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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 199 records · Page 11Linked to original sources

bioRxiv2017

Modeling neuron-glia interactions with the Brian 2 simulator

Despite compelling evidence that glial cells could crucially regulate neural network activity, the vast majority of available neural simulators ignores the possible contribution of glia to neuronal physiology. Here, we show how to model glial physiology and neuron-glia interactions in the Brian 2 simulator. Brian 2 offers facilities to explicitly describe any model in mathematical terms with limited and simple simulator-specific syntax, automatically generating high-performance code from the user-provided descriptions. The flexibility of this approach allows us to model not only networks of neurons, but also individual glial cells, electrical coupling of glial cells, and the interaction between glial cells and synapses. We therefore conclude that Brian 2 provides an ideal platform to efficiently simulate glial physiology, and specifically, the influence of astrocytes on neural activity.

neuroscience↗

bioRxiv2018

The UBR-1 Ubiquitin Ligase Regulates Glutamate Metabolism to Generate Coordinated Motor Pattern in C. elegans

UBR1 is an E3 ubiquitin ligase best known for its ability to target protein degradation by the N-end rule. The physiological functions of UBR family proteins, however, remain not fully understood. We found that the functional loss of C. elegans UBR-1 leads to a specific motor deficit: when adult animals generate reversal movements, A-class motor neurons exhibit synchronized activation, preventing body bending. This motor deficit is rescued by removing GOT-1, a transaminase that converts aspartate to glutamate. Both UBR-1 and GOT-1 are expressed and critically required in premotor interneurons of the reversal motor circuit to regulate the motor pattern. ubr-1 and got-1 mutants exhibit elevated and decreased glutamate level, respectively. These results raise an intriguing possibility that UBR proteins regulate glutamate metabolism, which is critical for neuronal development and signaling.\n\nAuthor SummaryUbiquitin-mediated protein degradation is central to diverse biological processes. The selection of substrates for degradation is carried out by the E3 ubiquitin ligases, which target specific groups of proteins for ubiquitination. The human genome encodes hundreds of E3 ligases; many exhibit sequence conservation across animal species, including one such ligase called UBR1. Patients carrying mutations in UBR1 exhibit severe systemic defects, but the biology behinds UBR1s physiological function remains elusive. Here we found that the C. elegans UBR-1 regulates glutamate level. When UBR-1 is defective, C. elegans exhibits increased glutamate; this leads to synchronization of motor neuron activity, hence defective locomotion when animals reach adulthood. UBR1-mediated glutamate metabolism may contribute to the physiological defects of UBR1 mutations.

neuroscience↗

bioRxiv2017

Superficial Slow Rhythms Integrate Cortical Processing in Humans

The neocortex is composed of six anatomically and physiologically specialized layers. It has been proposed that integration of activity across cortical areas is mediated anatomically by associative connections terminating in superficial layers, and physiologically by slow cortical rhythms. However, the means through which neocortical anatomy and physiology interact to coordinate neural activity remains obscure. Using laminar microelectrode arrays in 19 human participants, we found that most EEG activity is below 10-Hz (delta/theta) and generated by superficial cortical layers during both wakefulness and sleep. Cortical surface grid, grid-laminar, and dual-laminar recordings demonstrate that these slow rhythms are synchronous within upper layers across broad cortical areas. The phase of this superficial slow activity is reset by infrequent stimuli and coupled to the amplitude of faster oscillations and neuronal firing across all layers. These findings support a primary role of superficial slow rhythms in generating the EEG and integrating cortical activity.

neuroscience↗

bioRxiv2017

Reproductive mode, stem cells and regeneration in a freshwater cnidarian with post-reproductive senescence

In many basal metazoans both somatic and reproductive functions are performed by cellular derivatives of a single multipotent stem cell population. Reproduction can drain these stem cell pools, imposing a physiological cost with subsequent negative effects on somatic maintenance functions. In the freshwater cnidarian Hydra oligactis both asexual (budding) and sexual reproductive modes (production of resting eggs) are present, and both of these are dependent on a common pool of interstitial stem cells. Resting eggs tolerate abiotic conditions which neither the parental animals, nor asexual offspring can survive (e.g. freezing). Therefore, when facing unfavorable conditions and increased mortality risk, hydra polyps are expected to show higher differentiation of interstitial stem cells into germ cells (i.e. sexual reproduction), compared to other cell types needed for selfmaintenance or asexual reproduction. Here, by comparing sexually and asexually reproducing individuals to non-reproductives, we studied the physiological costs of reproduction (size of interstitial stem cell pools, their somatic derivatives and regeneration rate, which is dependent on these cell types) in H. oligactis polyps from a free-living Hungarian population prior to the onset of winter. Sexual individuals (but not asexuals) were characterized by significantly smaller interstitial stem cell pools, fewer somatic derivatives (nematoblasts involved in food capture) and lower regeneration ability compared to non-reproductives. We also found a negative correlation between germ cell counts and stem cell numbers in males (but not in females). These results show that the physiological costs of reproduction are higher for sexual individuals. They also suggest that increased differentiation of stem cells into gametes might limit investment into somatic functions in hydra polyps. Exhaustion of cellular resources (stem cells) could be a major mechanism behind the extreme post-reproductive senescence observed in this species.

evolutionary biology↗

bioRxiv2017

Multivariate analysis of the cotton seed ionome reveals integrated genetic signatures of abiotic stress response

To mitigate the effects of heat and drought stress, a better understanding of the genetic control of physiological responses to these environmental conditions is needed. To this end, we evaluated an upland cotton (Gossypium hirsutum L.) mapping population under water-limited and well-watered conditions in a hot, arid environment. The elemental concentrations (ionome) of seed samples from the population were profiled in addition to those of soil samples taken from throughout the field site to better model environmental variation. The elements profiled in seeds exhibited moderate to high heritabilities, as well as strong phenotypic and genotypic correlations between elements that were not altered by the imposed irrigation regimes. Quantitative trait loci (QTL) mapping results from a Bayesian classification method identified multiple genomic regions where QTL for individual elements colocalized, suggesting that genetic control of the ionome is highly interrelated. To more fully explore this genetic architecture, multivariate QTL mapping was implemented among groups of biochemically related elements. This analysis revealed both additional and pleiotropic QTL responsible for coordinated control of phenotypic variation for elemental accumulation. Machine learning algorithms that utilized only ionomic data predicted the irrigation regime under which genotypes were evaluated with very high accuracy. Taken together, these results demonstrate the extent to which the seed ionome is genetically interrelated and predictive of plant physiological responses to adverse environmental conditions.\n\nOne sentence summaryThe cotton seed ionome has a shared genetic basis that provides insight into the physiological status of the plant.

genetics↗

bioRxiv2017

Functional Plasticity of the gut and the Malpighian tubules underlies cold acclimation and mitigates cold-induced hyperkalemia in Drosophila melanogaster

At low temperatures, Drosophila, like most insects, lose the ability to regulate ion and water balance across the gut epithelia, which can lead to a lethal increase of [K+] in the hemolymph (hyperkalemia). Cold-acclimation, the physiological response to low temperature exposure, can mitigate or entirely prevent these ion imbalances, but the physiological mechanisms that facilitate this process are not well understood. Here, we test whether plasticity in the ionoregulatory physiology of the gut and Malpighian tubules of Drosophila may aid in preserving ion homeostasis in the cold. Upon adult emergence, D. melanogaster females were subjected to seven days at warm (25{degrees}C) or cold (10{degrees}C) acclimation conditions. The cold acclimated flies had a lower critical thermal minimum (CTmin), recovered from chill coma more quickly, and better maintained hemolymph K+ balance in the cold. The improvements in chill tolerance coincided with increased Malpighian tubule fluid secretion and better maintenance of K+ secretion rates in the cold, as well as reduced rectal K+ reabsorption in cold-acclimated flies. To test whether modulation of ion-motive ATPases, the main drivers of epithelial transport in the alimentary canal, mediate these changes, we measured the activities of Na+-K+-ATPase and V-type H+-ATPase at the Malpighian tubules, midgut, and hindgut. Na+/K+-ATPase and V-type H+-ATPase activities were lower in the midgut and the Malpighian tubules of cold-acclimated flies, but unchanged in the hindgut of cold acclimated flies, and were not predictive of the observed alterations in K+ transport. Our results suggest that modification of Malpighian tubule and gut ion and water transport likely prevents cold-induced hyperkalemia in cold-acclimated flies and that this process is not directly related to the activities of the main drivers of ion transport in these organs, Na+/K+- and V-type H+-ATPases.\n\nSummary StatementAt low temperatures, insects lose the ability to regulate ion and water balance and can experience a lethal increase in hemolymph [K+]. Previous exposure to low temperatures can mitigate this effect and improve chill tolerance. Here, we show that plasticity of ion and fluid transport across the Malpighian tubule and rectal epithelia likely drive this response.

zoology↗

bioRxiv2018

Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution without inducing undue stress on either. We combined lattice light sheet microscopy with two-channel adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages, and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.\n\nOne Sentence SummaryCombining lattice light sheet microscopy with adaptive optics enables high speed, high resolution in vivo 3D imaging of dynamic processes inside cells under physiological conditions within their parent organisms.

cell biology↗

bioRxiv2018

Olfactory Projection Neurons From the Moth Antennal Lobe Lateral Cluster Exhibit Diverse Morphological and Neurophysiological Characteristics

Olfactory projection neurons convey information from the insect antennal lobe (AL) to higher centers in the brain. Many studies on moths have reported excitatory projection neurons with cell bodies in the medial cell cluster (mcPNs) that predominantly send an axon from the AL to calyces of the mushroom body (CA) via the medial antennal lobe tract (mALT) and then to the lateral horn (LH) of the protocerebrum. These neurons tend to have dendritic arbors restricted to a single glomerulus (i.e. they are uniglomerular). In this study, we report on the physiological and morphological properties of a group of pheromone-responsive olfactory projection neurons with cell bodies in the moth AL lateral cell cluster (lcPNs) of two heliothine moth species. While mcPNs typically exhibit a narrow odor tuning range related to the restriction of their dendritic arbors within a single glomerulus, lcPNs exhibited an array of morphological and physiological configurations. Pheromone-responsive lcPNs varied in their associations with glomeruli (uniglomerular and multiglomerular), dendritic arborization structure and connections to higher brain centers with projections primarily through the lateral antennal lobe tract and to a lesser extent the mediolateral antennal lobe tract to a variety of protocerebral targets including ventrolateral and superior neuropils as well as LH. Physiological characterization of lcPNs also revealed a diversity of response profiles including those either enhanced by or reliant upon presentation of a pheromone blend. These responses manifested themselves as higher maximum firing rates and/or improved temporal resolution of pulsatile stimuli. lcPNs therefore participate in conveying a variety of olfactory information relating to qualitative and temporal facets of the pheromone stimulus to a more expansive number of protocerebral targets than their mcPN counterparts. The role of lcPNs in the overall scheme of olfactory processing is discussed.

neuroscience↗

bioRxiv2018

The Structural Basis of IKs Ion-Channel Activation: Mechanistic Insights from Molecular Simulations

Relating ion-channel (iCh) structural dynamics to physiological function remains a challenge. Current experimental and computational techniques have limited ability to explore this relationship in atomistic detail over physiological timescales. A framework associating iCh structure to function is necessary for elucidating normal and disease mechanisms. We formulated a modeling schema that overcomes the limitations of current methods through applications of Artificial Intelligence Machine Learning (ML). Using this approach, we studied molecular processes that underlie human IKs voltage mediated gating. IKs malfunction underlies many debilitating and life-threatening diseases. Molecular components of IKs that underlie its electrophysiological function include KCNQ1 (pore forming tetramer) and KCNE1 (auxiliary subunit). Simulations, using the IKs structure-function model, reproduced experimentally recorded saturation of gating charge displacement at positive membrane voltages, two-step voltage sensor (VS) movement shown by fluorescence, iCh gating statistics, and current-voltage (I-V) relationship. New mechanistic insights include - (1) pore energy profile determines iCh subconductance (SC), (2) entire protein structure, not limited to the pore, contributes to pore energy and channel SC, (3) interactions with KCNE1 result in two distinct VS movements, causing gating charge saturation at positive membrane voltages and current activation delay, and (4) flexible coupling between VS and pore permits pore opening at lower VS positions, resulting in sequential gating. The new modeling approach is applicable to atomistic scale studies of other proteins on timescales of physiological function.

biophysics↗

bioRxiv2018

Anti-HIV-1 B cell responses are dependent on B cell precursor frequency and antigen binding affinity

The discovery that humans can produce potent broadly neutralizing antibodies (bNAbs) to several different epitopes on the HIV-1 spike has reinvigorated efforts to develop an antibody based HIV-1 vaccine. Antibody cloning from single cells revealed that nearly all bNAbs show unusual features that could help explain why it has not been possible to elicit them by traditional vaccination, and instead that it would require a sequence of different immunogens. This idea is supported by experiments with genetically modified immunoglobulin knock-in mice. Sequential immunization with a series of specifically designed immunogens was required to shepherd the development of bNAbs. However, knock-in mice contain super-physiologic numbers of bNAb precursor expressing B cells and therefore how these results can be translated to a more physiologic setting remains to be determined. Here we make use of adoptive transfer experiments using knock-in B cells that carry a synthetic intermediate in the pathway to anti-HIV-1 bNAb development to examine how the relationship between B cell receptor affinity and precursor frequency affects germinal center B cell recrutiment and clonal expansion. Immunization with soluble HIV-1 antigens can recruit bNAb precursor B cells to the germinal center when there are as few as 10 such cells per mouse. However, at low precursor frequencies the extent of clonal expansion is directly proportional to the affinity of the antigen for the B cell receptor, and recruitment to germinal centers is variable and dependent on re-circulation.\n\nSignificance statementAn essential requirement for an HIV-vaccine is to elicit antibodies to conserved regions of the spike protein (Env) becasue these antibodies can protect against infection. Although broadly neutralizing antibodies develop naturally in rare individuals after prolongued HIV infection, eliciting them by vaccination has only been possible in artificial knock-in mouse models wherein the number of B cells expressing the antibody precursor is super-physiologic. To understand the relationship between precursor frequency, antigen affinity and germinal center recruitment we have performed adoptive transfer experiments in which fixed numbers of precursor cells are engrafted in wild type mice. Our results provide a framework for understanding how precursor frequency and antigen affinity shape humoral immunity to HIV.

immunology↗

bioRxiv2018

Serotonergic regulation of corticoamygdalar neurons in the mouse prelimbic cortex

Neuromodulatory transmitters, such as serotonin (5-HT), selectively regulate the excitability of subpopulations of cortical projection neurons to gate cortical output to specific target regions. For instance, in the mouse prelimbic cortex, 5-HT selectively excites commissurally projecting intratelencephalic (COM) neurons via activation of 5-HT2A (2A) receptors, while simultaneously inhibiting corticofugally projecting pyramidal neurons targeting the pons via 5-HT1A (1A) receptors. Here we characterize the physiology, morphology, and serotonergic regulation of corticoamygdalar (CAm) projection neurons in the mouse prelimbic cortex. Layer 5 CAm neurons shared a number of physiological and morphological characteristics with COM neurons, including higher input resistances, smaller HCN-channel mediated responses, and sparser dendritic arbors than corticopontine neurons. Across cortical lamina, CAm neurons also resembled COM neurons in their serotonergic modulation; focally applied 5-HT (100 {micro}M; 1 s) generated 2A-receptor-mediated excitation, or 1A- and 2A- dependent biphasic responses, in ipsilaterally and contralaterally projecting CAm neurons. Serotonergic excitation depended on extrinsic excitatory drive, as 5-HT failed to depolarize CAm neurons from rest, but could enhance the number of action potentials generated by simulated barrages of synaptic input. Finally, using dual tracer injections, we identified double-labeled CAm/COM neurons that displayed primarily excitatory or biphasic responses to 5-HT. Overall, our findings reveal that prelimbic CAm neurons overlap with COM neurons, and that both neuron subtypes exhibit 2A-dependent serotonergic excitation. Our findings suggest that 5-HT, acting at 2A receptors, will promote cortical output to the amygdala.\n\nSignificance StatementCortical projections to the amygdala allow for executive \"top-down\" control of emotional responses. Corticoamygdalar (CAm) neurons in the prelimbic cortex contribute to the learning and expression of conditioned fear responses, processes that may also be regulated by serotonin (5-HT). Our study provides a physiological and morphological characterization of prelimbic CAm neurons, and demonstrates that the excitability of CAm neurons is regulated by 5-HT acting at 5-HT2A receptors alone, or in combination with 5-HT1A receptors. Our results suggest that 5-HT may regulate corticoamygdalar circuits during the learning and expression of conditioned fear.

neuroscience↗

bioRxiv2018

Identification of a secretion-enhancing cis regulatory targeting element (SECReTE) involved in mRNA localization and protein synthesis

Earlier dogma states that mRNAs encoding secreted and membrane protein (mSMPs) reach the ER in a translation-dependent manner through the signal recognition particle (SRP) pathway. In this pathway, the signal sequence of the translation product is recognized by SRP and the mRNA-ribosome-nascent-chain-SRP complex is recruited to the ER via the interaction with an endoplasmic reticulum (ER)-localized SRP receptor. This model suggests that the translation product dictates the delivery of mRNAs to the ER and that the mRNA is a passive passenger. However, new evidence challenges this model and implies the existence of both translation - and SRP-independent mRNA localization to the ER, raising the possibility that mRNAs have an active role in determining their localization to the ER.\n\nBesides serving as a template for protein translation, mRNAs carry information required for other regulatory processes such as mRNA processing, translation and transcription efficiency, degradation and localization. In yeast, mRNA localization governed by cis-acting sequence elements has been characterized for asymmetrically (e.g. bud) localized mRNAs that localize to, and are transported with, cortical ER. Now, we identify a cis motif in mSMPs that targets mRNAs mainly to the nuclear ER in yeast and increases both protein synthesis and secretion. Termed SECReTE, for secretion-enhancing cis regulatory targeting element, this motif was identified by computational analysis of genes encoding secretome proteins. SECReTE consists of [≥]10 repetitive triplets enriched with pyrimidines (i.e. Cs and Us) every third base (i.e. NNY, N - any nucleotide, Y - pyrimidine), and is found particularly in mRNAs coding for cell wall proteins. To study the physiological relevance of SECReTE, we introduced synonymous mutations that either elevate or decrease its overall score in genes coding for secreted proteins, without changing the protein sequence, and examined the physiological effects in yeast. An increase in the SECReTE score elevated the synthesis and secretion of endogenous proteins while, in contrast, a reduction led to less secretion and physiological defects. Importantly, the addition of SECReTE to the 3UTR of an exogenous protein (e.g. SS-GFP) led to its increased secretion from yeast. SECReTE is present all through evolution and, thus, constitutes a novel RNA targeting motif found in both prokaryotes and eukaryotes.

cell biology↗

bioRxiv2018

Phylogenetically novel uncultured microbial cells dominate Earth microbiomes

To unequivocally determine a microbes physiology, including its metabolism, environmental roles, and growth characteristics, it must be grown in a laboratory culture. Unfortunately, many phylogenetically-novel groups have never been cultured, so their physiologies have only been inferred from genomics and environmental characteristics. Although the diversity, or number of different taxonomic groups, of uncultured clades has been well-studied, their global abundances, or number of cells in any given environment, have not been assessed. We quantified the degree of similarity of 16S rRNA gene sequences from diverse environments in publicly-available metagenome and metatranscriptome databases, which we show are largely free of the culture-bias present in primer-amplified 16S rRNA gene surveys, to their nearest cultured relatives. Whether normalized to scaffold read depths or not, the highest abundance of metagenomic 16S rRNA gene sequences belong to phylogenetically novel uncultured groups in seawater, freshwater, terrestrial subsurface, soil, hypersaline environments, marine sediment, hot springs, hydrothermal vents, non-human hosts, snow and bioreactors (22-87% uncultured genera to classes and 0-64% uncultured phyla). The exceptions were human and human-associated environments which were dominated by cultured genera (45-97%). We estimate that uncultured genera and phyla could comprise 7.3 x 1029 (81%) and 2.2 x 1029 (25%) microbial cells, respectively. Uncultured phyla were over-represented in meta transcript omes relative to metagenomes (46-84% of sequences in a given environment), suggesting that they are viable, and possibly more active than cultured clades. Therefore, uncultured microbes, often from deeply phylogenetically divergent groups, dominate non-human environments on Earth, and their undiscovered physiologies may matter for Earth systems.

microbiology↗

bioRxiv2018

Structural basis for relief of the sarcoplasmic reticulum Ca2+-ATPase inhibition by phospholamban at saturating Ca2+ conditions

We have performed extensive atomistic molecular dynamics simulations to probe the structural mechanism for relief of sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibition by phospholamban (PLB) at saturating Ca2+ conditions. Reversal of SERCA-PLB inhibition by saturating Ca2+ operates as a physiological rheostat to reactivate SERCA function in the absence of PLB phosphorylation. Simulation of the inhibitory complex at super-physiological Ca2+ concentrations ([Ca2+]=10 mM) revealed that calcium ions interact primarily with SERCA and the lipid headgroups, but not with the cytosolic domain of PLB or the cytosolic side of the SERCA-PLB interface. At this [Ca2+], a single Ca2+ ion is translocated from the cytosol to the transmembrane transport sites. We used this Ca2+-bound complex as an initial structure to simulate the effects of saturating Ca2+ at physiological conditions ([Ca2+]total{approx}400 M). At these conditions, ~30% of the Ca2+-bound complexes exhibit structural features that correspond to an inhibited state. However, in ~70% of the Ca2+-bound complexes, Ca2+ moves to transport site I, recruits Glu771 and Asp800, and disrupts key inhibitory contacts involving conserved PLB residue Asn34. Structural analysis showed that Ca2+ induces only local changes in interresidue inhibitory interactions, but does not induce dissociation, repositioning or changes in the structural dynamics of PLB. Upon relief of SERCA inhibition, Ca2+ binding produces a productive site I configuration that is sufficient for subsequent SERCA activation. We propose that at saturating [Ca2+] and in the absence of PLB phosphorylation, binding of a single Ca2+ ion in the transport sites rapidly shifts the equilibrium toward a non-inhibited SERCA-PLB complex.

biophysics↗

bioRxiv2018

Fine-tuning cellular levels of DprA ensures transformant fitness in the human pathogen Streptococcus pneumoniae

Natural genetic transformation is a widespread mechanism of bacterial horizontal gene transfer. Transformation involves the internalization of exogenous DNA as single strands, followed by chromosomal integration via homologous recombination, promoting acquisition of new genetic traits. Transformation occurs during a distinct physiological state called competence, during which all proteins required to transform are produced. In the human pathogen Streptococcus pneumoniae, competence is controlled by a two-component system ComDE, which is induced by an exported peptide pheromone. DprA is universal among transformable species, strongly and specifically induced during pneumococcal competence, and crucial for pneumococcal transformation. Pneumococcal DprA plays three crucial roles in transformation and competence. Firstly, DprA protects internalized single-stranded (ss) DNA from degradation. Secondly, DprA loads the homologous recombinase RecA onto transforming ssDNA to promote transformation. Finally, DprA interacts with the response regulator ComE to shut-off pneumococcal competence. Pneumococcal shut-off has been linked to physiology, with long growth delays in competent dprA- cells. Here, we explored the effect of altering the cellular levels of DprA on these three roles. High cellular levels of DprA were not required for the primary role of DprA as a transformation-dedicated recombinase loader or for protection of transforming ssDNA. In contrast, full expression of dprA was required for optimal competence shut-off. Full expression of dprA was also crucial for transformant fitness. High cellular levels of DprA in competent cells thus ensure the fitness of pneumococcal transformants by promoting competence shut-off. This promotes survival and propagation of transformants, thus maximizing the adaptive potential of this human pathogen.\n\nImportanceTransformation is a widespread mechanism of horizontal gene transfer that allows bacteria to acquire new genetic traits by internalizing foreign DNA and integrating it into their chromosomes. Transformation occurs during a transient physiological state called competence. DprA is conserved in transformable species and crucial for the protection and integration of transforming DNA. In the human pathogen Streptococcus pneumoniae, DprA is highly abundant and is also crucial for competence shut-off. Here, we show that high DprA expression is not required for transformation. In contrast, full expression of dprA was required for competence shut-off and transformant fitness. These findings thus link high cellular levels of DprA to survival and propagation of pneumococcal transformants, maximizing the adaptive potential of this human pathogen.

microbiology↗

bioRxiv2018

Repurposing the quinoline antibiotic nitroxoline to treat infections caused by the brain-eating amoeba Balamuthia mandrillaris

Balamuthia mandrillaris is a pathogenic free-living amoeba that causes a rare but almost always fatal infection of the central nervous system called granulomatous amoebic encephalitis (GAE). Two distinct forms of B. mandrillaris - a proliferative trophozoite form and a non-proliferative cyst form, which is highly resistant to harsh physical and chemical conditions - have been isolated from environmental samples worldwide and are both observed in infected tissue. Patients suffering from GAE are typically treated with aggressive and prolonged multi-drug regimens often including the antimicrobial agents miltefosine and pentamidine isethionate. However, survival rates remain low and studies evaluating the susceptibility of B. mandrillaris to these compounds and other potential therapeutics are limited. To address the need for more effective treatments, we screened 2,177 clinically-approved compounds for in vitro activity against B. mandrillaris. The quinoline antibiotic nitroxoline, which has safely been used in humans to treat urinary tract infections, was identified as a lead compound. We show that nitroxoline inhibits both trophozoites and cysts at low micromolar concentrations, which are within a physiologically relevant range. We compare the in vitro efficacy of nitroxoline to drugs currently used in the standard of care for GAE and find that nitroxoline is the most potent and selective inhibitor of B. mandrillaris tested. Furthermore, we demonstrate that nitroxoline prevents B. mandrillaris-mediated destruction of host cells in cultured fibroblast and primary brain explant models also at physiologically relevant concentrations. Together, our findings indicate that nitroxoline is a promising candidate for repurposing as a novel treatment of B. mandrillaris infections.\n\nImportanceBalamuthia mandrillaris is responsible for hundreds of reported cases of amoebic encephalitis, the majority of which have been fatal. Despite being an exceptionally deadly pathogen, B. mandrillaris is understudied, leaving many open questions regarding epidemiology, diagnosis, and treatment. Due to the lack of effective drugs to fight B. mandrillaris infections, mortality rates remain high even for patients receiving intensive care. This study addresses the need for new anti-amoebic drugs using a high-throughput screening approach to identify novel B. mandrillaris inhibitors. The most promising candidate identified was the quinoline antibiotic nitroxoline, which has a long history of safe use in humans. We show that nitroxoline kills B. mandrillaris at physiologically relevant concentrations and exhibits greater potency and selectivity than drugs commonly used in the current standard of care. The findings we present demonstrate the potential of nitroxoline to be an important new tool in the treatment of life threatening B. mandrillaris infections.

pharmacology and toxicology↗

bioRxiv2018

PorSignDB: a database of in vivo perturbation signatures for dissecting clinical outcome of PCV2 infection

Porcine Circovirus Type 2 (PCV2) is a pathogen that has the ability to cause often devastating disease manifestations in pig populations with major economic implications. How PCV2 establishes subclinical persistence and why certain individuals progress to lethal lymphoid depletion remain to be elucidated. Here we present PorSignDB, a gene signature database describing in vivo porcine tissue physiology that we generated from a large compendium of in vivo transcriptional profiles and that we subsequently leveraged for deciphering the distinct physiological states underlying PCV2-affected lymph nodes. This systems biology approach indicated that subclinical PCV2 infections shut down the immune system. A robust signature of PCV2 disease emphasized that immune activation is dysfunctional in subclinical infections, however, in contrast it is promoted in PCV2 patients with clinical manifestations. Functional genomics further uncovered IL-2 as a driver of PCV2-mediated disease and we identified STAT3 as a druggable PCV2 host factor candidate. Our systematic dissection of the mechanistic basis of PCV2 reveals that subclinical and clinical PCV2 display two diametrically opposed immunotranscriptomic recalibrations that represent distinct physiological states in vivo, which suggests a paradigm shift in this field. Finally, our PorSignDB signature database is publicly available as a community resource (http://www.vetvirology.ugent.be/PorSignDB/, included in Gene Sets from Community Contributors http://software.broadinstitute.org/gsea/msigdb/contributed_genesets.jsp) and provides systems biologists with a valuable tool for catalyzing studies of human and veterinary disease.\n\nAuthor SummaryPorcine Circovirus Type 2 (PCV2) is a small but economically important pathogen circulating endemically in pig populations. Although PCV2 causes mostly chronic subclinical infections, many individuals develop a lethal form of circoviral disease consisting of a collapse of lymphoid tissue. In order to provide a fresh look at how PCV2 reprograms host tissue, we created PorSignDB, a compendium of hundreds of transcriptomic gene-expression signatures derived from primary porcine tissue specimens of well over 1500 patients or lab animals. By leveraging PorSignDB on transcriptomic data of PCV2 patients, we uncover that subclinical PCV2 reprograms the host into a striking state of non-infection, which explains its failure to respond to an initial phase of circoviral presence. A PCV2 disease signature further demonstrates that the silenced immune system associated with subclinical PCV2 becomes fully activate in PCV2 patients, triggering severe circoviral disease. Further genomic and functional analysis demonstrate STAT3 as a druggable host factor and IL-2 as a disease driver. Together, this study demonstrates the mechanistic underpinnings of clinical outcome of PCV2 infections: subclinical and clinical PCV2 display two entirely opposing transcriptomic recalibrations of lymphoid tissue.

microbiology↗

bioRxiv2018

Resilience of Atlantic Slippersnail Crepidula fornicata Larvae in the Face of Severe Coastal Acidification

Globally, average oceanic pH is dropping, and it will continue to decline into the foreseeable future. This ocean acidification (OA) will exacerbate the natural fluctuations in pH that nearshore ecosystems currently experience daily, potentially pushing marine organisms to their physiological limits. Adults of Crepidula fornicata (the Atlantic slippersnail) have proven remarkably resilient to many environmental changes, which is perhaps not surprising considering that they are common intertidally, have a geographically large native range, and have been extremely successful at invading coastal water in many other parts of the world. However, the larvae of C. fornicata have been shown to be somewhat more vulnerable than adults to the effects of reduced pH. Research to date has focused on the physiological impacts of OA on C. fornicata larvae; few studies have explored shifts in gene expression resulting from changes in pH. In the present study, we examined the response of young (4- day old) C. fornicata larvae to two extreme OA treatments (pH 7.5 and 7.6) relative to pH 8.0, documenting both phenotypic and genome-wide gene expression responses. We found that rearing larvae at reduced pH had subtle influences on gene expression, predominantly involving downregulation of genes related to growth and metabolism, accompanied by significantly reduced shell growth rates only for larvae reared at pH 7.5. Additionally, 10-day old larvae that had been reared at the two lower pH levels were far less likely to metamorphose within six hours when exposed to inducer. However, all larvae eventually reached similarly high levels of metamorphosis 24 hours after settlement induction. Finally, there were no observed impacts of OA on larval mortality. Taken together, our results indicate that far future OA levels have observable, but not severe, impacts on C. fornicata larvae, which is consistent with the resilience of this invasive snail across rapidly changing nearshore ecosystems. We propose that future work should delve further into the physiological and transcriptomic responses of all life history stages to gain a more comprehensive understanding of how OA impacts the intertidal gastropod C. fornicata.

molecular biology↗