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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 217 records · Page 12Linked to original sources

bioRxiv2018

High-resolution mapping of cancer cell networks using co-functional interactions

Powerful new technologies for perturbing genetic elements have expanded the study of genetic interactions in model systems ranging from yeast to human cell lines. However, technical artifacts can confound signal across genetic screens and limit the immense potential of parallel screening approaches. To address this problem, we devised a novel PCA-based method for eliminating these artifacts and bolstering sensitivity and specificity for detection of genetic interactions. Applying this strategy to a set of >300 whole genome CRISPR screens, we report ~1 million pairs of correlated \"co-functional\" genes that provide finer-scale information about cell compartments, biological pathways, and protein complexes than traditional gene sets. Lastly, we employed a gene community detection approach to implicate core genes for cancer growth and compress signal from functionally related genes in the same community into a single score. This work establishes new algorithms for probing cancer cell networks and motivates the acquisition of further CRISPR screen data across diverse genotypes and cell types to further resolve the complexity of cell signaling processes.

genomics↗

bioRxiv2018

Plasmodium falciparum mature schizont transcriptome variation among clinical isolates and laboratory-adapted clones

Malaria parasite genes exhibit variation in both sequence and expression level. There is much information on sequence polymorphism, but less resolution on natural variation in transcriptomes of parasites at specific developmental stages. This is largely because it is challenging to obtain highly replicated sampling of transcriptomes to overcome potentially confounding technical and biological variation. We address the issue in the major human parasite Plasmodium falciparum by obtaining RNA-seq profiles of multiple independent replicate preparations of mature schizont-stage parasites from a panel of clinical isolates recently established in culture and from long-term laboratory-adapted clones. With a goal of robustly identifying variably expressed genes, we show that increasing the numbers of biological sample replicates greatly improves the discovery rate. Generally, six independent replicates of each parasite culture is recommendable as being significantly to lower numbers, although for highly expressed genes variable expression can be detected when fewer replicates are available. A broad comparison identifies genes differing in relative expression between cultured clinical isolates and laboratory-adapted clones. Genes more highly expressed in the laboratory-adapted clones include an AP2 transcription factor gene Pf3D7_0420300 and putative methyl transferase genes. The variable expression of several known merozoite invasion ligands is confirmed, and previously uncharacterised genes are shown to be differentially expressed among clinical isolates. New RT-qPCR assays validate the variation in transcript levels of these genes, and allow quantitation of expression to be extended to a wider panel of clinical isolate samples. These variably expressed genes are new candidates for investigation as potential determinants of alternative parasite developmental pathways or targets of immunity.\n\nAuthor summaryUnderstanding parasite diversity and adaptation may require characterisation of gene expression variation, and is vital if chemotherapeutic or vaccine development is to consider new candidate targets, but it is technically challenging to generate precise data on clinical isolates. Here, we analyse the transcriptomes of mature Plasmodium falciparum schizonts using RNA-sequencing, using large numbers of biological replicate samples to minimise the impact of inter-replicate variation on observed patterns of differential expression. This identifies genes that are differentially expressed in long term laboratory-adapted parasites and recently cultured clinical isolates, as well as among different clinical isolates. In additional samples of schizonts grown in the first cycle ex vivo prior to any erythrocyte invasion, expression levels of a selected panel of these genes vary among isolates, but mean levels are similar to those in the continuously cultured clinical isolates, indicating that the latter are useful for experimental studies requiring biological replication.

cell biology↗

bioRxiv2017

Pericyte ontogeny: the use of chimeras to track a cell lineage of diverse germ line origins

The goal of lineage tracing is to understand body formation over time by discovering which cells are the progeny of a specific, identified, ancestral progenitor. Subsidiary questions include unequivocal identification of what they have become, how many descendants develop, whether they live or die, and where they are located in the tissue or body at the end of the window examined. A classical approach in experimental embryology, lineage tracing continues to be used in developmental biology, stem cell and cancer research, wherever cellular potential and behavior need to be studied in multiple dimensions, of which one is time. Each technical approach has its advantages and drawbacks. This chapter, with some previously unpublished data, will concentrate non-exclusively on the use of interspecies chimeras to explore the origins of perivascular (or mural) cells, of which those adjacent to the vascular endothelium are termed pericytes for this purpose. These studies laid the groundwork for our understanding that pericytes derive from progenitor mesenchymal pools of multiple origins in the vertebrate embryo, some of which persist into adulthood. The results obtained through xenografting, like in the methodology described here, complement those obtained through genetic lineage tracing techniques within a given species.

developmental biology↗

bioRxiv2017

Real-time chromatin dynamics at the single gene levelduring transcription activation

Genome dynamics relate to regulation of gene expression, the most fundamental process in biology. Yet we still do not know whether the very process of transcription drives spatial organization and chromatin conformation at specific gene loci. To address this issue, we have optimized the ANCHOR/ParB DNA labeling system for real-time imaging and quantitative analysis of the dynamics of a single-copy transgene in human cells. Transcription of the transgene under the control of the endogenous Cyclin D1 promoter was induced by addition of 17{beta}-estradiol. Motion of the ANCHOR3-tagged DNA locus was recorded in the same cell prior to and during appearance of nascent mRNA visualized using the MS2 system. We found that transcription initiation resulted in rapid confinement of the mRNA-producing gene. The confinement was maintained even upon inhibition of pol2 elongation. It did not occur when recruitment of pol2 or transcription initiation was blocked by anti-estrogens or Triptolide. These results suggest that preinitiation complex formation and concomitant reorganization of the chromatin domain constrains freedom of movement of an induced genes promoter within minutes. Confined diffusion reflects assembly of functional protein hubs and DNA processing during the rate-limiting steps of transcription.

cell biology↗

bioRxiv2017

Improving Phenotypic Measurements in High-Content Imaging Screens

Image-based screening is a powerful technique to reveal how chemical, genetic, and environmental perturbations affect cellular state. Its potential is restricted by the current analysis algorithms that target a small number of cellular phenotypes and rely on expert-engineered image features. Newer algorithms that learn how to represent an image are limited by the small amount of labeled data for ground-truth, a common problem for scientific projects. We demonstrate a sensitive and robust method for distinguishing cellular phenotypes that requires no additional ground-truth data or training. It achieves state-of-the-art performance classifying drugs by similar molecular mechanism, using a Deep Metric Network that has been pre-trained on consumer images and a transformation that improves sensitivity to biological variation. However, our method is not limited to classification into predefined categories. It provides a continuous measure of the similarity between cellular phenotypes that can also detect subtle differences such as from increasing dose. The rich, biologically-meaningful image representation that our method provides can help therapy development by supporting high-throughput investigations, even exploratory ones, with more sophisticated and disease-relevant models.

cell biology↗

bioRxiv2017

Diverse functions of closely homologous actin isoforms are defined by their nucleotide, rather than their amino acid sequence.

{beta}- and {gamma}-cytoplasmic-actin are nearly indistinguishable in their amino acid sequence, but are encoded by different genes that play non-redundant biological roles. The key determinants that drive their functional distinction are unknown. Here we tested the hypothesis that {beta}- and {gamma}-actin functions are defined by their nucleotide, rather than their amino acid sequence, using targeted editing of the mouse genome. Although previous studies have shown that disruption of {beta}-actin gene critically impacts cell migration and mouse embryogenesis, we demonstrate here that generation of a mouse lacking {beta}-actin protein by editing {beta}-actin gene to encode {gamma}-actin protein, and vice versa, does not affect cell migration and/or organism survival. Our data suggest that the essential in vivo function of {beta}-actin is provided by the gene sequence independent of the encoded protein isoform. We propose that this regulation constitutes a global \"silent code\" mechanism that controls the functional diversity of protein isoforms.

cell biology↗

bioRxiv2018

Structural determinants and genetic modifications enhance BMP2 stability and extracellular secretion

The short half-life and use of recombinant bone morphogentic protein (BMP)-2 in large doses poses major limitations in the clinic. Events regulating post-translational processing and degradation of BMP2 in situ, linked to its secretion, have not been understood. Towards identifying mechanisms regulating intracellular BMP2 stability, we first discovered that inhibiting proteasomal degradation enhances both intracellular BMP2 level and its extracellular secretion. Next, we identified BMP2 degradation occurs through an ubiquitin-mediated mechanism. Since ubiquitination precedes proteasomal turnover and mainly occurs on lysine residues of nascent proteins, we systematically mutated individual lysine residues within BMP2 and tested them for enhanced stability. Results revealed that substitutions on four lysine residues within the pro-BMP2 region and three in the mature region increased both BMP2 turnover and extracellular secretion. Structural modeling revealed key lysine residues involved in proteasomal degradation occupy a lysine cluster near proprotein convertase cleavage site. Interestingly, mutations within these residues did not affect biological activity of BMP2. These data suggest preventing intracellular proteasomal loss of BMP2 through genetic modifications can overcome limitations related to its short half-life.

cell biology↗

bioRxiv2017

The collective influence of 1, 25-dihydroxyvitamin D3 with physiological fluid shear stress on osteoblasts

1, 25-dihydroxyvitamin D3 (1, 25 (OH)2 D3) and mechanical stimuli in physiological environment play an important role in the pathogenesis of osteoporosis. The effects of 1, 25-dihydroxyvitamin D3 alone and mechanical stimuli alone on osteoblasts have been widely investigated. This study reports the collective influences of 1, 25-dihydroxyvitamin D3 and flow shear stress (FSS) on biological functions of osteoblasts. 1, 25 (OH)2 D3 were constructed in various kinds of concentration (0, 1, 10, 100 nmmol/L), while physiological fluid shear stress (12 dynes/cm2) were produced by using a parallel-plate fluid flow system. 1, 25 (OH)2 D3 affects the responses of ROBs to FSS, including the inhibition of NO releases and cell proliferation as well as the promotion of PGE2 releases and cell differentiation. These findings provide a possible mechanism by which 1, 25(OH)2 D3 influences osteoblasts responses to FSS and may provide guidance for the selection of 1, 25(OH)2 D3 concentration and mechanical loading in order to in vitro produce functional bone tissues.

cell biology↗

bioRxiv2018

Representativeness and robustness of TCR repertoire diversity assessment by high-throughput sequencing

High-throughput sequencing (HTS) has the potential to decipher the diversity of T cell repertoires and their dynamics during immune responses. Applied to T cell subsets such as T effector and T regulatory cells, it should help identify novel biomarkers of diseases. However, given the extreme diversity of TCR repertoires, understanding how the sequencing conditions, including cell numbers, biological and technical sampling and sequencing depth, impact the experimental outcome is critical to properly use of these data. Here we assessed the representativeness and robustness of TCR repertoire diversity assessment according to experimental conditions. By comparative analyses of experimental datasets and computer simulations, we found that (i) for small samples, the number of clonotypes recovered is often higher than the number of cells per sample, even after removing the singletons; (ii) high sequencing depth for small samples alters the clonotype distributions, which can be corrected by filtering the datasets using Shannon entropy as a threshold; (iii) a single sequencing run at high depth does not ensure a good coverage of the clonotype richness in highly polyclonal populations, which can be better covered using multiple sequencing. Altogether, our results warrant better understanding and awareness of the limitation of TCR diversity analyses by HTS and justify the development of novel computational tools for improved modelling of the highly complex nature of TCR repertoires.

immunology↗

bioRxiv2018

Unbiased dynamic characterization of RNA-protein interactions by OOPS

Current methods for the identification of RNA-protein interactions require a quantity and quality of sample that hinders their application, especially for dynamic biological systems or when sample material is limiting. Here, we present a new approach to enrich RNA-Binding Proteins (RBPs): Orthogonal Organic Phase Separation (OOPS), which is compatible with downstream proteomics and RNA sequencing. OOPS enables recovery of RBPs and free protein, or protein-bound RNA and free RNA, from a single sample in an unbiased manner. By applying OOPS to human cell lines, we extract the majority of known RBPs, and importantly identify additional novel RBPs, including those from previously under-represented cellular compartments. The high yield and unbiased nature of OOPS facilitates its application in both dynamic and inaccessible systems. Thus, we have identified changes in RNA-protein interactions in mammalian cells following nocodazole cell-cycle arrest, and defined the first bacterial RNA-interactome. Overall, OOPS provides an easy-to-use and flexible technique that opens new opportunities to characterize RNA-protein interactions and explore their dynamic behaviour.

cell biology↗

bioRxiv2016

The ATP synthase subunit β (ATP5B) is an entry factor for the hepatitis E virus

Hepatitis E occurs sporadically and as outbreaks due to contamination of drinking water. The causative agent, hepatitis E virus (HEV) is a hepatotropic non-enveloped RNA virus, which grows poorly in vitro. Consequently, many aspects of HEV biology are poorly characterized, including its cellular receptor and entry mechanism(s). Previous studies from our laboratory have shown that heparan sulfate proteoglycans (HSPGs) act as attachment factors for the virus. In the absence of purified high titer infectious virus, we have used hepatitis E virus-like particles (HEV-LPs) expressed and purified from E. coli to identify HEV entry factor(s) on liver cells in culture. Using a pull down and mass spectrometric approach, we identified the ATP synthase subunit {beta} (ATP5B) to bind the HEV capsid protein. Its role in the entry of HEV was then validated using antibody and siRNA mediated approaches, and infectious HEV from the stools of a hepatitis E patient. Though ATP synthase is largely a mitochondrial protein, the cell surface expressed form of ATP5B is implicated in other viral infections.

Cell Biology↗

bioRxiv2017

A novel mathematical method for disclosing oscillations ingene transcription: a comparative study

Circadian rhythmicity, the 24-hour cycle responsive to light and dark, is determined by periodic oscillations in gene transcription. This phenomenon has broad ramifications in physiologic function. Recent work has disclosed more cycles in gene transcription, and to the uncovering of these we apply a novel signal processing methodology known as the pencil method and compare it to conventional parametric, nonparametric, and statistical methods. Methods: In order to assess periodicity of gene expression over time, we analyzed a database derived from livers of mice entrained to a 12-hour light/12-hour dark cycle. We also analyzed artificially generated signals to identify differences between the pencil decomposition and other alternative methods.\n\nResultsThe pencil decomposition revealed hitherto-unsuspected oscillations in gene transcription with 12-hour periodicity. The pencil method was robust in detecting the 24-hour circadian cycle that was known to exist, as well as confirming the existence of shorter-period oscillations. A key consequence of this approach is that orthogonality of the different oscillatory components can be demonstrated. thus indicating a biological independence of these oscillations, that has been subsequently confirmed empirically by knocking out the gene responsible for the 24-hour clock.\n\nConclusionSystem identification techniques can be applied to biological systems and can uncover important characteristics that may elude visual inspection of the data. Significance: The pencil method provides new insights on the essence of gene expression and discloses a wide variety of oscillations in addition to the well-studied circadian pattern. This insight opens the door to the study of novel mechanisms by which oscillatory gene expression signals exert their regulatory effect on cells to influence human diseases.

cell biology↗

bioRxiv2018

RAD51 AND MITOTIC FUNCTION OF MUS81 ARE ESSENTIAL FOR RECOVERY FROM LOW-DOSE OF CAMPTOTHECIN IN THE ABSENCE OF THE WRN EXONUCLEASE

Stabilisation of the stalled replication fork is crucial to prevent excessive fork reversal or degradation, which can undermine genome integrity. The WRN protein is a human RecQ helicase that participates in the processing and recovery of perturbed replication forks. WRN is unique among the other human RecQ family members to possess exonuclease activity. However, the biological role of the WRN exonuclease is poorly defined, and little is known about an involvement in the response to perturbed replication. Recently, the WRN exonuclease has been linked to protection of stalled forks from MRE11-dependent degradation in response to clinically-relevant nanomolar doses of the Topoisomerase I inhibitor camptothecin. Alternative processing of perturbed forks has been associated to chemoresistance of BRCA-deficient cancer cells, thus, we used WRN exonuclease-deficiency as a model to investigate the fate of perturbed replication forks undergoing degradation, but in a BRCA wild-type condition. We find that, upon nanomolar doses of camptothecin, loss of WRN exonuclease stimulates fork inactivation and accumulation of parental gaps, which engages RAD51. Such alternative mechanism affects reinforcement of CHK1 phosphorylation and causes persistence of RAD51 during recovery from treatment. Notably, in WRN exonuclease-deficient cells, persistence of RAD51 correlates with elevated mitotic phosphorylation of MUS81 at Serine 87, which is essential to avoid accumulation of mitotic abnormalities. Altogether, these findings indicate that aberrant fork degradation, in the presence of a wild-type RAD51 axis, stimulates RAD51-mediated post-replicative repair and engagement of the MUS81 complex to limit genome instability and cell death.\n\nAUTHOR SUMMARYCorrect progression of the molecular machine copying the chromosomes is threatened by multiple causes that induce its delay or arrest. Once the replication machinery is arrested, the cell needs to stabilise it to prevent DNA damage. Many proteins contribute to this task and the Werners syndrome protein, WRN, is one of them.\n\nDefining what happens to replication machineries when they are blocked is highly relevant. Indeed, destabilised replication machineries may form upon treatment with anticancer drugs and influence the efficacy of some of them in specific genetic backgrounds. We used cells that lack one of the two enzymatic functions of WRN, the exonuclease activity, to investigate the fate of destabilised replication machineries. Our data show that they are handled by a repair pathway normally involved in fixing DNA breaks but, in this case, recruited to deal with regions of the genome that are left unreplicated after their destabilisation. This alternative mechanism involves a protein, RAD51, which tries to copy DNA from the sister chromosome. In so doing, however, RAD51 produces a lot of DNA interlinking that requires upregulation of a complex, called MUS81/EME1, which resolves this interlinking prior cell division and prevents accumulation of mitotic defects and cell death.

cell biology↗

bioRxiv2018

Ostreococcus tauri is a high-lipid content green algae that extrudes clustered lipid droplets

Lipid droplet biogenesis, accumulation and secretion is an important field of research spanning biofuel feedstock production in algae and yeast to plant-microbe symbiosis or human metabolic disorders and other diseases. Here we evaluate the critical elements that influence lipid accumulation in the highly simplified and smallest known eukaryote Ostreococcus tauri and identify several conditions that satisfy its classification as an oleaginous green alga. In addition, these experiments revealed the release of excess lipids in pea-pod like structures where many dense lipid droplets are clustered in a linear fashion surrounded by an enveloping membrane which contrasts with known mechanisms from other eukaryotes. These results highlight the potential for Ostreococcus tauri to probe the evolution of lipid droplet dynamics as an emerging model organism with a compacted eukaryotic genome and also to impact lipid feedstock bioproduction applications either directly or using synthetic biology.\n\nOne Sentence SummaryThe smallest known eukaryote Ostreococcus tauri is oleaginous and sheds lipid droplets as pea-pod like membrane enclosed clusters.

cell biology↗

bioRxiv2018

MICU1 modulates MCU ion selectivity and tolerance to manganese stress

The mitochondrial calcium uniporter is a highly selective ion channel composed of species-and tissue-specific structural and regulatory subunits. However, the contribution of each component to uniporter-mediated activity still remains unclear. Here, we employ an evolutionary and synthetic biology approach to investigate the functional inter-dependence between the pore-forming subunit MCU and the EF-hand protein MICU1. Using phylogenetic profiling and genetic complementation analyses, we show that MCU and MICU1 constitute the minimal eukaryotic unit of the uniporter, pointing towards a strong selective pressure behind their co-occurrence. Heterologous reconstitution of MCU-mediated and MICU1-gated mitochondrial calcium entry in vivo in yeast cells demonstrates that MICU1 per se is essential to protect yeast from MCU-dependent manganese cytotoxicity. Accordingly, MICU1 deletion significantly sensitizes human HEK-293 cells to manganese-induced stress. Our study identifies a critical role of MICU1 in the regulation of MCU ion selectivity, with potential implications for patients with MICU1 deficiency.

cell biology↗

bioRxiv2018

Enhanced super-resolution microscopy by extreme value based emitter recovery

Super-resolution localization microscopy allows visualization of biological structure at nanoscale resolution. However, the presence of heterogeneous background can degrade the nanoscale resolution by tens of nanometers and introduce significant image artifacts. Here we develop a new approach, referred to as extreme value based emitter recovery (EVER), to accurately recover the distorted fluorescent emitters from heterogeneous background. Through numerical simulation and biological experiments, we demonstrate that EVER significantly improves the accuracy and fidelity of the reconstructed super-resolution image for a wide variety of imaging characteristics. EVER requires no manual adjustment of parameters and is implemented as an easy-to-use ImageJ plugin that can immediately enhance the quality of super-resolution images. Our method paves the way for accurate nanoscale imaging of samples with heterogeneous background fluorescence, such as thicker tissue and cells.

cell biology↗

bioRxiv2016

Towards comprehensive and quantitative proteomics for diagnosis and therapy of human disease

Abbreviations\n\nAbstractDespite superior analytical features, mass spectrometry proteomics remains seldom used for the basic investigation and clinical treatment of human disease. This need is particularly pressing for childhood diseases that can be rare in incidence and variable in presentation. Modern mass spectrometry enables detailed functional characterization of the pathogenic biochemical processes, as achieved by accurate and comprehensive quantification of proteins and their regulatory chemical modifications. Here, we describe how high-accuracy mass spectrometry in combination with high-resolution chromatographic separations can be leveraged to meet these analytical requirements in a mechanism-focused manner. We review the quantification methods capable of producing accurate measurements of protein abundance and post-translational modification stoichiometries. We then discuss how experimental design and chromatographic resolution can be leveraged to achieve comprehensive functional characterization of biochemical processes in complex biological proteomes. Finally, we describe current approaches for quantitative analysis of a common functional protein modification: reversible phosphorylation. In all, current instrumentation and methods of high-resolution chromatography and mass spectrometry proteomics are poised for immediate translation into improved diagnostic and therapeutic strategies for pediatric and adult diseases.

Cell Biology↗

bioRxiv2018

A multi-omic atlas of the human frontal cortex for aging and Alzheimer's disease research

We initiated the systematic profiling of the dorsolateral prefrontal cortex obtained from a subset of autopsied individuals enrolled in the Religious Orders Study (ROS) or the Rush Memory and Aging Project (MAP), which are jointly designed and belong to a very few prospective studies of aging and dementia with detailed, longitudinal cognitive phenotyping during life and a quantitative, structured neuropathologic examination after death for >3,322 subjects. Here, we outline the first generation of data including genome-wide genotypes (n=2,090), whole genome sequencing (n=1,179), DNA methylation (n=740), chromatin immunoprecipitation with sequencing using an anti-Histone 3 Lysine 9 acetylation (H3K9Ac) antibody (n=712), RNA sequencing (n=638), and miRNA profile (n=702). Generation of other omic data including ATACseq, proteomic and metabolomics profiles is ongoing. Thanks to its prospective design and recruitment of older, non-demented individuals, these data can be repurposed to investigate a large number of syndromic and quantitative neuroscience phenotypes. The many subjects that are cognitively non-impaired at death also offer insights into the biology of the human brain in older non-impaired individuals.

cell biology↗