitness.\n\nIMPORTANCEEnterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen which can cause diarrhea, vomiting and in some cases, severe complications such as kidney problems in humans. Up to 30% of cattle are colonized with EHEC, which can enter the food-chain through contaminated meat, dairy and vegetables. In order to control infections and stop transmission, it is important to understand what factors allow EHEC to colonize its hosts, cause virulence and aid transmission. Since this cannot be systematically studied in humans, it is important to develop animal models of infection and transmission. We developed a model which allows us to study food-borne infection in zebrafish, a vertebrate host that is transparent and genetically tractable. Using the zebrafish host, we can follow the bacterial infection cycle in real time, and gain important information regarding bacterial physiology and microbe-host interactions. This will allow us to identify potential new targets for infection control and prevention.

microbiology↗

bioRxiv2017

Power and shift invariant detection of dynamically coupled networks (PSIICOS) from non-invasive MEG data

MEG reflects electrical activity of neuronal assemblies that coalesce and decoalesce in time providing for massively parallel and dynamic flow of information exchange in the brain. There is a growing evidence that our behavior is mediated by simultaneous activity of several interacting and simultaneously active cortical networks. Not only the location of network nodes but also the temporal profiles of such interaction are of interest. The existing methods are primarily concerned with proposing novel measures of synchrony to be applied to the cortical region activity signals extracted from the MEG data using beamforming or other spatial filtering approaches.\n\nIn this work we propose to render the task of estimating network topology and temporal profiles as a source estimation problem but in the space of interacting topographies. The proposed point of view has a number of benefits as it allows to use the wealth of techniques and intuition accumulated in the community when dealing with source estimation problems. Operating in the interacting topographies space we first propose subspace projection method to significantly reduce the spatial leakage effect in the cross-spectral matrix. We then use subspace matching metrics to extract a set of networks with their synchronicity profiles that explain the variance in the sensor space cross-spectral matrix, much like regular dipoles and their activations explain the variance in the regular evoked responses.\n\nThe preliminary results of application of this method to a simple self-paced voluntary finger movement paradigm comparing allowed to observe functional coupling between primary motor and supplementary motor area and establish that this coupling can be characterized with near-zero phase delay between the oscillatory activity of these two regions. This observed synchrony pattern is physiologically plausible and is in agreement with the current hypothesis about the neural mechanics of the motor acts.

neuroscience↗

bioRxiv2017

Pooled CRISPR interference screens enable high-throughput functional genomics study and elucidate new rules for guide RNA library design in Escherichia coli

Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology provides potential advantages in high-throughput functional genomics analysis in prokaryotes over previously established platforms based on recombineering or transposon mutagenesis. In this work, as a proof-of-concept to adopt CRISPR/Cas9 method as a pooled functional genomics analysis platform in prokaryotes, we developed a CRISPR interference (CRISPRi) library consisting of 3,148 single guide RNAs (sgRNAs) targeting the open reading frame (ORF) of 67 genes with known knockout phenotypes and performed pooled screens under two stressed conditions (minimal and acidic medium) in Escherichia coli. Our approach confirmed most of previously described gene-phenotype associations while maintaining < 5% false positive rate, suggesting that CRISPRi screen is both sensitive and specific. Our data also supported the ability of this method to narrow down the candidate gene pool when studying operons, a unique structure in prokaryotic genome. Meanwhile, assessment of multiple loci across treatments enables us to extract several guidelines for sgRNA design for such pooled functional genomics screen. For instance, sgRNAs locating at the first 5% upstream region within ORF exhibit enhanced activity and 10 sgRNAs per gene is suggested to be enough for robust identification of gene-phenotype associations. We also optimized the hit-gene calling algorithm to identify target genes more robustly with even fewer sgRNAs. This work showed that CRISPRi could be adopted as a powerful functional genomics analysis tool in prokaryotes and provided the first guideline for the construction of sgRNA libraries in such applications.\n\nImportanceTo fully exploit the valuable resource of explosive sequenced microbial genomes, high-throughput experimental platform is needed to associate genes and phenotypes at the genome level, giving microbiologists the insight about the genetic structure and physiology of a microorganism. In this work, we adopted CRISPR interference method as a pooled high-throughput functional genomics platform in prokaryotes with Escherichia coli as the model organism. Our data suggested that this method was highly sensitive and specific to map genes with previously known phenotypes, potent to act as a new strategy for high-throughput microbial genetics study with advantages over previously established methods. We also provided the first guideline for the sgRNA library design by comprehensive analysis of the screen data. The concept, gRNA library design rules and open-source scripts of this work should benefit prokaryotic genetics community to apply high-throughput mapping of defined gene set with phenotypes in a broad spectrum of microorganisms.

microbiology↗

bioRxiv2017

Ophiuroid Phylotranscriptomics Enables Discovery Of Novel Echinoderm Representatives Of Bilaterian Neuropeptide Families And Reconstruction Of Neuropeptide Precursor Evolution Over ~270 Million Years

BackgroundNeuropeptides are a diverse class of intercellular signaling molecules that mediate neuronal regulation of many physiological and behavioural processes, including feeding, reproduction and locomotion. Recent advances in genome/transcriptome sequencing are enabling identification of neuropeptide precursor proteins in species from a growing variety of animal taxa, providing new insights into the evolution of neuropeptide signaling. Here we report a phylo-transcriptomic analysis of neuropeptide precursors in over fifty species of brittle stars (Class Ophiuroidea; Phylum Echinodermata).\n\nResultsDetailed analysis of transcriptome sequence data from three brittle star species, Ophionotus victoriae, Amphiura filiformis and Ophiopsila aranea, enabled the first comprehensive identification of neuropeptide precursors in ophiuroids. Representatives of over thirty bilaterian neuropeptide precursor families were identified, some of which occur as paralogs (e.g. thyrotropin-releasing hormone, corticotropin-releasing hormone, cholecystokinin, somatostatin and pedal peptide). Furthermore, homologs of endothelin/CCHamide, eclosion hormone, neuropeptide-F/Y and nucleobinin/nesfatin were discovered here in a deuterostome/echinoderm for the first time. The majority of ophiuroid neuropeptide precursors contain a single copy of a neuropeptide, but several precursors comprise multiple copies of identical or non-identical, but structurally-related, neuropeptides. Here we performed an unprecedented investigation of the evolution of neuropeptide copy-number over a period of ~270 million years by analysing sequence data from over fifty ophiuroid species, with reference to a robust phylogeny. Interestingly, the number of neuropeptide copies in the majority of precursors was constant across all the species examined, but examples of clade-specific losses/gains of neuropeptides were also observed.\n\nConclusionsWe report here the most comprehensive analysis to date of neuropeptide precursors in the phylum Echinodermata, with novel representatives of several bilaterian neuropeptide families discovered for the first time in echinoderms. Furthermore, analysis of precursor proteins comprising multiple copies of identical or related neuropeptides across ~270 million years of ophiuroid evolution indicates that the composition of neuropeptide \"cocktails\" is functionally important, but with plasticity over long evolutionary time scales.

neuroscience↗

bioRxiv2017

The closed-loop pathways of signaling molecules

The pathways of signaling molecules are important to understanding how signaling molecules regulate physiological function and also in predicting the pathological development which is important to therapeutic strategy, however the thorough knowledge of these pathways is still lack. In this paper, we used the big data concept to analyze the pathways of signaling molecules and categorize these molecules into five groups according to their origin and effect on the five organs of heart-spleen-lung-kidney-liver. Heart group includes IGF, Ang and Mg; spleen group includes ANP, aldosterone, retinoic acid and ghrelin; lung group includes FGF7, VEGF, ascorbic acid and HIF; kidney group includes calcitonin, PTHrP, Wnt and NO; and liver group includes EPO, renin, SOD, AKR and GSH. We found that each group of molecules have assisting effect on the other organ in the order of heart-spleen-lung-kidney-liver-heart, and have regulating effect on the other organ in the order of heart-lung-liver-spleen-kidney-heart. Moreover, the pathways of molecules of each group also follow these two arrangements, in which the pathways of molecules form a closed-loop that may lead to new therapeutic strategies.

bioinformatics↗

bioRxiv2017

A Versatile Genetic Tool For Post-Translational Control Of Gene Expression With A Small Molecule In Drosophila melanogaster

Several techniques have been developed in Drosophila to control gene expression temporally. While some of these techniques are incompatible with existing GAL4 lines, others suffer from side effects on physiology or behavior. Here, we describe a method of post-translational temporal control of gene expression which is compatible with the current library of transgenic reagents. We adopted a strategy to regulate protein degradation by fusing a protein of interest to a destabilizing domain (DD) derived from the Escherichia coli dihydrofolate reductase (ecDHFR). Trimethoprim (TMP), a stabilizing small molecule, binds to DD and blocks degradation of the chimeric protein. With a GFP-DD reporter, we show that this system is effective across different tissues and developmental stages in the fly. Notably, feeding flies with TMP can increase the expression level of GFP-DD up to 34 times in a dosage-dependent and reversible manner without altering the lifespan or behavior of the animal. To broaden the utility of our method, we engineered GAL80-DD flies that can be crossed to the available GAL4 lines to control the temporal pattern of gene expression with TMP. We also developed an inducible recombinase, FLP-DD, for high-efficiency sparse labeling and intersectional lineage analysis. Finally, we demonstrated the utility of the DD system in manipulating neuronal activity of sensory neurons. In summary, we have developed a system to control in vivo gene expression levels with negligible background, large dynamic range, and in a reversible manner, all by feeding a small molecule to Drosophila melanogaster.

genetics↗

bioRxiv2017

Modulation Of Type IV Pili Phenotypic Plasticity Through A Novel Chaperone-Usher System In Synechocystis sp 6803

Controlling the transition from a multicellular motile state to a sessile biofilm is an important eco-physiological decision for most prokaryotes, including cyanobacteria. Photosynthetic and bio geochemically significant cyanobacterium Synechocystis sp. PCC6803 (Syn6803) uses Type IV pili (TFP) for surface-associated motility and light-directed phototaxis. We report the identification of a novel Chaperone-Usher (CU) system in Syn6803 that regulate secretion of minor pilins as a means of stabilizing TFP morphology. These secreted minor-pilins aid in modifying TFP morphology to suit the adhesion state by forming cell to surface contacts when motility is not required. This morphotype is structurally distinct from TFP assembled during motile phase. We further demonstrate by examining mutants lacking either the CU system or the minor-pilins, which produce aberrant TFP, that are morphologically and functionally distinct from wild-type (WT). Thus, here we report that in Syn6803, CU system work independent of TFP biogenesis machinery unlike reported for other pathogenic bacterial systems and contributes to provide multifunctional plasticity to TFP. cAMP levels play an important role in controlling this switch. This phenotypic plasticity exhibited by the TFP, in response to cAMP levels would allow cells and cellular communities to adapt to rapidly fluctuating environments by dynamically transitioning between motile and sessile states.\n\nSignificance of this workHow cyanobacterial communities cope with fluctuating or extreme environments is crucial in understanding their role in global carbon and nitrogen cycles. This work addresses the key question: how do cyanobacteria modulate external appendages, called Type IV pili, to effectively switch between motile and sessile biofilm states? We demonstrate that cells transition between forming strong cell-surface interactions indispensable for biofilm formation to forming cell-cell interactions that allow for coordinated movement crucial for social motility by functional/ structural modification of same TFP appendage. The second messenger, cAMP and a Chaperone-Usher secretion are indispensible to achieve these structural modifications of TFP and control the complex phenotypic transition. We have uncovered a strategy that Syn6803 has evolved to deal with molecular decision-making under uncertainty, which we call phenotypic plasticity. Here we demonstrate how a single motility appendage can be structurally modified to attain two antagonistic functions in order to meet the fluctuating environmental demands.

microbiology↗