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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Continuous and bolus intraventricular topotecan prolong survival in a mouse model of leptomeningeal medulloblastoma

Leptomeningeal metastasis remains a difficult clinical challenge. Some success has been achieved by direct administration of therapeutics into the cerebrospinal fluid (CSF) circumventing limitations imposed by the blood brain barrier. Here we investigated continuous infusion versus bolus injection of therapy into the CSF in a preclinical model of human Group 3 medulloblastoma, the molecular subgroup with the highest incidence of leptomeningeal disease. Initial tests of selected Group 3 human medulloblastoma cell lines in culture showed that D283 Med and D425 Med were resistant to cytarabine and methotrexate. D283 Med cells were also resistant to topotecan, whereas 1 M topotecan killed over 99% of D425 Med cells. We therefore introduced D425 Med cells, modified to express firefly luciferase, into the CSF of immunodeficient mice. Mice were then treated with topotecan or saline in five groups: continuous intraventricular (IVT) topotecan via osmotic pump (5.28 g/day), daily bolus IVT topotecan injections with a similar daily dose (6 g/day), systemic intraperitoneal injections of a higher daily dose of topotecan (15 g/day), daily IVT pumped saline and daily intraperitoneal injections of saline. Bioluminescence analyses revealed that both IVT topotecan treatments effectively slowed leptomeningeal tumor growth in the brains, although histological analysis showed that they were associated with localized brain necrosis. In the spines, bolus IVT topotecan showed a trend towards slower tumor growth compared to continuous (pump) IVT topotecan, as measured by bioluminescence. Both continuous and bolus topotecan IVT showed similar survival that was longer compared to other groups. Thus, both direct IVT topotecan CSF delivery methods produced better anti-medulloblastoma effect compared to systemic therapy at the dosages used here.

cancer biology↗

bioRxiv2018

Persistence of microcystin production of Planktothrix agardhii exposed to different salinity concentrations.

Recent reports tend to predict the increase of harmful cyanobacteria in water systems worldwide due to the climatic and environmental changes, which would compromise water quality and public health. Among abiotic changes, the higher salinities are expected to promote the growth of some harmful species such as Planktothrix agardhii, which is known to build up blooms in brackish areas. Since P. agardhii is a common cyanotoxin producer (microcystin-producing), we investigated here the growth and tolerance of this species when exposed in vitro to a range of salinity levels, while assessing its microcystins variation and production in batch cultures during a time-frame experiment of 18 days. The study revealed a salt acclimation of the brackish P. agardhii that still produced microcystins in salty cultures while maintaining its growth ability in low to medium salinities (ranged from 0 to 7.5 g L-1). For higher salinity concentrations (10 to 12.5 g L-1), microcystins were still detected, while significantly lower growth rates were obtained during the exponential growth phase. This suggests that moderate to high salt ranges do not inhibit the microcystins production of P. agardhii at least for several weeks. Finally, the predicted remediation perspectives in a context of environment salinization assumed by environmental policies may be insufficient to eradicate this potential toxic cyanobacteria, especially when this species is already dominant in the waterbodies.

microbiology↗

bioRxiv2018

A fully-automated, robust, and versatile algorithm for long-term budding yeast segmentation and tracking

Live cell time-lapse microscopy, a widely-used technique to study gene expression and protein dynamics in single cells, relies on segmentation and tracking of individual cells for data generation. The potential of the data that can be extracted from this technique is limited by the inability to accurately segment a large number of cells from such microscopy images and track them over long periods of time. Existing segmentation and tracking algorithms either require additional dyes or markers specific to segmentation or they are highly specific to one imaging condition and cell morphology and/or necessitate manual correction. Here we introduce a fully automated, fast and robust segmentation and tracking algorithm for budding yeast that overcomes these limitations. Full automatization is achieved through a novel automated seeding method, which first generates coarse seeds, then automatically fine-tunes cell boundaries using these seeds and automatically corrects segmentation mistakes. Our algorithm can accurately segment and track individual yeast cells without any specific dye or biomarker. Moreover, we show how existing channels devoted to a biological process of interest can be used to improve the segmentation. The algorithm is versatile in that it accurately segments not only cycling cells with smooth elliptical shapes, but also cells with arbitrary morphologies (e.g. sporulating and pheromone treated cells). In addition, the algorithm is largely independent of the specific imaging method (bright-field/phase) and objective used (40X/63X). We validate our algorithms performance on 9 cases each entailing a different imaging condition, objective magnification and/or cell morphology. Taken together, our algorithm presents a powerful segmentation and tracking tool that can be adapted to numerous budding yeast single-cell studies.

cell biology↗

bioRxiv2018

A Computational Framework To Assess Genome-Wide Distribution Of Polymorphic Human Endogenous Retrovirus-K In Human Populations

Human Endogenous Retrovirus type K (HERV-K) is the only HERV known to be insertionally polymorphic. It is possible that HERV-Ks contribute to human disease because people differ in both number and genomic location of these retroviruses. Indeed viral transcripts, proteins, and antibody against HERV-K are detected in cancers, auto-immune, and neurodegenerative diseases. However, attempts to link a polymorphic HERV-K with any disease have been frustrated in part because population frequency of HERV-K provirus at each site is lacking and it is challenging to identify closely related elements such as HERV-K from short read sequence data. We present an integrated and computationally robust approach that uses whole genome short read data to determine the occupation status at all sites reported to contain a HERV-K provirus. Our method estimates the proportion of fixed length genomic sequence (k-mers) from whole genome sequence data matching a reference set of k-mers unique to each HERV-K loci and applies mixture model-based clustering to account for low depth sequence data. Our analysis of 1000 Genomes Project Data (KGP) reveals numerous differences among the five KGP super-populations in the frequency of individual and co-occurring HERV-K proviruses; we provide a visualization tool to easily depict the prevalence of any combination of HERV-K among KGP populations. Further, the genome burden of polymorphic HERV-K is variable in humans, with East Asian (EAS) individuals having the fewest integration sites. Our study identifies population-specific sequence variation for several HERV-K proviruses. We expect these resources will advance research on HERV-K contributions to human diseases.\n\nAuthor summaryHuman Endogenous Retrovirus type K (HERV-K) is the youngest of retrovirus families in the human genome and is the only group that is polymorphic; a HERV-K can be present in one individual but absent from others. HERV-Ks could contribute to disease risk but establishing a link of a polymorphic HERV-K to a specific disease has been difficult. We develop an easy to use method that reveals the considerable variation existing among global populations in the frequency of individual and co-occurring polymorphic HERV-K, and in the total number of HERV-K that any individual has in their genome. Our study provides a global reference set of HERV-K genomic diversity and tools needed to determine the genomic landscape of HERV-K in any patient population.

genomics↗

bioRxiv2018

The sequence dependent search mechanism of EcoRI

One-dimensional search is an essential step in DNA target recognition. Theoretical studies have suggested that the sequence dependence of one-dimensional diffusion can help resolve the competing demands of fast search and high target affinity, a conflict known as the speed-selectivity paradox. The resolution requires that the diffusion energy landscape is correlated with the underlying specific binding energies. In this work, we report observations of one-dimensional search by QD labeled EcoRI. Our data supports the view that proteins search DNA via rotation coupled sliding over a corrugated energy landscape. We observed that while EcoRI primarily slides along DNA at low salt concentrations, at higher concentrations its diffusion is a combination of sliding and hopping. We also observed long-lived pauses at genomic star sites which differ by a single nucleotide from the target sequence. To reconcile these observations with prior biochemical and structural data, we propose a model of search in which the protein slides over a sequence independent energy landscape during fast search, but rapidly interconverts with a \"hemi-specific\" binding mode in which a half site is probed. This half site interaction stabilizes the transition to a fully specific mode of binding which can then lead to target recognition.

biophysics↗

bioRxiv2018

Hypomorphic mutation of the mouse Huntingtons disease gene orthologue

Rare individuals with hypomorphic inactivating mutations in the Huntingtons Disease (HD) gene (HTT), identified by CAG repeat expansion in the eponymous neurodegenerative disorder, exhibit variable abnormalities that imply HTT essential roles during organ development. Here we report phenotypes produced when increasingly severe hypomorphic mutations in Htt, the murine HTT orthologue (in HdhneoQ20, HdhneoQ50, HdhneoQ111 mice), were placed over a null allele (Hdhex4/5). The most severe hypomorphic allele failed to rescue null lethality at gastrulation, while the intermediate alleles yielded perinatal lethality and a variety of fetal abnormalities affecting body size, skin, skeletal and ear formation, and transient defects in hematopoiesis. Comparative molecular analysis of wild-type and Htt-null retinoic acid-differentiated cells revealed gene network dysregulation associated with organ development and proposed polycomb repressive complexes and miRNAs as molecular mediators. Together these findings demonstrate that the HD gene acts both pre- and post-gastrulation and possibly suggest pleiotropic consequences of HTT-lowering therapeutic strategies.\n\nAuthor SummaryThe HTT gene product mutated in Huntingtons Disease (HD) has essential roles during normal organism development, however, still not fully predictable are the functional consequences of its partial inactivation. Our genetic study provides a comprehensive effects description of progressively stronger suppression of Htt gene, the murine HTT counterpart. The most severe Htt reduction leads to embryo lethality, while intermediate Htt dosages yield a variety of developmental abnormalities affecting body size, skin, skeletal and ear formation, and hematopoiesis. Complementing molecular analysis in differentiating cells depleted of a functional Htt gene further elucidates genes networks dysregulated during organ development and proposes chromatin regulators and short non-coding RNAs as key molecular mediators. Together these findings demonstrate that the HD gene acts both at early and later stages of development, thus possibly suggesting long-term consequences associated to the newest HD therapeutic strategies aimed at lowering the HTT gene product.

genetics↗

bioRxiv2018

MxB restricts HIV-1 by targeting the tri-hexamer interface of the viral capsid

Myxovirus resistance protein B (MxB) is an interferon-inducible restriction factor of HIV-1 that blocks nuclear import of the viral genome. Evidence suggests that MxB recognizes higher-order interfaces of the HIV capsid lattice, but the mechanistic details of this interaction are not known. Previous studies have mapped the restriction activity of MxB to its N-terminus encompassing a triple arginine motif 11RRR13. Here we demonstrate a direct and specific interaction between the MxB N-terminus and helical assemblies of HIV-1 capsid protein (CA) using highly purified recombinant proteins. We performed thorough mutagenesis to establish the detailed molecular requirements for the CA interaction with MxB. The results map MxB binding to the interface of three CA hexamers, specifically interactions between positively charged MxB N-terminal residues and negatively charged CA residues. Our crystal structures show that the CA mutations affecting MxB interaction and restriction do not alter the conformation of capsid assembly. In addition, 30 microsecond long all-atom molecular dynamics (MD) simulations of the complex between the MxB N-terminus and the HIV CA tri-hexamer interface show persistent MxB binding and identify a MxB-binding pocket surrounded by three CA hexamers. These results establish the molecular details of the binding of a lattice-sensing host factor onto HIV capsid, and provide insight into how MxB recognizes HIV capsid for the restriction of HIV-1 infection.\n\nAuthor summaryThe human antiviral protein MxB is a restriction factor that fights HIV infection. Previous experiments have demonstrated that MxB targets the HIV capsid, a protein shell that protects the viral genome. To make the conical shaped capsid, HIV CA proteins are organized into a lattice composed of hexamer and pentamer building blocks, providing many interfaces for host proteins to recognize. Through extensive biochemical and biophysical studies and molecular dynamics simulations, we show that MxB is targeting the HIV capsid by recognizing the region created at the intersection of three CA hexamers. We are further able to map this interaction to a few CA residues, located in a negatively-charged well at the interface between the three CA hexamers. This work provides detailed residue-level mapping of the targeted capsid interface and how MxB interacts. This information could inspire the development of capsid-targeting therapies for HIV.

biochemistry↗

bioRxiv2018

Brain-wide cellular resolution imaging of Cre transgenic zebrafish lines for functional circuit-mapping

Decoding the functional connectivity of the nervous system is facilitated by transgenic methods that express a genetically encoded reporter or effector in specific neurons; however, most transgenic lines show broad spatiotemporal and cell-type expression. Increased specificity can be achieved using intersectional genetic methods which restrict reporter expression to cells that co-express multiple drivers, such as Gal4 and Cre. To facilitate intersectional targeting in zebrafish, we have generated more than 50 new Cre lines, and co-registered brain expression images with the Zebrafish Brain Browser, a cellular resolution atlas of 264 transgenic lines. Lines labeling neurons of interest can be identified using a web-browser to perform a 3D spatial search (zbbrowser.com). This resource facilitates the design of intersectional genetic experiments and will advance a wide range of precision circuit-mapping studies.

neuroscience↗

bioRxiv2018

Lymph-Circulating Tumor Cells show distinct properties to Blood-Circulating Tumor Cells and constitute extraordinary efficient metastatic precursors

The molecular properties of tumor cells as they exit the primary tumor into the afferent lymphatics en route to the sentinel lymph nodes (SLNs) are not yet known. We developed an innovative technique that enables the collection of lymph and lymph-circulating tumor cells (LCTCs) en route to the SLN in immunocompetent animal model of breast cancer metastasis. We found that LCTCs and blood circulating tumor cells (BCTCs) as exited the primary tumor shared similar gene and protein expression profiles that were distinct from those of primary tumors and lymph node metastases (LNMs) despite their common parental cell origin. LCTCs but not BCTC exist in clusters, display a hybrid epithelial/mesenchymal phenotype and cancer stem cell-like properties and constitute extraordinarily efficient metastatic precursors. These results demonstrate that tumor cell metastasizing through the lymphatic are different from those spread by the blood circulation. The contribution of these cells to overall peripheral blood CTC is important in cancer therapy. Whether these two types of cells occur in cancer patients remain to be determined.\n\nStatement of significanceThe presence of tumor cells in the SLN denotes poor prognosis and worse patient outcomes. We have developed a capability to routinely collect these tumor cells before they reach their first stop, SLN, in their metastatic journey to distant sites. Examination of these cells and their lymph microenvironment revealed that they are molecularly different from their tumor of origin, their LNMs and their counterpart cells in the blood. This is the first time these cells are captured and studied. The approach will provide a new level of information that is highly relevant to our understanding of metastasis.

cancer biology↗

bioRxiv2018

Using pan RNA-seq analysis to reveal the ubiquitous existence of 5′ end and 3′ end small RNAs

In this study, we used pan RNA-seq analysis to reveal the ubiquitous existence of 5 end and 3 end small RNAs. 5 and 3 sRNAs alone can be used to annotate mitochondrial with 1-bp resolution and nuclear non-coding genes and identify new steady-state RNAs, which are usually from functional genes. Using 5, 3 and intronic sRNAs, we revealed that the enzymatic dsRNA cleavage and RNAi could involve in the RNA degradation and gene expression regulation of U1 snRNA in human. The further study of 5, 3 and intronic sRNAs help rediscover double-stranded RNA (dsRNA) cleavage, RNA interference (RNAi) and the regulation of gene expression, which challenges the classical theories. In this study, we provided a simple and cost effective way for the annotation of mitochondrial and nuclear non-coding genes and the identification of new steady-state RNAs, particularly long non-coding RNAs (lncRNAs). We also provided a different point of view for cancer and virus, based on the new discoveries of dsRNA cleavage, RNAi and the regulation of gene expression.

bioinformatics↗

bioRxiv2018

Breakdown of gametophytic self-incompatibility in subdivided populations

Many hermaphroditic flowering plants species possess a genetic self-incompatibility (SI) system that prevents self-fertilization and is typically controlled by a single multiallelic locus, the S-locus. The conditions under which SI can be stably maintained in single isolated populations are well known and depend chiefly on the level of inbreeding depression and the number of SI alleles segregating at the S-locus. However, while both the number of SI alleles and the level of inbreeding depression are potentially affected by population subdivision, the conditions for the maintenance of SI in subdivided populations remain to be studied. In this paper, we combine analytical predictions and two different individual-based simulation models to show that population subdivision can severely compromise the maintenance of SI. Under the conditions we explored, this effect is mainly driven by the decrease of the local diversity of SI alleles rather than by a change in the dynamics of inbreeding depression. We discuss the implications of our results for the interpretation of empirical data on the loss of SI in natural populations.\n\nData accessibility statementNo data to be archived

evolutionary biology↗

bioRxiv2018

Targeting MYC Overexpressing Leukemia with Cardiac Glycoside Proscillaridin Through Downregulation of Histone Acetyltransferases

Targeting MYC oncogene remains a major therapeutic goal in cancer chemotherapy. Here, we demonstrate that proscillaridin, a cardiac glycoside approved for heart failure treatment exhibit anticancer selectivity towards high MYC expressing leukemic cell lines and leukemia stem cells. At a clinically relevant concentration, proscillaridin induced a rapid downregulation of MYC protein level, due to a significant decrease in MYC protein half-life. Proscillaridin treatment induced a downregulation of gene sets involved in MYC pathway, and a concomitant upregulation of genes involved in hematopoietic differentiation. Proscillaridin induced a significant loss of lysine acetylation in histone H3 (K9, K14, K18 and K27) and in non-histone proteins such as MYC, MYC target proteins, and a series of histone acetylation regulators. Loss of lysine acetylation correlated with a rapid downregulation of histone acetyltransferase protein levels, involved in histone and MYC acetylation (CBP, P300, GCN5, TIP60, and MOZ), preferentially in MYC overexpressing leukemia as compared to other cancer cells. These results support the repurposing of proscillaridin in MYC overexpressing leukemia and propose a novel strategy to target MYC in cancer.

pharmacology and toxicology↗

bioRxiv2018

Enhanced fitness relates to reduced cerebrovascular reactivity and perfusion in a sample of very healthy older adults

Aging is accompanied by decreased grey matter volume (GMV), cerebral blood flow (CBF), and cerebrovascular reactivity (CVR), where the latter tends to decline the earliest in aging. Enhanced fitness in aging has been related to preservation of GMV and CBF, and in some cases CVR, although there are contradictory relationships reported between CVR and fitness. Therefore, to gain a better understanding of the complex interplay with fitness and GMV, CBF and CVR, it is necessary to study them concurrently. Here, we aimed to disentangle the interactions between these outcomes in healthy older adults. MRI acquisitions collected anatomical, CBF and CVR information in all participants, as well as VO2,max. Results revealed decreased CVR was associated with increased fitness throughout large areas of the cerebral cortex. Within these regions it was found that lower fitness was associated with higher CBF and a slower hemodynamic response to hypercapnia. Overall, results indicate that the relationship between age, cerebral health and cerebral hemodynamics are complex. Future studies should collect other physiological outcomes in parallel with quantitative imaging, such as measures of carbon dioxide sensitivity and autoregulation, to further understand the intricacy of the effects fitness has on the aging brain, and how this may bias quantitative measures of cerebral health.

neuroscience↗

bioRxiv2018

Molecular anatomy and plasticity of the long noncoding RNA HOTAIR

Long noncoding RNA molecules (lncRNAs) are estimated to account for the majority of eukaryotic genomic transcripts, and have been associated with multiple diseases in humans. However, our understanding of their structure-function relationships is scarce, with structural evidence coming mostly from indirect biochemical approaches or computational predictions. Here we describe the hypothetical molecular anatomy of the lncRNA HOTAIR (HOx Transcript AntIsense RNA) inferred from direct, high-resolution visualization by atomic force microscopy (AFM) in nucleus-like conditions at 37 degrees. Our observations reveal that HOTAIR has a distinct anatomy with a high degree of plasticity. Fast AFM scanning enabled the quantification of this plasticity, and provided visual evidence of physical interactions with genomic DNA segments. Our report provides the first biologically-plausible hypothetical description of the anatomy and intrinsic properties of HOTAIR, and presents a framework for studying the structural biology of lncRNAs.

biophysics↗

bioRxiv2018

High resolution atlasing of the venous brain vasculature from 7T quantitative susceptibility

The vascular organization of the human brain can determine neurological and neurophysiological functions, yet thus far it has not been comprehensively mapped. Aging and diseases such as dementia are known to be associated with changes to the vasculature and normative data could help detect these vascular changes in neuroimaging studies. Furthermore, given the well-known impact of venous vessels on the blood oxygen level dependent (BOLD) signal, information about the common location of veins could help detect biases in existing datasets. In this work, a quantitative atlas of the venous vasculature using quantitative susceptibility maps (QSM) acquired with a 0.6 mm isotropic resolution is presented. The Venous Neuroanatomy (VENAT) atlas was created from 5 repeated 7 Tesla MRI measurements in young and healthy volunteers (n = 20, 10 females, mean age = 25.1 {+/-} 2.5 years) using a two-step registration method on 3D segmentations of the venous vasculature. This cerebral vein atlas includes the average vessel location, diameter (mean: 0.84 {+/-} 0.33 mm) and curvature (0.11 {+/-} 0.05 mm-1) from all participants and provides an in vivo measure of the angio-architectonic organization of the human brain and its variability. This atlas can be used as a basis to understand changes in the vasculature during aging and neurodegeneration, as well as vascular and physiological effects in neuroimaging.

neuroscience↗

bioRxiv2018

Deep Recurrent Attention Models for Histopathological Image Analysis

Histopathology defines the gold standard in oncology. Automatic analysis of pathology images could thus have a significant impact on diagnoses, prognoses and treatment decisions for cancer patients. Recently, convolutional neural networks (CNNs) have shown strong performance in computational histopathology tasks. However, given it remains intractable to process pathology slides in their entirety, CNNs have traditionally performed inference on small individual patches extracted from the image. This often requires a significant amount of computation and can result in ignoring potentially relevant spatial and contextual information. Being able to process larger input patches and locating discriminatory regions more efficiently could help improve both computational and task specific performance. Inspired by the recent success of Deep Recurrent Attention Models (DRAMs) in image recognition tasks, we propose a novel attention-based architecture for classification in histopathology. Similar to CNNs, DRAMs have a degree of translation invariance built-in, but the amount of computation performed can be controlled independently from the input image size. The model is a deep recurrent neural network trained with reinforcement learning to attend to the most relevant areas of large input patches. We evaluate our model on histological and molecular subtype classification tasks for the glioma cohorts of The Cancer Genome Atlas (TCGA). Our results suggest that the DRAM has comparable performance to state-of-the-art CNNs despite only processing a select number of patches.

bioinformatics↗

bioRxiv2018

Intricate structure of the interphase chromocenter revealed by the analysis of a factor involved in species formation

In higher eukaryotes centromeres often coalesce into a large intranuclear domain called the chromocenter. Chromocenters are important for the organization of pericentric heterochromatin and a disturbance of their formation results in an upregulation of repetitive elements and causes defects in chromosome segregation. Mutations in the gene encoding for the centromere associated Drosophila speciation factor HMR show very similar phenotypes suggesting a role of HMR in chromocenter architecture and function. We performed confocal and super resolution microscopy as well as proximity based biotinylation experiments of HMR and its associated protein HP1a to generate a molecular map of HMR and HP1a bound chromatin. Our work reveals an intricate internal structure of the centromeric chromatin region, which suggests a role of HMR in separating heterochromatin from centromeric chromatin.

biochemistry↗

bioRxiv2018

DNA and RNA binding mediate the toxicity of arginine-rich peptides encoded by C9ORF72 GGGGCC repeats

Due to their capability to transport chemicals or proteins into target cells, cell-penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting properties, arginine-rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic-lateral sclerosis (ALS) as a model of an arginine-rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA- and DNA-binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids such as RNA transcription, translation, splicing and degradation or DNA replication and repair are impaired by the presence of the CPP. Interestingly, the effects of (PR)n are fully mimicked by PROTAMINE, a small arginine-rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA- and DNA-binding factors from chromatin and mRNA accounts for the toxicity of arginine-rich CPPs, including those that have been recently associated to the onset of ALS.

neuroscience↗