Search bioRxiv⌕ Search

SEARCH · Search bioRxiv

Results for “Physiology”

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 901 records · Page 50Linked to original sources
bioRxiv2017

Genome-Enabled Insights Into The Ecophysiology Of The Comammox Bacterium Candidatus Nitrospira nitrosa

The recently discovered comammox bacteria have the potential to completely oxidize ammonia to nitrate. These microorganisms are part of the Nitrospira genus and are present in a variety of environments, including Biological Nutrient Removal (BNR) systems. However, the physiological traits within and between comammox- and nitrite oxidizing bacteria (NOB)-like Nitrospira species have not been analyzed in these ecosystems. In this study, we identified Nitrospira strains dominating the nitrifying community of a sequencing batch reactor (SBR) performing BNR under micro-aerobic conditions. We recovered metagenomes-derived draft genomes from two Nitrospira strains: (1) Nitrospira sp. UW-LDO-01, a comammox-like organism classified as Candidatus Nitrospira nitrosa, and (2) Nitrospira sp. UW-LDO-02, a nitrite oxidizing strain belonging to the Nitrospira defluvii species. A comparative genomic analysis of these strains with other Nitrospira-like genomes identified genomic differences in Ca. Nitrospira nitrosa mainly attributed to each strains niche adaptation. Traits associated with energy metabolism also differentiate comammox from NOB-like genomes. We also identified several transcriptionally regulated adaptive traits, including stress tolerance, biofilm formation and micro-aerobic metabolism, which might explain survival of Nitrospira under multiple environmental conditions. Overall, our analysis expanded our understanding of the genetic functional features of Ca. Nitrospira nitrosa, and identified genomic traits that further illuminate the phylogenetic diversity and metabolic plasticity of the Nitrospira genus.

microbiology↗
bioRxiv2017

Contextual Tumor Suppressor Function Of T Cell Death-Associated Gene 8 (TDAG8) In Hematological Malignancies

Key points1. TDAG8 (GPR65) gene expression is down-regulated in hematological malignancies.\n\n2. Restoration of TDAG8 gene expression in blood cancer cells suppresses tumor growth and metastasis.\n\nAbstractExtracellular acidosis is a condition found within the tumor microenvironment due to inadequate blood perfusion, hypoxia, and altered tumor cell metabolism. Acidosis has pleiotropic effects on malignant progression; therefore it is essential to understand how acidosis exerts its diverse effects. In this study a bioinformatics analysis revealed the expression of the proton sensing G-protein-coupled receptor TDAG8 is significantly reduced in human blood cancers in comparison to normal blood cells. To understand how TDAG8 functions in hematological malignancies, TDAG8 expression was restored in U937 acute myeloid leukemia cells and other blood cancer cells. It was discovered that severe acidosis, pH 6.4, inhibited U937 cell proliferation while mild acidosis, pH 6.9, stimulated cell proliferation. However, restoring TDAG8 gene expression modulated the U937 cell response to mild extracellular acidosis and physiological pH by reducing cell proliferation. Tumor xenograft experiments further revealed that restoring TDAG8 expression in U937 and Ramos cancer cells reduced tumor growth. It was also shown U937 cells with restored TDAG8 expression attached less to Matrigel, migrated slower toward a chemoattractant, and metastasized less in severe combined immunodeficient mice. These effects correlated with a reduction in c-myc oncogene expression. The mechanistic investigation indicated that G13/RhoA signaling arbitrated the TDAG8-mediated c-myc oncogene repression in response to acidosis. Overall, this study provides compelling data to support the concept that TDAG8 functions as a contextual tumor suppressor in hematological malignancies and sensitizes blood cancer cells to acidotic stress.

cancer biology↗
bioRxiv2017

Classifying Drosophila Olfactory Projection Neuron Subtypes by Single-cell RNA Sequencing

How a neuronal cell type is defined and how this relates to its transcriptome are still open questions. The Drosophila olfactory projection neurons (PNs) are among the best-characterized neuronal types: Different PN classes target dendrites to distinct olfactory glomeruli and PNs of the same class exhibit indistinguishable anatomical and physiological properties. Using single-cell RNA-sequencing, we comprehensively characterized the transcriptomes of 40 PN classes and unequivocally identified transcriptomes for 6 classes. We found a new lineage-specific transcription factor that instructs PN dendrite targeting. Transcriptomes of closely-related PN classes exhibit the largest difference during circuit assembly, but become indistinguishable in adults, suggesting that neuronal subtype diversity peaks during development. Genes encoding transcription factors and cell-surface molecules are the most differentially expressed, indicating their central roles in specifying neuronal identity. Finally, we show that PNs use highly redundant combinatorial molecular codes to distinguish subtypes, enabling robust specification of cell identity and circuit assembly.

genomics↗
bioRxiv2017

Genetically modified human type II collagen for N- and C-terminal covalent tagging

Collagen is the predominant structural protein in vertebrates, where it contributes to connective tissues and the extracellular matrix; it is also widely used in biomaterials and tissue engineering. Dysfunction of this protein and its processing can lead to a wide variety of developmental disorders and connective tissue diseases. Recombinantly engineering the protein is challenging due to posttranslational modifications generally required for its stability and secretion from cells. Introducing end labels into the protein is problematic, because the N- and C-termini of the physiologically relevant tropocollagen lie internal to the initially flanking N- and C-propeptide sequences. Here, we introduce mutations into human type II procollagen in a manner that address these concerns, and purify the recombinant protein from a stably transfected HT1080 human fibrosarcoma cell line. Our approach introduces chemically addressable groups into the N- and Ctelopeptide termini of tropocollagen. Simultaneous overexpression of formylglycine generating enzyme (FGE) allows the endogenous production of an aldehyde tag in a defined, substituted sequence in the N-terminus of the mutated collagen, while the C-terminus of each chain presents a sulfhydryl group from an introduced cysteine. These modifications are designed to enable specific covalent end-labelling of collagen. We find that the doubly-mutated protein folds and is secreted from cells, while higher-order assembly into well-ordered collagen fibrils is demonstrated through transmission electron microscopy. Chemical tagging of thiols is successful, however background from endogenous aldehydes present in wildtype collagen has thus far obscured the desired specific N-terminal labelling. Strategies to overcome this challenge are proposed.

biochemistry↗
bioRxiv2017

Bacterial lipopolysaccharide reduces the stability of avian and human influenza viruses

Commensal bacteria can promote or reduce the severity of viral infection and disease progression in their hosts depending on the specific viral pathogen1. Influenza A virus (IAV) has a broad host-range, comprises many subtypes, and utilizes different routes of transmission including the fecal-oral route in wild birds2. It has been previously demonstrated that commensal bacteria can interact with the hosts immune system to protect against IAV pathogenesis3,4. However, it is unclear whether bacteria and their products may be interacting directly with IAV to impact virion stability. Herein we show that gastrointestinal (GI) tract bacterial isolates in an in vitro system significantly reduce the thermal stability of IAV. Moreover, bacterial lipopolysaccharide (LPS), found on the exterior surfaces of bacteria, was sufficient to significantly decrease the stability of both human and avian viral strains at the physiological temperatures of their respective hosts, as well as in the aquatic environment. Subtype and host-origin of the viruses were shown to affect the extent to which IAV was susceptible to LPS. Furthermore, using a receptor-binding assay and transmission electron microscopy, we observed that LPS binds to and affects the morphology of influenza virions.

microbiology↗
bioRxiv2017

Cooling Induced by Uphill Energy Transport in Plant Photosystems

The uphill energy transfer in photosystems implies input energy at higher wavelength leading to energy output at lower wavelength. Briefly, energy is uphill transported from photosystem I (PSI) to photosystem II (PSII), the latter having a lower wavelength emission. This uphill energy transport involves absorption of thermal energy from the surroundings. While such cooling effects have been reported in laser systems we report for the first time a white light driven cooling in thylakoid suspension. The cooling of the surrounding medium by appropriate illumination was illustrated using thermal measurements. Again cooling is inhibited by agents like 3-(3,4-Dichlorophenyl)-1,1-dimethylurea,that block the linear electron flow between the photocenters, implying a dependence of the cooling on interplay between such centers. Furthermore, it is possible to modulate the cooling pattern by addition of external agents like nanopaticles, some favoring further cooling (e.g., Ag nanoparticle) and some like Au or chlorophyll nanoparticles, showing insignificant or even reverse trends. Interestingly, the cooling is invariably associated with the 77K spectra of the thylakoid suspension. With reference to the dark control, an agent causing cooling always increases PSII to PSI ratio and vice versa i.e.,the uphill energy transport. Importantly, the cooling effect, apart from its import role in plant physiology can be exploited artificially for energy saving in post-harvest or food preservation.

bioengineering↗
bioRxiv2017

Analytical solution of a bilateral Brown-type central pattern generator for symmetric and asymmetric locomotion

The coordinated activity of muscles is produced in part by spinal rhythmogenic neural circuits, termed central pattern generators (CPGs). A classical CPG model, proposed conceptually by T.G. Brown in 1911, is a system of coupled oscillators that transform locomotor drive into coordinated and gait-specific patterns of muscle recruitment. A system of ordinary differential equations with a physiologically-inspired coupling locus of interactions captures the timing relationship for bilateral coordination of limbs in locomotion and is typically solved numerically. Consequently, it is intriguing to have a full analytical description of this plausible CPG architecture to illuminate the functionality within this structure. Here, we provided a closed-form analytical solution contrasted against the previous numerical method. The computational load of the analytical solution was decreased by an order of magnitude when compared to the numerical approach (relative errors, <0.01%). The analytical solution tested and supported the previous finding that the input to the model can be expressed in units of the desired limb locomotor speed. Furthermore, we performed parametric sensitivity analysis in the context of controlling asymmetric locomotion and documented two possible mechanisms associated with either an external drive or intrinsic CPG parameters. The results support the idea that many different combinations of network states, even within the same anatomical CPG structure, may generate the same behavioral outcomes.\n\nAuthor SummaryUsing a simple process of leaky integration, we developed an analytical solution to a robust model of spinal pattern generation. We analyzed the ability of this neural element to exert locomotor control of the signal associated with limb speeds, which represent high-level modality within the motor system. Furthermore, we tested the ability of this simple structure to embed steering control using the velocity signal in the models inputs or within the internal connectivity of its elements. Both mechanisms can produce the same behavioral outcome, pointing to the methodological challenges of modeling central pattern generators and demonstrating the possibility of spinal circuit adaptations to asymmetric short- or long-term conditions in health and disease.

neuroscience↗
bioRxiv2017

Non-invasive laminar inference with MEG: Comparison of methods and source inversion algorithms

Magnetoencephalography (MEG) is a direct measure of neuronal current flow; its anatomical resolution is therefore not constrained by physiology but rather by data quality and the models used to explain these data. Recent simulation work has shown that it is possible to distinguish between signals arising in the deep and superficial cortical laminae given accurate knowledge of these surfaces with respect to the MEG sensors. This previous work has focused around a single inversion scheme (multiple sparse priors) and a single global parametric fit metric (free energy). In this paper we use several different source inversion algorithms and both local and global, as well as parametric and non-parametric fit metrics in order to demonstrate the robustness of the discrimination between layers. We find that only algorithms with some sparsity constraint can successfully be used to make laminar discrimination. Importantly, local t-statistics, global cross-validation and free energy all provide robust and mutually corroborating metrics of fit. We show that discrimination accuracy is affected by patch size estimates, cortical surface features, and lead field strength, which suggests several possible future improvements to this technique. This study demonstrates the possibility of determining the laminar origin of MEG sensor activity, and thus directly testing theories of human cognition that involve laminar- and frequency- specific mechanisms. This possibility can now be achieved using recent developments in high precision MEG, most notably the use of subject-specific head-casts, which allow for significant increases in data quality and therefore anatomically precise MEG recordings.

neuroscience↗
bioRxiv2017

Subgraphs of functional brain networks identify dynamical constraints of cognitive control

Brain anatomy and physiology support the human ability to navigate a complex space of perceptions and actions. To maneuver across an ever-changing landscape of mental states, the brain invokes cognitive control - a set of dynamic processes that engage and disengage different sets of brain regions to modulate attention, switch between tasks, and inhibit prepotent responses. Current theory suggests that cooperative and competitive interactions between brain areas may mediate processes of network reorganization that support transitions between dynamical states. In this study, we used a quantitative approach to identify distinct topological states of functional interactions and examine how their expression relates to cognitive control processes and behavior. In particular, we acquired fMRI BOLD signal in twenty-eight healthy subjects as they performed two cognitive control tasks - a local-global perception switching task using Navon figures and a Stroop interference task - each with low cognitive control demand and high cognitive control demand conditions. Based on these data, we constructed dynamic functional brain networks and used a parts-based network decomposition technique called non-negative matrix factorization to identify putative cognitive control subgraphs whose temporal expression captured key dynamical states involved in control processes. Our results demonstrate that the temporal expression of these functional subgraphs reflect cognitive demands and are associated with individual differences in task-based performance. These findings offer insight into how coordinated changes in the cooperative and competitive roles of distributed brain networks map trajectories between cognitively demanding brain states.

neuroscience↗
bioRxiv2017

Complex spatio-temporal distribution and genogeographic affinity of mitochondrial DNA haplogroups in 24,216 Danes

Mitochondrial DNA (mtDNA) haplogroups (hgs) are evolutionarily conserved sets of mtDNA SNP-haplotypes with characteristic geographical distribution. Associations of hgs with disease and physiological characteristics have been reported, but have frequently not been reproducible. Using 418 mtDNA SNPs on the PsychChip (Illumina), we assessed the spatio-temporal distribution of mtDNA hgs in Denmark in DNA isolated from 24,642 geographically un-biased dried blood spots (DBS), collected from 1981 to 2005 through the Danish National Neonatal Screening program. Geno-geographic affinity (ancestry background) was established with ADMIXTURE using a reference of 100K+ autosomal SNPs in 2,248 individuals from nine populations. The hg distribution was typically Northern European, and hgs were highly variable based on median-joining analysis, suggesting multiple founder events. Considerable heterogeneity and variation in autosomal geno-geographic affinity was observed. Thus, individuals with hg H exhibited 95 %, and U hgs 38.2 % - 92.5 %, Danish ancestry. Significant clines between geographical regions and rural and metropolitan populations were found. Over 25 years, macro-hg L increased from 0.2 % to 1.2 % (p = 1.1*E-10), and M from 1 % to 2.4 % (p = 3.7*E-8). Hg U increased among the R macro-hg from 14.1 % to 16.5 % (p = 1.9*E-3). Geno-geographic affinity, geographical skewedness, and sub-hg distribution suggested that the L, M and U increases are due to immigration. The complex spatio-temporal dynamics and geno-geographic heterogeneity of mtDNA in the Danish population reflect repeated migratory events and, in later years, net immigration. Such complexity may explain the often contradictory and population-specific reports of mito-genomic association with disease.

genomics↗
bioRxiv2017

Parallel epigenetic modifications induced by hatchery rearing in a Pacific Salmon

Highlights- First study to highlight parallel epigenetic modifications induced by hatchery rearing as a potential explanatory mechanism for rapid change in fitness\n\nSummaryA puzzling question in conservation biology is how to maintain overall fitness of individuals bred in captive environment upon release into the wild, especially for rehabilitating declining or threatened species [1,2]. For salmonid species, a heritable change in fitness related traits and gene expression has been reported to occur in a single generation of captivity in hatchery environment [3-5]. Such rapid changes are congruent with models of inadvertent domestication selection which may lead to maladaptation in the natural environment [4]. Arguably, the underlying mechanism by which captivity may induce fitness difference between wild and captive congeners is still poorly understood. Short-term selection on complex phenotypic traits is expected to induce subtle changes in allele frequency over multiple loci [7-9]. Yet, most studies investigating the molecular basis for rapid change in fitness related traits occurring in hatchery have concentrated their effort on finding evidence for selection at the genome level by identifying loci with large effect.\n\nNumerous wild stocks of Pacific anadromous salmon and trout (genus Oncorhynchus and Salmo) have experienced fluctuating abundance over the past century, with a series of sharp declines [6-8]. With the objectives of preserving ecosystem integrity, enhancing declining populations and sustaining fisheries, conservation hatcheries have been flourishing. This is particularly true along the North American Pacific coast where billions of salmonids, all species included, are released each year. Despite substantial improvement of production management, the beneficial ecological role of hatcheries in enhancing and restoring wild stocks is still debated, mainly because of the reduced fitness and maladaptation of hatchery-fish when released in the wild [3,5,9]. Although previous studies showed that domestication selection was involved in such fitness impairment, they also observed that different environmental conditions (e.g. reduced fish density) significantly modulated the physiological acclimation to hatchery environment [4].\n\nEnvironmental stimuli are especially relevant during early embryonic development, which also correspond to a sensitive methylation reprogramming window in vertebrates [10,11]. It is therefore plausible that differences in rearing environment during early development may result in epigenetic modifications that could in turn impact on fitness. However, the only epigenetic study to date pertaining to captive rearing in salmonids and performed using methylation-sensitive amplified fragments (MSAP) failed to identify significant changes in methylation profile associated with hatchery rearing [12]\n\nHere, we used a higher resolution approach to compare the genome-wide pattern of methylation in hatchery-reared juvenile (smolt) Coho Salmon with that of their wild counterparts in two geographically distant rivers in British Columbia, Canada. Using a reduced representation bisulfite sequencing (RRBS) approach covering an average per individual of about 70 million cytosines in CpG context, we identified 100 methylated regions (DMRs) that differed in parallel between hatchery and natural origin salmon in both rivers. The total variance of epigenetic variation among individuals explained by river or origin and rearing environment in a RDA model was 16% (adj.R2=0.16), and both variables equally explained about 8% of the variance after controlling for each other. The gene ontology analysis revealed that regions with different methylation levels between hatchery and natural origin salmon showed enrichment for ion homeostasis, synaptic and neuromuscular regulation, immune and stress response, and control of locomotion functions. We further identified 15,044 SNPs that allowed detection of significant differences between either rivers or sexes. However, no effect of rearing environment was observed, confirming that hatchery and natural origin fish of a given river belong to the same panmictic population, as expected based on the hatchery programs applied in these rivers (see Supplementary experimental procedures). Moreover, neither a standard genome-scan approach nor a polygenic statistical framework allowed detection of selective effects within a single generation between hatchery and natural origin salmon. Therefore, this is the first study to demonstrate that parallel epigenetic modifications induced by hatchery rearing during early development may represent a potential explanatory mechanism for rapid change in fitness-related traits previously reported in salmonids.

genomics↗
bioRxiv2017

Sperm chemotaxis is driven by the slope of the chemoattractant concentration field

Spermatozoa of marine invertebrates are attracted to their conspecific female gamete by diffusive molecules, called chemoattractants, released from the egg investments in a process known as chemotaxis. The information from the egg chemoattractant concentration field is decoded into intracellular Ca2+ concentration ([Ca2+]i) changes that regulate the internal motors that shape the flagellum as it beats. By studying sea urchin species-specific differences in sperm chemoattractant-receptor characteristics we show that receptor density constrains the steepness of the chemoattractant concentration gradient detectable by spermatozoa. Through analyzing different chemoattractant gradient forms, we demonstrate for the first time that Strongylocentrotus purpuratus sperm are chemotactic and this response is consistent with frequency entrainment of two coupled physiological oscillators: i) the stimulus function and ii) the [Ca2+]i changes. We demonstrate that the slope of the chemoattractant gradients provides the coupling force between both oscillators, arising as a fundamental requirement for sperm chemotaxis.

biophysics↗
bioRxiv2017

The Ras-like GTPase Rem2 is a potent endogenous inhibitor of calcium/calmodulin-dependent kinase II activity

CaMKII is a well-characterized, abundant protein kinase that regulates a diverse set of functions in a tissue specific manner. For example, in heart muscle, CaMKII regulates Ca2+ homeostasis while in neurons CaMKII regulates activity-dependent dendritic remodeling and Long Term Potentiation (LTP), a biological correlate of learning and memory. Previously, we identified the noncanonical GTPase Rem2 as a critical regulator of dendrite branching and synapse formation in the vertebrate nervous system. Here, we report that Rem2 directly interacts with CaMKII and potently inhibits the activity of the intact holoenzyme, a previously undescribed function for the Rem2 protein. To date, only one other endogenous inhibitor of CaMKII has been described: CaMKIIN, which blocks CaMKII activity through binding to the catalytic domain. Our data suggest that Rem2 inhibits CaMKII through a novel mechanism, as inhibition requires the presence of the association domain of CaMKII. Our biochemical finding that Rem2 is a direct, endogenous inhibitor of CaMKII activity, coupled with known functions of Rem2 in neurons, provides a framework which will enable future experiments probing the physiological role of CaMKII inhibition in a cellular context.

biochemistry↗
bioRxiv2017

A vacuum-actuated microtissue stretcher for long-term exposure to oscillatory strain within a 3D matrix

Although our understanding of cellular behavior in response to extracellular biological and mechanical stimuli has greatly advanced using conventional 2D cell culture methods, these techniques lack physiological relevance. We developed the microtissue vacuum-actuated stretcher (MVAS) to probe cellular behavior within a 3D multicellular environment composed of innate matrix protein, and in response to continuous uniaxial stretch. The MVAS consists of an array of fifty self-assembled microtissues bordered by vacuum chambers. When a vacuum is applied, the microtissues stretch in plane allowing live imaging. The MVAS is highly suitable for biomedical research and pharmaceutical discovery due to a high-throughput array format and scalable fabrication steps outlined in this paper. We validated our approach by characterizing the bulk microtissue strain, the microtissue strain field and single cell strain, and by assessing F-actin expression in response to chronic cyclic strain of 10%. The MVAS was shown to be capable of delivering reproducible dynamic bulk strain amplitudes up to 13% and the strain field had local maxima around each of the cantilevers. The strain at the single cell level was found to be 10.4% less than the microtissue axial strain due to cellular rotation. Chronic cyclic strain produced a 35% increase in F-actin expression consistent with previously observed cytoskeletal reinforcement in 2D cell culture. The MVAS may further our understanding of the reciprocity shared between cells and their environment, which is critical to meaningful biomedical research and successful therapeutic approaches.

bioengineering↗
bioRxiv2017

Assessing tACS-induced phosphene perception using closed-loop Bayesian optimization

Transcranial alternating current stimulation (tACS) can evoke illusory flash-like visual percepts known as phosphenes. The perception of phosphenes represents a major experimental challenge when studying tACS-induced effects on cognitive performance. Besides growing concerns that retinal phosphenes themselves could potentially have neuromodulatory effects, the perception of phosphenes may also modify the alertness of participants. Past research has shown that stimulation intensity, frequency and electrode montage affect phosphene perception. However, to date, the effect of an additional tACS parameter on phosphene perception has been completely overlooked: the relative phase difference between stimulation electrodes. This is a crucial and timely topic given the confounding nature of phosphene perception and the increasing number of studies reporting changes in cognitive function following tACS phase manipulations. However, studying phosphene perception for different frequencies and phases simultaneously is not tractable using standard approaches, as the physiologically plausible range of parameters results in a combinatorial explosion of experimental conditions, yielding impracticable experiment durations. To overcome this limitation, here we applied a Bayesian optimization approach to efficiently sample an exhaustive tACS parameter space. Moreover, unlike conventional methodology, which involves subjects judging the perceived phosphene intensity on a rating scale, our study leveraged the strength of human perception by having the optimization driven based on a subjects relative judgement. Applying Bayesian optimization for two different montages, we found that phosphene perception was affected by differences in the relative phase between cortical electrodes. The results were replicated in a second study involving new participants and validated using computational modelling. In summary, our results have important implications for the experimental design and conclusions drawn from future tACS studies investigating the effects of phase on cognition.

neuroscience↗
bioRxiv2017

Producing Hfq/Sm Proteins and sRNAs for Structural and Biophysical Studies of Ribonucleoprotein Assembly

Hfq is a bacterial RNA-binding protein that plays key roles in the post-transcriptional regulation of gene expression. Like other Sm proteins, Hfq assembles into toroidal discs that bind RNAs with varying affinities and degrees of sequence specificity. By simultaneously binding to a regulatory small RNA (sRNA) and an mRNA target, Hfq hexamers facilitate productive RNA[···]RNA interactions; the generic nature of this chaperone-like functionality makes Hfq a hub in many sRNA-based regulatory networks. That Hfq is crucial in diverse cellular pathways--including stress response, quorum sensing and biofilm formation-- has motivated genetic and RNAomic studies of its function and physiology (in vivo), as well as biochemical and structural analyses of Hfq[···]RNA interactions (in vitro). Indeed, crystallographic and bio-physical studies first established Hfq as a member of the phylogenetically-conserved Sm superfamily. Crystallography and other biophysical methodologies enable the RNA-binding properties of Hfq to be elucidated in atomic detail, but such approaches have stringent sample requirements, viz.: reconstituting and characterizing an Hfq*RNA complex requires ample quantities of well-behaved (sufficient purity, homogeneity) specimens of Hfq and RNA (sRNA, mRNA fragments, short oligoribonucleotides, or even single nucleotides). The production of such materials is covered in this Chapter, with a particular focus on recombinant Hfq proteins for crystallization experiments.\n\nAbbreviations\n\nJournal formatMethods in Molecular Biology (Springer Protocols series); this volume is entitled \"Bacterial Regulatory RNA: Methods and Protocols\"; an author guide is linked at http://www.springer.com/series/7651

biochemistry↗
bioRxiv2017

RNA-mediated Genomic Arrangements in Mammalian Cells

One of the hallmarks of cancer is the formation of oncogenic fusion genes as a result of chromosomal translocations. Fusion genes are presumed to occur prior to fusion RNA expression. However, studies have reported the presence of fusion RNAs in individuals who were negative for chromosomal translocations. These observations give rise to \"the cart before the horse\" hypothesis, in which fusion RNA precedes the fusion gene and guides the genomic rearrangements that ultimately result in gene fusions. Yet RNA-mediated genomic rearrangement in mammalian cells has never been demonstrated. Here we provide evidence that expression of a chimeric RNA drives formation of a specified gene fusion via genomic rearrangement in mammalian cells. The process is (1) specified by the sequence of chimeric RNA involved, (2) facilitated by physiological hormone levels, (3) permissible regardless of intra-chromosomal (TMPRSS2-ERG) or inter-chromosomal (TMPRSS2-ETV1) fusion, and (4) can occur in normal cells prior to malignant transformation. We demonstrate that, contrary to \"the cart before the horse\" model, it is the antisense rather than sense chimeric RNAs that effectively drive gene fusion, and that this disparity can be explained by transcriptional conflict. Furthermore, we identified an endogenous RNA AZI1 that acts as the initiator RNA to induce TMPRSS2-ERG fusion. RNA-driven gene fusion demonstrated in this report provides important insight in early disease mechanism, and could have fundamental implications in the biology of mammalian genome stability, as well as gene editing technology via mechanisms native to mammalian cells.

cancer biology↗
bioRxiv2017

Photosynthesis is heavily chlororespiration-sensitive under fluctuating light conditions

Photosynthesis needs to adjust to dynamically changing light intensities in order to maximize its efficiency, notably by the employment of alternative electron pathways. One of them is chlororespiration - initially described in Chlamydomonas reinhardtii. This electron transfer pathway, found in all photosynthetic lineages, consists of a reduction of plastoquinone (PQ) through an NAD(P)H:PQ oxidoreductase and quinol (PQH2) oxidation by Plastid Terminal Oxidase, PTOX. Hence, chlororespiration constitutes an electron pathway potentially antagonistic to the linear photosynthetic electron flow from H2O to CO2. However, the limited flow chlororespiratory enzymes can sustain suggests that their relative contribution, at least in the light and in steady-state conditions, is insubstantial. Here, we focused on the involvement of PTOX in Chlamydomonas reinhardtii during transitions from dark to light and vice versa. We show that the kinetics of redox relaxation of the chloroplast in the dark was greatly affected when PTOX2, the major plastoquinol oxidase in Chlamydomonas, is lacking. Importantly, we show that this has a direct physiological relevance, as the growth of a PTOX2-lacking mutant is markedly slower in intermittent light. The latter can be rationalized in terms of a decreased flux sustained by photosystem II due to a redox limitation at the acceptor side of the PSI during the illumination periods. We finally show that the long-term regulation of cyclic electron flow around PSI is strongly affected in the PTOX2 mutant, substantiating an important role of chlororespiration in the maintenance of chloroplast redox balance.

plant biology↗