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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Revealing the nanoscale morphology of the primary cilium using super-resolution fluorescence microscopy

Super-resolution (SR) microscopy has been used to observe structural details beyond the diffraction limit of ~250 nm in a variety of biological and materials systems. By combining this imaging technique with both computer-vision algorithms and topological methods, we reveal and quantify the nanoscale morphology of the primary cilium, a tiny tubular cellular structure (~2-6 m long and 200-300 nm diameter). The cilium in mammalian cells protrudes out of the plasma membrane and is important in many signaling processes related to cellular differentiation and disease. After tagging individual ciliary transmembrane proteins, specifically Smoothened (SMO), with single fluorescent labels in fixed cells, we use three-dimensional (3D) single-molecule SR microscopy to determine their positions with a precision of 10-25 nm. We gain a dense, pointillistic reconstruction of the surfaces of many cilia, revealing large heterogeneity in membrane shape. A Poisson surface reconstruction (PSR) algorithm generates a fine surface mesh, allowing us to characterize the presence of deformations by quantifying the surface curvature. Upon impairment of intracellular cargo transport machinery by genetic knockout or small-molecule treatment of cells, our quantitative curvature analysis shows significant morphological differences not visible by conventional fluorescence microscopy techniques. Furthermore, using a complementary SR technique, 2-color, 2D STimulated Emission Depletion (STED) microscopy, we find that the cytoskeleton in the cilium, the axoneme, also exhibits abnormal morphology in the mutant cells, similar to our 3D results on the SMO-measured ciliary surface. Our work combines 3D SR microscopy and computational tools to quantitatively characterize morphological changes of the primary cilium under different treatments and uses STED to discover correlated changes in the underlying structure. This approach can be useful for studying other biological or nanoscale structures of interest.

biophysics↗

bioRxiv2018

Genetic variability in response to Aβ deposition influences Alzheimer’s risk

Genetic analysis of late-onset Alzheimers disease risk has previously identified a network of largely microglial genes that form a transcriptional network. In transgenic mouse models of amyloid deposition we have previously shown that the expression of many of the mouse orthologs of these genes are co-ordinately up-regulated by amyloid deposition. Here we investigate whether systematic analysis of other members of this mouse amyloid-responsive network predicts other Alzheimers risk loci. This statistical comparison of the mouse amyloid-response network with Alzheimers disease genome-wide association studies identifies 5 other genetic risk loci for the disease (OAS1, CXCL10, LAPTM5, ITGAM and LILRB4). This work suggests that genetic variability in the microglial response to amyloid deposition is a major determinant for Alzheimers risk.\n\nOne Sentence SummaryIdentification of 5 new risk loci for Alzheimers by statistical comparison of mouse A{beta} microglial response with gene-based SNPs from human GWAS

neuroscience↗

bioRxiv2018

MetaCell: analysis of single cell RNA-seq data using k-NN graph partitions

Single cell RNA-seq (scRNA-seq) has become the method of choice for analyzing mRNA distributions in heterogeneous cell populations. scRNA-seq only partially samples the cells in a tissue and the RNA in each cell, resulting in sparse data that challenge analysis. We develop a methodology that addresses scRNA-seqs sparsity through partitioning the data into metacells: disjoint, homogenous and highly compact groups of cells, each exhibiting only sampling variance. Metacells constitute local building blocks for clustering and quantitative analysis of gene expression, while not enforcing any global structure on the data, thereby maintaining statistical control and minimizing biases. We illustrate the MetaCell framework by re-analyzing cell type and transcriptional gradients in peripheral blood and whole organism scRNA-seq maps. Our algorithms are implemented in the new MetaCell R/C++ software package.

bioinformatics↗

bioRxiv2018

Distinct waking states for strong evoked responses in primary visual cortex and optimal visual detection performance

Variability in cortical neuronal responses to sensory stimuli and in perceptual decision making performance is substantial. Moment-to-moment fluctuations in waking state or arousal can account for much of this variability. Yet, the nature of this variability across the full spectrum of waking states is often not completely characterized, leaving the characteristics of the optimal state for sensory processing unresolved. Using pupillometry in concert with extracellular multiunit and intracellular whole-cell recordings, we found that the magnitude and reliability of visually evoked responses in primary visual cortex (V1) of awake, passively behaving male mice increase as a function of arousal and are largest during sustained locomotion periods. During these high-arousal, sustained locomotion periods, cortical neuronal membrane potential was at its most depolarized and least variable. Contrastingly, behavioral performance of mice on two distinct visual detection tasks was generally best at a range of intermediate arousal levels, but worst during locomotion. These results suggest that large, reliable responses to visual stimuli in V1 occur at a distinct arousal level from that associated with optimal visual detection performance. Our results clarify the relation between neuronal responsiveness and the continuum of waking states, and suggest new complexities in the relation between primary sensory cortical activity and behavior.

neuroscience↗

bioRxiv2018

Flavor compounds in fermented Chinese alcoholic beverage alter gut microbiota and attenuate ethanol-induced liver

Alcoholic beverages which are consumed widely in most parts of the world have long been identified as a major risk factor for all liver diseases, particularly alcohol-induced liver disease (ALD). Recent compositional analyses suggest that Chinese Baijiu (CB), a clear alcoholic liquid distilled from fermented grains, contains large amounts of small molecule bioactive compounds in addition to a significant amount of ethanol. Here, in an experimental mouse model, we show that CB caused lower degrees of liver injury than pure ethanol by protecting against the decrease of the relative abundance of Akkermansia and increased relative abundance of Prevotella in the gut thereby preventing the destruction of the intestinal barrier. Furthermore, we demonstrated that ethanol-induced alteration of the gut microbiota profoundly affected the host metabolome. Compared with ethanol feeding, CB feeding resulted in higher concentrations of functional saturated LCFAs and SCFAs. Our results provide supporting evidence that ALD was profoundly influenced by host-gut microbiota metabolic interactions and that small molecule organic compounds in CB could attenuate ALD.

microbiology↗

bioRxiv2018

Developmental tuning of mineralization drives morphological diversity of gill cover bones in sculpins and their relatives

The role of osteoblast placement in skeletal morphological variation is relatively wellunderstood, but alternative developmental mechanisms affecting bone shape remain largely unknown. Specifically, very little attention has been paid to variation in later mineralization stages of intramembranous ossification as a driver of morphological diversity. We discover the occurrence of specific, sometimes large regions of nonmineralized osteoid within bones that also contain mineralized tissue. We show through a variety of histological, molecular, and tomographic tests that this \"extended\" osteoid material is most likely nonmineralized bone matrix. This tissue type is a significant determinant of gill cover bone shape in the teleostean suborder Cottoidei. We demonstrate repeated evolution of extended osteoid in Cottoidei through ancestral state reconstruction and test for an association between its presence and habitat differences among species. Through measurement of extended osteoid at various stages of gill cover development in species across the phylogeny, we gain insight into possible evolutionary developmental origins of the trait. We conclude that this finetuned developmental regulation of bone matrix mineralization reflects heterochrony at multiple biological levels and is a novel mechanism for the evolution of diversity in skeletal morphology. This research lays the groundwork for a new model in which to study bone mineralization and evolutionary developmental processes, particularly as they may relate to adaptation during a prominent evolutionary radiation of fishes.

evolutionary biology↗

bioRxiv2018

A tagging method for very small fish

Identifying individuals over time and across contexts is essential in many scientific fields. There are a variety of well-established methods for uniquely marking individuals (e.g., visible implant elastomer, barcodes, paint). However, for some species, life history stages, and/or experiments, existing methods are not sufficient. Here, we describe procedures for how two tagging methods - a tattoo ink injection method and a fishing line piercing method - can be used with the youngest, smallest juveniles of the African cichlid fish, Astatotilapia burtoni, which are too small for the methods used with adults. With the tattoo method, we injected tattoo ink into the dorsal muscle. Different colors and injection locations can be used to distinguish among individuals over a period of weeks (up to 4 weeks, average 2.5-3 weeks under our conditions). Because fish this young and small are sensitive to handling and injection, we also include physiological data showing fish recover well from anesthetization and tagging. With the piercing method, very thin fishing line is threaded through the dorsal muscle and tied into a barbell or loop. Unique colors and patterns can be used to distinguish among individuals over a period of months. Because a physical tag might impede normal movement in a very small fish, we also include data from an open field exploration test showing similar behavior between tagged and control (non-tagged) juveniles. We expect these effective and inexpensive methods to be useful for a variety of small species and will facilitate early-life, developmental, and longitudinal research.

animal behavior and cognition↗

bioRxiv2018

Reconstitution of the equilibrium state of dynamic actin networks

Principles of regulation of actin network dimensions, fundamentally important for cell functions, remain unclear. We studied in vitro and in silico the effect of key parameters, actin density, ADF/Cofilin concentration and network width on the network length. In the presence of ADF/Cofilin, networks reached equilibrium and became globally treadmilling. At the trailing edge, the network disintegrated into large fragments. A mathematical model predicts the network length as a function of width, actin and ADF/Cofilin concentrations. Local depletion of ADF/Cofilin by binding to actin is significant, leading to wider networks growing longer. A single rate of breaking network nodes, proportional to ADF/Cofilin density and inversely proportional to the square of the actin density, can account for the disassembly dynamics. Selective disassembly of heterogeneous networks by ADF/Cofilin controls steering during motility. Our results establish general principles on how the dynamic equilibrium state of actin network emerges from biochemical and structural feedbacks.

cell biology↗

bioRxiv2018

Tau accumulation activates STAT1 triggering memory deficits via suppressing NMDA receptor expression

Intracellular tau accumulation forming neurofibrillary tangles is hallmark pathology of Alzheimer's disease (AD), but how tau accumulation induces synapse impairment is elusive. By overexpressing human full-length wildtype tau (termed hTau) to mimic tau abnormality as seen in the brain of sporadic AD patients, we found that hTau accumulation activated JAK2 to phosphorylate STAT1 (Signal Transducer and Activator of Transcription 1) at Tyr701 leading to STAT1 dimerization, nuclear translocation and its activation. STAT1 activation suppressed expression of N-methyl-D-aspartate receptors (NMDARs) through direct binding to the specific GAS element of GluN1, GluN2A and GluN2B promoters, while knockdown STAT1 by AAV-Cre in STAT1flox/flox mice or expressing dominant negative Y701F-STAT1 efficiently rescued hTau-induced suppression of NMDARs expression with amelioration of synaptic functions and memory performance. These findings indicate that hTau accumulation impairs synaptic plasticity through JAK2/STAT1-induced suppression of NMDARs expression, revealing a novel mechanism for hTau-associated synapse and memory deficits.

neuroscience↗

bioRxiv2018

Kinetic models of metabolism that consider alternative steady-state solutions of intracellular fluxes and concentrations

Large-scale kinetic models are used for designing, predicting, and understanding the metabolic responses of living cells. Kinetic models are particularly attractive for the biosynthesis of target molecules in cells as they are typically better than other types of models at capturing the complex cellular biochemistry. Using simpler stoichiometric models as scaffolds, kinetic models are built around a steady-state flux profile and a metabolite concentration vector that are typically determined via optimization. However, as the underlying optimization problem is underdetermined, even after incorporating available experimental omics data, one cannot uniquely determine the operational configuration in terms of metabolic fluxes and metabolite concentrations. As a result, some reactions can operate in either the forward or reverse direction while still agreeing with the observed physiology. Here, we analyze how the underlying uncertainty in intracellular fluxes and concentrations affects predictions of constructed kinetic models and their design in metabolic engineering and systems biology studies. To this end, we integrated the omics data of optimally grown Escherichia coli into a stoichiometric model and constructed populations of non-linear large-scale kinetic models of alternative steady-state solutions consistent with the physiology of the E. coli aerobic metabolism. We performed metabolic control analysis (MCA) on these models, highlighting that MCA-based metabolic engineering decisions are strongly affected by the selected steady state and appear to be more sensitive to concentration values rather than flux values. To incorporate this into future studies, we propose a workflow for moving towards more reliable and robust predictions that are consistent with all alternative steady-state solutions. This workflow can be applied to all kinetic models to improve the consistency and accuracy of their predictions. Additionally, we show that, irrespective of the alternative steady-state solution, increased activity of phosphofructokinase and decreased ATP maintenance requirements would improve cellular growth of optimally grown E. coli.

systems biology↗

bioRxiv2018

Differential effects of corticosteroids and anti-TNF on tumor-specific immune responses - implications for the management of irAEs

Up to 60% of patients treated with cancer immunotherapy develop severe or life threatening immune-related adverse events (irAEs). Immunosuppression with high doses of corticosteroids or, in refractory cases, with tumor necrosis factor (TNF) antagonists, are the mainstay of treatment for irAEs. It is currently unknown what is the impact of corticosteroids and anti-TNF on the activity of antitumor T cells. In this study, the influences of clinically relevant doses of dexamethasone (corresponding to an oral dose of 10 to 125 mg prednisolone) and infliximab (anti-TNF) on the activation and killing ability of tumor-infiltrating lymphocytes (TILs) was tested in vitro. Overall, dexamethasone at low or intermediate/high dose impaired the activation (respectively -46% and -62%) and tumor-killing ability (respectively -48% and -53%) of tumor-specific TILs. In contrast, a standard clinical dose of infliximab only had a minor effect on T cell activation (-20%) and tumor killing (-10%). A brief resting following exposure to dexamethasone was sufficient to rescue the in vitro activity of TILs. In conclusion, clinically-relevant doses of infliximab only influenced to a lesser extent the activity of tumor-specific TILs in vitro, whereas even low doses of corticosteroids markedly impaired the antitumor activity of TILs. These data support steroid-sparing strategies and early initiation of anti-TNF for the treatment of irAEs in immuno-oncology.

cancer biology↗

bioRxiv2018

Psychological resilience correlates with EEG source-space brain network flexibility

ObjectiveWe aimed at identifying the potential relationship between the dynamical properties of the human functional network at rest and one of the most prominent traits of personality, namely resilience.\n\nApproachTo tackle this issue, we used resting-state EEG data recorded from 45 healthy subjects. Resilience was quantified using the 10-items Connor-Davidson Resilience Scale (CD-RISC). Using a sliding windows approach, brain networks in each EEG frequency band (delta, theta, alpha and beta) were constructed using the EEG source-space connectivity method. Brain networks dynamics were evaluated using the network flexibility, linked with the tendency of a given node to change its modular affiliation over time.\n\nMain ResultsThe results revealed a negative correlation between the psychological resilience and the brain network flexibility for a limited number of brain regions within the delta, alpha and beta bands.\n\nSignificanceThis study provides evidence that network flexibility, a metric of dynamic functional networks, is strongly correlated with psychological resilience as assessed from personality testing. Beyond this proof-of-principle that reliable EEG-based quantities representative of personality traits can be identified, this motivates further investigation regarding the full spectrum of personality aspects and their relationship with functional networks.

neuroscience↗

bioRxiv2018

A bacteria-derived tail anchor localizes to peroxisomes in yeast and mammalian cells

Prokaryotes can provide new genetic information to eukaryotes by horizontal gene transfer (HGT), and such transfers are likely to have been particularly consequential at the dawn of eukaryogenesis. Since eukaryotes are highly compartmentalized, it is worthwhile to consider the mechanisms by which newly transferred proteins might reach diverse organellar destinations. Toward this goal, we have focused our attention upon the behavior of bacteria-derived tail anchors (TAs) expressed in the eukaryote Saccharomyces cerevisiae. In this study, we report that a predicted membrane-associated domain of the Escherichia coli YgiM protein is specifically trafficked to peroxisomes in budding yeast, can be found at a pre-peroxisomal compartment (PPC) upon disruption of peroxisomal biogenesis, and can functionally replace an endogenous peroxisome-directed TA. Furthermore, the YgiM(TA) can also localize to peroxisomes in mammalian cells. Since the YgiM(TA) plays no endogenous role in peroxisomal function or assembly, this domain is likely to serve as an excellent tool toward illumination of the mechanisms by which TAs can travel to peroxisomes. Moreover, our findings emphasize the ease with which bacteria-derived sequences might target to organelles in eukaryotic cells following HGT, and we discuss the importance of flexible recognition of organelle targeting information during and after eukaryogenesis.

evolutionary biology↗

bioRxiv2018

Transcriptomic metaanalyses of autistic brains reveals shared gene expression and biological pathway abnormalities with cancer.

Epidemiological and clinical evidence points to cancer as a comorbidity in people with autism spectrum disorders (ASD). A significant overlap of genes and biological processes between both diseases has also been reported. Here, for the first time, we compared the gene expression profiles of ASD frontal cortex tissues and 22 cancer types obtained by differential expression meta-analysis. Four cancer types (brain, thyroid, kidney, and pancreatic cancers) presented a significant overlap in gene expression deregulations in the same direction as ASD whereas two cancer types (lung and prostate cancers) showed differential expression profiles significantly deregulated in the opposite direction from ASD. Functional enrichment and LINCS L1000 based drug set enrichment analyses revealed the implication of several biological processes and pathways that were affected jointly in both diseases, including impairments of the immune system, and impairments in oxidative phosphorylation and ATP synthesis among others. Our data also suggest that brain and kidney cancer have patterns of transcriptomic dysregulation in the PI3K/AKT/MTOR axis that are similar to those found in ASD. These shared transcriptomic alterations could help explain epidemiological observations suggesting direct and inverse comorbid associations between ASD and particular cancer types.

systems biology↗

bioRxiv2018

Crystal Toxins and the volunteer’s dilemma in bacteria

1The growth and virulence of the bacteria Bacillus thuringiensis depends on the production of Cry toxins, which are used to perforate the gut of its host. Successful invasion of the host relies on producing a threshold amount of toxin, after which there is no benefit from producing more toxin. Consequently, the production of Cry toxin appears to be a different type of social problem compared with the public goods scenarios that bacteria often encounter. We show that selection for toxin production is a volunteers dilemma. We make the specific predictions that: (1) selection for toxin production depends upon an interplay between the number of bacterial cells that each host ingests, and the genetic relatedness between those cells; (2) cheats that do not produce toxin gain an advantage when at low frequencies, and at high bacterial density, allowing them to be maintained in a population alongside toxin producing cells. More generally, our results emphasise the diversity of the social games that bacteria play.

evolutionary biology↗

bioRxiv2018

Genome-wide transcription during early wheat meiosis is independent of synapsis, ploidy level and the Ph1 locus

Polyploidization is a fundamental process in plant evolution. One of the biggest challenges faced by a new polyploid is meiosis, particularly discriminating between multiple related chromosomes so that only homologous chromosomes synapse and recombine to ensure regular chromosome segregation and balanced gametes. Despite its large genome size, high DNA repetitive content and similarity between homoeologous chromosomes, hexaploid wheat completes meiosis in a shorter period than diploid species with a much smaller genome. Therefore, during wheat meiosis, mechanisms additional to the classical model based on DNA sequence homology, must facilitate more efficient homologous recognition. One such mechanism could involve exploitation of differences in chromosome structure between homologues and homoeologues at the onset of meiosis. In turn, these chromatin changes, can be expected to be linked to transcriptional gene activity. In this study, we present an extensive analysis of a large RNA-Seq data derived from six different genotypes: wheat, wheat-rye hybrids and newly synthesized octoploid triticale, both in the presence and absence of the Ph1 locus. Plant material was collected at early prophase, at the transition leptotene-zygotene, when the telomere bouquet is forming and synapsis between homologues is beginning. The six genotypes exhibit different levels of synapsis and chromatin structure at this stage; therefore, recombination and consequently segregation, are also different. Unexpectedly, our study reveals that neither synapsis, whole genome duplication nor the absence of the Ph1 locus are associated with major changes in gene expression levels during early meiotic prophase. Overall wheat transcription at this meiotic stage is therefore highly resilient to such alterations, even in the presence of major chromatin structural changes. This suggests that post-transcriptional and post-translational processes are likely to be more important. Thus, further studies will be required to reveal whether these observations are specific to wheat meiosis, and whether there are significant changes in post-transcriptional and post-translational modifications in wheat and other polyploid species associated with their polyploidisation.

genomics↗

bioRxiv2018

The Hox Transcription Factor Ubx stabilizes Lineage Commitment by Suppressing Cellular Plasticity

During development cells become gradually restricted in their differentiation potential by the repression of alternative cell fates. While we know that the Polycomb complex plays a crucial role in this process, it still remains unclear how alternative fate genes are specifically targeted for silencing in different cell lineages. We address this question by studying Ultrabithorax (Ubx), a multi-lineage transcription factor (TF) of the Hox class, in the mesodermal and neuronal lineages using sorted nuclei of Drosophila embryos and by interfering with Ubx in mesodermal cells that have already initiated differentiation. We find that Ubx is a key regulator of lineage development, as its mesoderm-specific depletion leads to the de-repression of many genes normally expressed in other lineages. Ubx silences expression of alternative fate genes by interacting with and retaining the Polycomb Group (PcG) protein Pleiohomeotic (Pho) at Ubx targeted genomic regions, thereby setting repressive chromatin marks in a lineage-dependent manner. In sum, our study demonstrates that Ubx stabilizes lineage choice by suppressing the multi-potency encoded in the genome in a lineage-specific manner via its interaction with Pho. This mechanism may explain why the Hox code is maintained throughout the lifecycle, since it seems to set a block to transdifferentiation in many adult cells.

developmental biology↗

bioRxiv2018

Exploring integument transcriptomes, cuticle ultrastructure, and cuticular hydrocarbons profiles in eusocial and solitary bee species displaying heterochronic adult cuticle maturation

Differences in the timing of exoskeleton melanization and sclerotization are evident when comparing eusocial and solitary bees. This cuticular maturation heterochrony may be associated with life style, considering that eusocial bees remain protected inside the nest for many days after emergence, while the solitary bees immediately start outside activities. To address this issue, we characterized gene expression using large-scale RNA sequencing (RNA-seq), and quantified cuticular hydrocarbon (CHC) through gas chromatography-mass spectrometry in comparative studies of the integument (cuticle plus its underlying epidermis) of two eusocial and a solitary bee species. In addition, we used transmission electron microscopy (TEM) for studying the developing cuticle of these and other three bee species also differing in life style. We found 13,200, 55,209 and 30,161 transcript types in the integument of the eusocial Apis mellifera and Frieseomelitta varia, and the solitary Centris analis, respectively. In general, structural cuticle proteins and chitin-related genes were upregulated in pharate-adults and newly-emerged bees whereas transcripts for odorant binding proteins, cytochrome P450 and antioxidant proteins were overrepresented in foragers. Consistent with our hypothesis, a distance correlation analysis based on the differentially expressed genes suggested delayed cuticle maturation in A. mellifera in comparison to the solitary bee. However, this was not confirmed in the comparison with F. varia. The expression profiles of 27 of 119 genes displaying functional attributes related to cuticle formation/differentiation were positively correlated between A. mellifera and F. varia, and negatively or non-correlated with C. analis, suggesting roles in cuticular maturation heterochrony. However, we also found transcript profiles positively correlated between each one of the eusocial species and C. analis. Gene co-expression networks greatly differed between the bee species, but we identified common gene interactions exclusively between the eusocial species. Except for F. varia, the TEM analysis is consistent with cuticle development timing adapted to the social or solitary life style. In support to our hypothesis, the absolute quantities of n-alkanes and unsaturated CHCs were significantly higher in foragers than in the earlier developmental phases of the eusocial bees, but did not discriminate newly-emerged from foragers in C. analis. By highlighting differences in integument gene expression, cuticle ultrastructure, and CHC profiles between eusocial and solitary bees, our data provided insights into the process of heterochronic cuticle maturation associated to the way of life.\n\nAuthor SummaryFrom our previous observation that bees with distinct habits of life, eusocial and solitary, exhibit different degrees of cuticle melanization and sclerotization at the emergence, we decided to analyze the genetic signatures and ultrastructure of the integument, as well as the CHC profiles that could be involved in cuticle maturation. The expression patterns of certain genes involved in the melanization/sclerotization pathway, chitin metabolism, cuticle structure, and also regulators of cuticle renewal and tanning, in addition to other genes, might be grounded the slow process of cuticle maturation in the eusocial bees in comparison to the solitary ones. The electron micrographs revealed differences in the timing of cuticle deposition for the eusocial and solitary species. Among the identified CHCs, the proportions and quantities of n-alkanes in the developing cuticle are consistent with the faster cuticular maturation in the solitary bee, thus supporting our hypothesis.

genetics↗