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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 955 records · Page 53Linked to original sources

bioRxiv2017

Comparing Caenorhabditis elegans gentle and harsh touch response behavior using a multiplexed hydraulic microfluidic device

The roundworm Caenorhabditis elegans is an important model system for understanding the genetics and physiology of touch. Classical assays for C. elegans touch, which involve manually touching the animal with a probe and observing its response, are limited by their low throughput and qualitative nature. We developed a microfluidic device in which several dozen animals are subject to spatially localized mechanical stimuli with variable amplitude. The device contains 64 sinusoidal channels through which worms crawl, and hydraulic valves that deliver touch stimuli to the worms. We used this assay to characterize the behavioral responses to gentle touch stimuli and the less well studied harsh (nociceptive) touch stimuli. First, we measured the relative response thresholds of gentle and harsh touch. Next, we quantified differences in the receptive fields between wild type worms and a mutant with non-functioning posterior touch receptor neurons. We showed that under gentle touch the receptive field of the anterior touch receptor neurons extends into the posterior half of the body. Finally, we found that the behavioral response to gentle touch does not depend on the locomotion of the animal immediately prior to the stimulus, but does depend on the location of the previous touch. Responses to harsh touch, on the other hand, did not depend on either previous velocity or stimulus location. Differences in gentle and harsh touch response characteristics may reflect the different innervation of the respective mechanosensory cells. Our assay will facilitate studies of mechanosensation, sensory adaptation, and nociception.

bioengineering↗

bioRxiv2017

Platform for Closed Loop Neuromodulation Based on Dual Mode Biosignals

Closed loop neuromodulation, where the stimulation is controlled autonomously based on physiological events, has been more effective than open loop techniques. In the few existing closed loop implementations which have a feedback, indirect non-neurophysiological biomarkers have been typically used (e.g. heart rate, stomach distension). Although these biomarkers enable automatic initiation of neural stimulation, they do not enable intelligent control of stimulation dosage. In this paper, we present a novel closed loop neuromodulation platform based on a dual signal mode that is detecting electrical and chemical signatures of neural activity. We demonstrated it on a case of vagus nerve stimulation (VNS). Vagal chemical (pH) signal is detected and used for initiatisng VNS and vagal compound action potential (CAP) signals are used to determine the stimulation dosage and pattern. Although we used the paradigm of appetite control and neurometabolic therapies, the platform developed here can be utilised for prototyping closed loop neuromodulation systems before adapting the final System-on-Chip (SoC) design.

bioengineering↗

bioRxiv2017

Forward Genetic ENU Mutagenesis Screen for Mouse Models of Chronic Fatigue Identifies a Novel Mutation in Slc2a4 (GLUT4)

In a screen of voluntary wheel-running behavior designed to identify genetic mouse models of chronic fatigue in ENU mutagenized C57BL/6J mice, we discovered two lines that showed aberrant wheel-running patterns. These lines both stem from a single original founder identified as a low body-weight candidate in a recessive screen. Progeny from both of these lines showed the abnormal wheel-running behavior, with affected mice showing significantly lower daily activity levels than unaffected mice. They also exhibited low amplitude circadian rhythms, consisting of lower activity levels during the normal active phase, and increased levels of activity during the rest or light phase, but only a modest alteration in free-running period. Their activity is not consolidated into longer bouts, but is frequently interrupted with periods of inactivity throughout the dark phase of the light-dark (LD) cycle. As seen with the low body weight, expression of the behavioral phenotypes in offspring of strategic crosses was consistent with a recessive heritance pattern. Mapping of these phenotypic abnormalities showed linkage to a single locus on chromosome 11, and whole exome sequencing (WES) identified a single point mutation in the Slc2a4 gene encoding the GLUT4 insulin-responsive glucose transporter. The single nucleotide change (A to T) was found in the distal end of exon 10, and results in a premature stop (Y440*). To our knowledge, this is the first time a mutation in this gene has been shown to result in extensive changes in general behavioral patterns.\n\nSIGNIFICANCE STATEMENTChronic fatigue is a debilitating and devastating disorder with widespread consequences for both the patient and the persons around them, but effective treatment strategies are lacking. The identification of novel genetic mouse models of chronic fatigue may prove invaluable for the study of its underlying physiological mechanisms and for the testing of treatments and interventions. A novel mutation in Slc2a4 (GLUT4) was identified in a forward mutagenesis screen because affected mice showed abnormal daily patterns and levels of wheel running consistent with chronic fatigue. This new mouse model may shed light on the pathophysiology of chronic fatigue.

genetics↗

bioRxiv2017

ScreenCube: A 3D Printed System For Rapid and Cost-Effective Chemical Screening in Adult Zebrafish

Phenotype-based small molecule screens in zebrafish embryos and larvae have been successful in accelerating pathway and therapeutic discovery for diverse biological processes. Yet, the application of chemical screens to adult physiologies has been relatively limited due to additional demands on cost, space, and labor associated with screens in adult animals. Here, we present a 3D printed system and methods for intermittent drug dosing that enable rapid and cost-effective chemical administration in adult zebrafish. Using pre-filled screening plates, the system enables dosing of 96 fish in {small tilde}3min, with a tenfold-reduction in drug quantity compared to that used in previous chemical screens in adult zebrafish. We characterize water quality kinetics during immersion in the system, and use these kinetics to rationally design intermittent dosing regimens that result in 100% fish survival. As a demonstration of system fidelity, we show the potential to identify two known chemical inhibitors of adult tail fin regeneration, cyclopamine and dorsomorphin. By developing methods for rapid and cost-effective chemical administration in adult zebrafish, this study expands the potential for small molecule discovery in post-embryonic models of development, disease, and regeneration.

pharmacology and toxicology↗

bioRxiv2017

Advanced processing and analysis of conventional confocal microscopy generated scanning FCS data

Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

biophysics↗

bioRxiv2017

Light and growth form interact to shape stomatal ratio among British angiosperms

O_LIIn most plants, stomata are located only on the abaxial leaf surface (hypostomy), but many plants have stomata on both surfaces (amphistomy). High light and herbaceous growth form have been hypothesized to favor amphistomy, but these hypotheses have not been rigourously tested together using phylogenetic comparative methods.\nC_LIO_LII leveraged a large dataset including stomatal ratio, Ellenberg light indicator value, growth form, and phylogenetic relationships for 372 species of British angiosperms. I used phylogenetic comparative methods to test how light and/or growth form influence stomatal ratio and density.\nC_LIO_LIHigh light and herbaceous growth form are correlated with amphistomy, as predicted, but they also interact; the effect of light is pronounced in therophytes (annuals) and perennial herbs, but muted in phanerophytes (shrubs and trees). Furthermore, amphistomy and stomatal density evolve together in response to light.\nC_LIO_LIComparative analyses of British angiosperms reveal two major insights into physiological evolution. First, light and growth form interact to shape variation in stomatal ratio; amphistomy is common under high light, but mostly for herbs. Second, coordinated evolution of adaxial stomatal density and light tolerance indicates that amphistomy is an important adaptation to optimally balance light acquisition with gas exchange. These results advance our understanding of why stomatal ratio evolves and its potential as a functional trait for paleoecology and crop improvement.\nC_LI

evolutionary biology↗

bioRxiv2017

Dark period transcriptomic and metabolic profiling of two diverse Eutrema salsugineum accessions

Eutrema salsugineum is a model species for the study of plant adaptation to abiotic stresses. Two accessions of E. salsugineum, Shandong (SH) and Yukon (YK), exhibit contrasting morphology, biotic, and abiotic stress tolerance. Transcriptome and metabolic profiling from tissue samples collected during the dark period were used to investigate the molecular and metabolic bases of these contrasting phenotypes. RNA sequencing identified 17,888 expressed genes, of which 157 were not in the published reference genome and 65 were detected for the first time. Differential expression was detected for only 31 genes. The RNA sequencing data contained 14,808 single nucleotide polymorphisms (SNPs) in transcripts, 3,925 of which are newly identified. Among the differentially expressed genes, there were no obvious candidates for the physiological or morphological differences between SH and YK. Metabolic profiling indicated that YK accumulates free fatty acids and long-chain fatty acid derivatives as compared to SH; whereas sugars are more abundant in SH. Metabolite levels suggest that carbohydrate and respiratory metabolism, including starch degradation, is more active during the first half of the dark period in SH. These metabolic differences may explain the greater biomass accumulation in YK over SH. The accumulation of 56% of the identified metabolites was lower in F1 hybrids than the mid-parent averages and the accumulation of 17% of the metabolites in F1 plants transgressed the level in both parents. Concentrations of several metabolites in F1 hybrids agree with previous studies and suggest a role for primary metabolism in heterosis. The improved annotation of the E. salsugineum genome and newly-identified high-quality SNPs will permit accelerated studies using the standing variation in this species to elucidate the mechanisms of its diverse adaptations to the environment.

plant biology↗

bioRxiv2017

Songbird organotypic culture as an in vitro model for interrogating sparse sequencing networks

Sparse sequences of neuronal activity are fundamental features of neural circuit computation; however, the underlying homeostatic mechanisms remain poorly understood. To approach these questions, we have developed a method for cellular-resolution imaging in organotypic cultures of the adult zebra finch brain, including portions of the intact song circuit. These in vitro networks can survive for weeks, and display mature neuron morphologies. Neurons within the organotypic slices exhibit a diversity of spontaneous and pharmacologically induced activity that can be easily monitored using the genetically encoded calcium indicator GCaMP6. In this study, we primarily focus on the classic song sequence generator HVC and the surrounding areas. We describe proof of concept experiments including physiological, optical, and pharmacological manipulation of these exposed networks. This method may allow the cellular rules underlying sparse, stereotyped neural sequencing to be examined with new degrees of experimental control.\n\nHighlightsO_LIOrganotypic brain slices from adult zebra finch (Taeniopygia guttata), expressing the calcium indicator GCaMP6, can be cultured and maintained for at least several weeks and display spontaneous and evoked calcium transients.\nC_LI

neuroscience↗

bioRxiv2018

HRS1/HHOs GARP transcription factors and reactive oxygen species are regulators of Arabidopsis nitrogen starvation response.

Plants need to cope with strong variations in the nitrogen content of the soil solution. Although many molecular actors are being discovered concerning how plants perceive NO3- provision, it is less clear how plants recognize a lack of Nitrogen. Indeed, following N removal plants activate their Nitrogen Starvation Response (NSR) being characterized in particular by the activation of very high affinity nitrate transport systems (NRT2.4, NRT2.5) and other sentinel genes such as GDH3. Here we show using a combination of functional genomics (via TF perturbation) and molecular physiology studies, that the GARP Transcription Factors (TFs) belonging the HHO sub-family are important regulators of the NSR through two potential mechanisms. First, HHOs directly repress NRT2.4 and NRT2.5 high-affinity nitrate transporters. Genotypes affected in HHO genes (mutants and overexpressors) display modified high-affinity nitrate transport activities opening interesting perspectives in biotechnology applications. Second, we show that Reactive Oxygen Species (ROS) are important to control NSR in wild type plants and that HRS1 and HHO1 overexpressors are affected in their ROS content, defining a potential feedforward branch of the signaling pathway. Taken together our results define two new classes of molecular actors in the control of NSR including ROS and the first transcription factors to date. This work (i) opens perspectives on a poorly understood nutrient related signaling pathway, and (ii) defines targets for molecular breeding of plants with enhanced NO3- uptake.

plant biology↗

bioRxiv2017

Engineered CRISPR-Cas9 system enables noiseless, fine-tuned and multiplexed repression of bacterial genes

Over the past few years, tools that make use of the Cas9 nuclease have led to many breakthroughs, including in the control of gene expression. The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. Here, we reveal that the level of complementarity between the guide RNA and the target controls the rate at which dCas9 successfully blocks the RNA polymerase. We use this mechanism to precisely and robustly reduce gene expression by defined relative amounts. We demonstrate broad applicability of this method to the study of genetic regulation and cellular physiology. First, we characterize feedback strength of a model auto-repressor. Second, we study the impact of copy-number variations of cell-wall synthesizing enzymes on cell morphology. Finally, we demonstrate that this system can be multiplexed to obtain any combination of fractional repression of two genes.

synthetic biology↗

bioRxiv2017

Elemental Accumulation in Kernels of the Maize Nested Association Mapping Panel Reveals Signals of Gene by Environment Interactions

Elemental accumulation in seeds is the product of a combination of environment and a wide variety of genetically controlled physiological processes. We measured the kernel elemental composition of the Nested Association Mapping (NAM) of maize (Zea mays L.) grown in 4 different environments. Analysis of variance revealed strong effects of genotype, environment and genotype by environment interactions. Using Joint-linkage mapping on a set of 7000 markers we identified 354 quantitative trait loci (QTL) across 20 elements, four environments and a combination of the environments. Leveraging 20 M SNPs derived from genome resequencing on the parents of the population, genome-wide association mapping studies (GWAS) detected 8573 loci. While most of the GWAS SNPs were located near genes not previously implicated in elemental regulation, several SNPs were located next to orthologs of well-characterized elemental regulation genes.

plant biology↗

bioRxiv2017

Dendritic processing of spontaneous neuronal sequences for one-shot learning

Spontaneous firing sequences colloquially called \"preplay\" are fundamental features of hippocampal network physiology. Preplay sequences have been hypothesized to participate in hippocampal learning and memory, but such functional roles and their potential cellular mechanisms remain unexplored. Here, we report a computational model based on the functional propagation of preplay sequences in the CA3 neuronal network. The model instantiates two synaptic pathways in CA3 neurons, one for proximal dendrite-somatic interactions to generate intrinsic preplay sequences and the other for distal dendritic processing of extrinsic sensory signals. The core dendritic computation is the maximization of matching between patterned activities in the two compartments through nonlinear spike generation. The model performs robust one-shot learning with long-term stability and independence that are modulated by the plasticity of dendrite-targeted inhibition. This model demonstrates that learning models combined with dendritic computations can enable preplay sequences to act as templates for rapid and stable memory formation.

neuroscience↗

bioRxiv2017

Structure of the deactive state of mammalian respiratory complex I

Complex I (NADH:ubiquinone oxidoreductase) is central to energy metabolism in mammalian mitochondria. It couples NADH oxidation by ubiquinone to proton transport across the energy-conserving inner membrane, catalyzing respiration and driving ATP synthesis. In the absence of substrates, active complex I gradually enters a pronounced resting or deactive state. The active-deactive transition occurs during ischemia and is crucial for controlling how respiration recovers upon reperfusion. Here, we set a highly-active preparation of Bos taurus complex I into the biochemically-defined deactive state, and used single-particle electron cryomicroscopy to determine its structure to 4.1 [A] resolution. The deactive state arises when critical structural elements that form the ubiquinone-binding site become disordered, and we propose reactivation is induced when substrate binding templates their reordering. Our structure both rationalizes biochemical data on the deactive state, and offers new insights into its physiological and cellular roles.

biochemistry↗

bioRxiv2017

SANe: The Seed Active Network For Mining Transcriptional Regulatory Programs of Seed Development

Developing seeds undergo coordinated physiological and morphological changes crucial for development of the embryo, dormancy and germination. The metabolic changes that occur during seed development are regulated by interconnected network of Transcription Factors (TFs) that regulate gene expression in a spatiotemporal manner. The complexity of these networks is such that the TFs that play key regulatory roles during seed development are largely unknown. In this study, we created a genome-scale regulatory network dedicated to describing regulation of biological processes within various compartments and developmental stages of Arabidopsis seeds. Differential network analysis revealed key TFs that rewire their targeting patterns specifically during seed development, many of which were already known, and a few novel ones that we verified experimentally. Our method shows that a high-resolution tissue-specific transcriptome dataset can be accurately modeled as a functional regulatory network predictive of related TFs. We provide an easy to use webtool using which researchers can upload a newly generated transcriptome and identify key TFs important to their dataset as well as gauge their regulatory effect on phenotypes observed in the experiment. We refer to this network as Seed Active Network (SANe) and made it accessible at https://plantstress-pereira.uark.edu/SANe/. We anticipate SANe will facilitate the discovery of TFs yet unknown for their involvement in seed related metabolic pathways and provide an interface to generate new hypothesis for experimentation.

bioinformatics↗

bioRxiv2017

Naked mole-rat acid-sensing ion channel 3 forms non-functional homomers, but functional heteromers

Acid-sensing ion channels (ASICs) form both homotrimeric and heterotrimeric ion channels that are activated by extracellular protons and are involved in a wide range of physiological and pathophysiological processes, including pain and anxiety. ASIC proteins can form both homotrimeric and heterotrimeric ion channels. The ASIC3 subunit has been shown to be of particular importance in the peripheral nervous system with pharmacological and genetic manipulations demonstrating a role in pain. Naked mole-rats, despite having functional ASICs, are insensitive to acid as a noxious stimulus and show diminished avoidance of acidic fumes, ammonia and carbon dioxide. Here we cloned naked mole-rat ASIC3 (nmrASIC3) and used a cell surface biotinylation assay to demonstrate that it traffics to the plasma membrane, but using whole-cell patch-clamp electrophysiology we observed that nmrASIC3 is insensitive to both protons and the non-proton ASIC3 agonist 2-Guanidine-4-methylquinazoline (GMQ). However, in line with previous reports of ASIC3 mRNA expression in dorsal root ganglia (DRG) neurons, we found that the ASIC3 antagonist APETx2 reversibly inhibits ASIC-like currents in naked mole-rat DRG neurons. We further show that like the proton-insensitive ASIC2b and ASIC4, nmrASIC3 forms functional, proton sensitive heteromers with other ASIC subunits. An amino acid alignment of ASIC3s between 9 relevant rodent species and human identified unique sequence differences that might underlie the proton insensitivity of nmrASIC3. However, introducing nmrASIC3 differences into rat ASIC3 (rASIC3) produced only minor differences in channel function, and replacing nmrASIC3 sequence with that of rASIC3 did not produce a proton-sensitive ion channel. Our observation that nmrASIC3 forms nonfunctional homomers may reflect a further adaptation of the naked mole-rat to living in an environment with high-carbon dioxide levels.

neuroscience↗

bioRxiv2017

Limited genetic variation for male mating success reveals low evolutionary potential for thermal plasticity in Drosophila melanogaster

Populations respond to novel environmental challenges either through genetic changes, through adaptive phenotypic plasticity for the traits in question, or by a combination of these factors. Here, we investigated the evolutionary potential of phenotypic plasticity for male mating success, locomotory ability, and heating rate (a physiological performance trait) in the fruitfly Drosophila melanogaster, using isogenic male lines from the Drosophila Reference Genome Panel (DGRP) and hemi-clonal males. We quantified thermal reaction norms of how male mating success changed in relation to a temperate gradient, ranging from cold (18 {degrees}C) via optimal (24 {degrees}C) to hot and stressful environments (either 30 {degrees}C or 36 {degrees}C). We found significant differences in male mating success and locomotory performance between different lines, as well as significant main effects of temperature, but no significant genotype-by-environment interactions (GEI:s). A statistical power analysis revealed that the variance explained by GEI:s for thermal plasticity using this sample size is likely to be modest or very small, and represent only 4% of the total variation in male mating success. The lack of strong GEI:s for these two behavioral traits contrast with the presence of significant GEI:s for male heating rate, as measured by thermal imaging (infrared camera technology). These results suggest that sexual selection through male mating success is not likely to be efficient in mediating evolutionary rescue through changed plasticity in response to changing temperatures.

evolutionary biology↗

bioRxiv2017

ESCRT-Independent Surface Receptor And Transporter Protein Degradation By The ILF Pathway

Surface receptor and transporter protein down-regulation drives cell signaling, quality control and metabolism underlying diverse physiology. After endocytosis, proteins are delivered to endosomes where ESCRTs package them into intralumenal vesicles, which are degraded by acid hydrolases upon fusion with lysosomes. However, reports of ESCRT-independent surface protein degradation are emerging suggesting that alternative, non-canonical pathways exist. Using Saccharomyces cerevisiae as a model, here we show that in response to substrates, protein misfolding or TOR signaling, some internalized surface transporters (Hxt3, Itr1, Aqr1) bypass ESCRTs en route to the lysosome membrane where they are sorted into an area that is internalized as an intralumenal fragment (ILF) and degraded upon organelle fusion. This ILF pathway also degrades typical ESCRT client proteins (Mup1, Can1, Ste3) when ESCRT function is impaired. As the underlying machinery is conserved, we speculate that the ILF pathway is an important contributor to receptor and transporter down-regulation in all eukaryotes.

cell biology↗

bioRxiv2017

The photocycle of orange carotenoid protein conceals distinct intermediates and asynchronous changes in the carotenoid and protein components

The 35 kDa water-soluble Orange Carotenoid Protein (OCP) is responsible for photoprotection in cyanobacteria. It acts as a light intensity sensor that simultaneously serves as efficient quencher of phycobilisome excitation energy as well as of reactive oxygen species. Photoactivation triggers large-scale conformational rearrangements to convert OCP from the orange OCPO state to the red active signaling state OCPR, as demonstrated by various structural methods. Eventually, such rearrangements imply complete yet reversible separation of structural domains (C- and N-terminal domain) and significant translocation of the carotenoid cofactor. Very recently, dynamic crystallography of OCPO crystals suggested the existence of photocycle intermediates with small-scale rearrangements that may trigger further transitions in the protein. However, the currently existing gap between the ultra-fast picosecond and 100 millisecond time scale of spectroscopic and structural data precludes knowledge about distinct intermediate states. In this study, we took advantage of single 7 ns laser pulses to study carotenoid absorption transients in OCP on the time-scale from 100 ns to 10 s, which allowed us to detect a red intermediate state preceding the red signaling state OCPR. In addition, time-resolved fluorescence spectroscopy and following assignment of carotenoid-induced quenching of different tryptophan residues revealed a novel orange intermediate state, which appears during back-relaxation of photoactivated OCPR to OCPO. Our results show asynchronous changes in the carotenoid and protein components and provide refined mechanistic information about the OCP photocycle as well as introduce new kinetic signatures for future studies of OCP photoactivity and photoprotection.\n\nSignificance statementCyanobacteria utilize the Orange Carotenoid Protein (OCP) to protect their photosynthetic apparatus from the harmful effects of intense sunlight. OCP is a blue light-triggered photoswitch, which undergoes photoconversion from its dark adapted orange to the active red state, the latter being able to interact with the phycobilisome antennae and quench their fluorescence, thus avoiding excessive energy flow to the photosystems. With the help of the fluorescence recovery protein (FRP), OCP detaches from phycobilisomes and can return faster into the orange state. Until now, only the thermodynamically stable orange state and the metastable red state are established in a primitive photocycle. In this work, we apply transient absorption and fluorescence spectroscopy and identify two novel photocycle intermediates of physiological relevance.

biophysics↗