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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Single-particle tracking of Nucleotide Excision Repair proteins inside living bacteria

Single-particle tracking (SPT) combined with Photoactivated Localization Microscopy (sptPALM) provides an opportunity to perform complex molecular biology experiments inside living cells. By tracking the motion of DNA repair proteins in vivo, information can be extracted not only about their diffusion, but also about the kinetics and spatial distribution of DNA binding1-3. From a methodological point of view, a Total Internal Reflection Microscope equipped with a sensitive detector (usually an EM-CCD camera4) is commonly used, allowing detection of individual fluorophores. The signal from individual emitters can be analysed and the position of a given fluorophore established with high accuracy (up to a single nm) by Gaussian fitting. To determine the mobility of each fluorophore, the positions of individual molecules are linked into trajectories over multiple frames using a tracking algorithm5.

biophysics↗

bioRxiv2018

The molecular pathogenesis of superoxide dismutase 1-linked ALS is promoted by low oxygen tension

Mutations that destabilize superoxide dismutase 1 (SOD1) are a cause of amyotrophic lateral sclerosis (ALS). SOD1, which is located in the reducing cytosol, contains an oxidized disulfide bond required for stability. We show that the bond is an Achilles heel of the protein because it is sensitive to the oxygen tension. Culture of ALS patient-derived fibroblasts, astrocytes and induced pluripotent stem cell-derived mixed motor neuron and astrocyte cultures (MNACs) under lowered oxygen tensions caused reductive bond cleavage and misfolding. The effects were greatest in cells expressing mutant SOD1s, but also occurred in wild type SOD1 in cultures derived from patients carrying ALS-linked mutations in C9orf72, FUS and TBK1, as well as from controls. MNACs showed a greater response than the other cell types, including enhanced SOD1 aggregation, in line with the vulnerability of the motor system. Our results show that oxygen tension is a principal determinant of SOD1 stability and shed light on how risk factors for ALS, such as aging and other conditions causing reduced vascular perfusion, could lead to disease initiation and progression.\n\nSubject categories Neuroscience; Molecular Biology of Disease

neuroscience↗

bioRxiv2018

Deciphering the metabolic perturbation in hepatic alveolar echinococcosis: a 1H NMR-based metabolomics study

Hepatic alveolar echinococcosis (HAE) is a chronic and potentially lethal parasitic disease. It is caused by growth of Echinococcus multilocularis larvae in liver. To date, early-stage diagnosis for the disease is not mature due to its long asymptomatic incubation period. In this study, a proton nuclear magnetic resonance (1H NMR) -based metabolomics approach was applied in conjunction with multivariate statistical analysis to investigate the altered metabolic profiles in blood serum and urine samples from HAE patients and to identify characteristic metabolic markers associated with HAE. The current results identified 21 distinctive metabolic difference between the HAE patients and healthy individuals, which can be associated with perturbations in energy metabolism, amino acid metabolism, oxidative stress, and neurotransmitter imbalance. In addition, the Fischer ratio, which is the molar ratio of branched-chain amino acids to aromatic amino acids was found significantly lower (p<0.001) in blood serum from HAE patients. The ratio, together with changes in other metabolic pathways may provide new insight into mechanistic understanding of HAE pathogenesis, and may be useful for early-stage HAE diagnosis.\n\nAuthor SummaryHepatic alveolar echinococcosis (HAE) is a life-threatening disease caused by Echinococcus multilocularis infection. The disease has a long asymptomatic early stage (5~15 years), which complicates effective diagnosis of early-stage HAE even with advanced imaging techniques. Metabolomics is an emerging analytical platform that comprises of analysis of all small molecule metabolites that are present within an organism. The applications of metabolomics method on HAE may help to reveal the molecular biology mechanisms of HAE. In the current study, we had used 1H NMR-based metabolomics technique to investigate blood serum and urine samples from HAE patients. Altered metabolic responses and characteristic differential metabolites for HAE were identified. The metabolic profiling of human biofluids provided valuable information for early-stage HAE diagnosis and for therapeutic interventions, without having to extract HAE vesicles from patients. By featuring global and comprehensive metabolic status, the metabolomics approach holds considerable promise as a noninvasive, dynamic, and effective tool for probing the underlying mechanism of HAE.

systems biology↗

bioRxiv2018

Improved G-AgarTrap: A highly efficient transformation method for intact gemmalings of the liverwort Marchantia polymorpha

Liverworts are key species for studies of plant evolution, occupying a basal position among the land plants. Marchantia polymorpha has emerged as a highly studied model liverwort, and many relevant techniques, including genetic transformation, have been established for this species. Agrobacterium-mediated transformation is widely used in many plant species because of its low cost. Recently, we developed a simplified Agrobacterium-mediated method for transforming M. polymorpha, known as AgarTrap (agar-utilized transformation with pouring solutions). The AgarTrap procedure, which involves culturing the liverwort tissue in various solutions on a single solid medium, yields up to a hundred independent transformants. AgarTrap is a simple procedure, requiring minimal expertise, cost, and time.\n\nHere, we investigated four factors that influence AgarTrap transformation efficiency: (1) humidity, (2) surfactant in the transformation buffer, (3) Agrobacterium strain, and (4) light/dark condition. We adapted the AgarTrap protocol for transforming intact gemmalings, achieving an exceptionally high transformation efficiency of 97%. The improved AgarTrap method will enhance the molecular biological study of M. polymorpha. The present study also provides new possibilities for improving transformation techniques for a variety of plant species.

plant biology↗

bioRxiv2018

Molecular characterization of beast’s maguey (Agave antillarum Descourt), in the Dominican Republic

BackgroundThis research project was proposed to determine the genetic variability (if any) of beast maguey (Agave antillanarum Decourt) populations, by means of molecular markers of the AFLPs (Amplified Fragment Length Polymorphism) type. For the determination of genetic variability, samples were collected from wild populations of maguey growing in some regions of the Dominican Republic, mainly the north, northwest, south and southwest regions, using statistical techniques already established for these cases. These samples were processed and analyzed in the plant molecular biology laboratory of the Centtro de Biotecnologia Vegetal (CEBIVE) of Instituto de Innovacion en Biotecnologia e Industria (IIBI) in Santo Domingo, Dominican Republic.\n\nResultsAs for the genetic diversity is concerned, it was found that several of the populations studied differ from each other without taking into account the geographic distance where they grow. We concluded that there is a possibility that more than one species of Agave antillarun could be found in the wild in the Dominican Republic.

plant biology↗

bioRxiv2018

Electrical-charge accumulation enables integrative quality control during B. subtilis sporulation

Quality control of offspring is important for the survival of cells. However, the mechanism by which quality of offspring cells may be monitored while running genetic programs of cellular differentiation remains largely unclear. Here we investigated a quality control system during Bacillus subtilis spore formation by combining single-cell time-lapse microscopy, molecular biology and mathematical modelling. Our results revealed that the quality-control system via premature germination is coupled with the accumulation of cations on the surface of developing forespores. Specifically, the forespores accumulating less cations on their surface are more likely to be aborted. This charge accumulation system enables the projection of multidimensional information about the external environment and morphological development of the forespore onto a one-dimensional information of cation accumulation. Based on the insight we gain, we propose a novel use of Nernstian chemicals for reducing the yield and quality of Bacillus endospores.

biophysics↗

bioRxiv2018

A framework for analysis of real-time nucleic acid amplification data using novel multidimensional standard curves

Research into improving methods for absolute quantification of nucleic acids using standard curves has plateaued despite its positive, far-reaching impact on biomedical applications and clinical diagnostics. Currently, the mathematics involved in this mature area is restricted by the simplicity of conventional standard curves such as the gold standard cycle-threshold (Ct) method. Here, we propose a novel framework that expands current methods into multidimensional space and opens the door for more complex mathematical techniques, signal processing and machine learning to be implemented. The heart of this work revolves around two new concepts: the multidimensional standard curve and its home - the feature space. This work has been validated using phage lambda DNA and standard qPCR instruments. We show that the capabilities of standard curves can be extended in order to simultaneously: enhance absolute quantification, detect outliers and provide insights into the intersection between molecular biology and amplification data. This work and its vision aims to maximise the information extracted from amplification data using current instruments without increasing the cost or complexity of existing diagnostic settings.

bioinformatics↗

bioRxiv2018

Designing robust N-of-1 studies for precision medicine

Recent advances in molecular biology, sensors, and digital medicine have led to an explosion of products and services for high-resolution monitoring of individual health. The N-of-1 study has emerged as an important methodological tool for harnessing these new data sources, enabling researchers to compare the effectiveness of health interventions at the level of a single individual. We have developed a stochastic time series model that simulates an N-of-1 study, facilitating rapid optimization of N-of-1 study designs and increasing the likelihood of study success while minimizing participant burden. Using simulation, we demonstrate how the number of treatment blocks, ordering of treatments within blocks, duration of each treatment, and sampling frequency affect our ability to detect true differences in treatment efficacy. We provide a set of recommendations for study designs based on treatment, outcome, and instrument parameters, and provide our simulation software as a supplement to the paper.

bioinformatics↗

bioRxiv2018

Long-read Sequencing Uncovers a Complex Transcriptome Topology in Varicella Zoster Virus

BackgroundVaricella zoster virus (VZV) is a human pathogenic alphaherpesvirus harboring a relatively large DNA molecule. The VZV transcriptome has already been analyzed by microarray and short-read sequencing analyses. However, both approaches have substantial limitations when used for structural characterization of transcript isoforms, even if supplemented with primer extension or other techniques. Among others, they are inefficient in distinguishing between embedded RNA molecules, transcript isoforms, including splice and length variants, as well as between alternative polycistronic transcripts. It has been demonstrated in several studies that long-read sequencing is able to circumvent these problems.\n\nResultsIn this work, we report the analysis of VZV lytic transcriptome using the Oxford Nanopore Technologies sequencing platform. These investigations have led to the identification of 114 novel transcripts, including mRNAs, non-coding RNAs, polycistronic RNAs and complex transcripts, as well as 10 novel spliced transcripts and 27 novel transcription start site isoforms and transcription end site isoforms. A novel class of transcripts, the nroRNAs are described in this study. These transcripts are encoded by the genomic region located in close vicinity to the viral replication origin. We also show that the VZV latency transcript (VLT) exhibits a more complex structural variation than formerly believed. Additionally, we have detected RNA editing in a novel non-coding RNA molecule.\n\nConclusionsOur investigations disclosed a composite transcriptomic architecture of VZV, including the discovery of novel RNA molecules and transcript isoforms, as well as a complex meshwork of transcriptional read-throughs and overlaps. The results represent a substantial advance in the annotation VZV transcriptome and in understanding the molecular biology of the herpesviruses in general.

genomics↗

bioRxiv2018

Pervasive contaminations in sequencing experiments are a major source of false genetic variability: a Mycobacterium tuberculosis meta-analysis

Contaminant DNA is a well-known confounding factor in molecular biology and in genomic repositories. Strikingly, analysis workflows for whole-genome sequencing (WGS) data usually neglect the errors introduced by potential contaminations. We performed a comprehensive evaluation of the extent and impact of contaminant DNA in WGS by analyzing more than 4,000 bacterial samples from 20 different studies. We found that contaminations are pervasive and can introduce large biases in variant analysis. We showed that these biases can translate in hundreds of false positive and negative SNPs, even for samples with slight contaminations. Studies investigating complex biological traits from sequencing data can be completely biased if contaminations are neglected during the bioinformatic analysis. We used both real and simulated data to evaluate and implement reliable, contamination-aware analysis pipelines. Our results urge for the implementation of such pipelines as sequencing technologies consolidate as a precision tool in the research and clinical context.

genomics↗

bioRxiv2018

Trimitomics: An efficient pipeline for mitochondrial assembly from transcriptomic reads in non-model species

Mitochondrial resources are of known utility to many fields of phylogenetic, population and molecular biology. Their combination of faster and slower-evolving regions and high copy number enables them to be used in many situations where other loci are unsuitable, with degraded samples and after recent speciation events.\n\nThe advent of next-generation sequencing technologies (and notably the Illumina platform) has led to an explosion in the number of samples that can be studied at transcriptomic level, at relatively low cost. Here we describe a robust pipeline for the recovery of mitochondrial genomes from these RNA-seq resources. This pipeline can be used on sequencing of a variety of depths, and reliably recovers the protein coding and ribosomal gene complements of mitochondria from almost any transcriptomic sequencing experiment. The complete sequence of the mitochondrial genome can also be recovered when sequencing is performed in sufficient depth. We evidence the efficacy of our pipeline using data from a number of non-model invertebrates of four disparate phyla, namely Porifera, Nemertea, Mollusca and Annelida. Interestingly, among our poriferan data, where microbiological symbionts are known empirically to make mitochondrial assembly difficult, this pipeline proved especially useful.\n\nOur pipeline will allow the recovery of mitochondrial data from a variety of previously-sequenced samples, and add an additional angle of enquiry to future RNA-seq efforts, simplifying the process of mitochondrial genome assembly for even the most recalcitrant clades and adding this data to the scientific record for a range of future uses.

genomics↗

bioRxiv2018

Freshwater Adaptation at the Molecular Scale in the Unique Sponges of Lake Baikal

Freshwater sponges (Spongillida) are a unique lineage of demosponges that secondarily colonized lakes and rivers and are now found ubiquitously in these ecosystems. They developed specific adaptations to freshwater systems, including the ability to survive extreme thermal ranges, long-lasting dessication, anoxia, and resistance to a variety of pollutants. While spongillids have colonized all freshwater systems, the family Lubomirskiidae is endemic to Lake Baikal, and plays a range of key roles in this ecosystem. Our work compares the genomic content and microbiome of individuals of three species of the Lubomirskiidae, providing hypotheses for how molecular evolution has allowed them to adapt to their unique environments. We have sequenced deep (>92% of the metazoan Benchmarking Universal Single-Copy Orthologs (BUSCO) set) transcriptomes from three species of Lubomirskiidae and a draft genome resource for Lubomirskia baikalensis. We note Baikal sponges contain unicellular algal and bacterial symbionts, as well as the dinoflagellate Gyrodinium. We investigated molecular evolution, gene duplication and novelty in freshwater sponges compared to marine lineages. Sixty one orthogroups have consilient evidence of positive selection. Transporters (e.g. zinc transporter-2), transcription factors (aristaless-related homeobox) and structural proteins (for example actin-3), alongside other genes, are under strong evolutionary pressure in freshwater, with duplication driving novelty across the Spongillida, but especially in the Lubomirskiidae. This addition to knowledge of freshwater sponge genetics provides a range of tools for understanding the molecular biology and, in the future, the ecology (for example, colonization and migration patterns) of these key species.

zoology↗

bioRxiv2018

Rat bone marrow derived mesenchymal stem cells differentiate to germ cell like cells

Summary StatementThe in vitro differentiation of rMSCs provides an excellent model for studying cell commitment and their potential in stem cell technology. We have demonstrated that rat bone marrow derived MSCs are able to differentiate into germ-line cells in vitro which has an enormous scope in the advancement of fertility research. In future, this technique can be utilized in different domestic animal species for propagation of elite germ plasm.\n\nAbstractGerm cells undergo distinctive male or female pathways to produce spermatozoa or oocyte respectively essential for sexual reproduction. Mesenchymal stem cells (MSCs) have the capacity of trans-differentiation to form the multiple lineages of cells of mesoderm, endoderm, and ectoderm origin. Herein, MSCs were isolated from rat bone marrow and characterized by their morphological features, expression of surface markers by immunophenotyping and molecular biology tools as well as self renewal and differentiation capability. Thereafter, by inducing these cells with retinoic acid we could able to show that bone marrow derived MSCs are able to trans-differentiate into male germ cell-like cells which were further characterized by the expressions of germ cell specific markers. This in vitro study for the generation of germ-like cells suggests that bone marrow MSCs can be a potential source of germ cells that could be a sustainable source of sperm / oocyte production for potential therapeutic applications in future. Moreover, this technique can be applied in different domestic animal species for propagation of elite germ plasm.

cell biology↗

bioRxiv2018

A Hybrid de novo Assembly of the Sea Pansy (Renilla muelleri) Genome

BackgroundOver 3,000 species of octocorals (Cnidaria, Anthozoa) inhabit an expansive range of environments, from shallow tropical seas to the deep-ocean floor. They are important foundation species that create coral \"forests\" which provide unique niches and three-dimensional living space for other organisms. The octocoral genus Renilla inhabits sandy, continental shelves in the subtropical and tropical Atlantic and eastern Pacific Oceans. Renilla is especially interesting because it produces secondary metabolites for defense, exhibits bioluminescence, and produces a luciferase that is widely used in dual-reporter assays in molecular biology. Although several cnidarian genomes are currently available, the majority are from hexacorals. Here, we present a de novo assembly of the R. muelleri genome, making this the first complete draft genome from an octocoral.\n\nFindingsWe generated a hybrid de novo assembly using the Maryland Super-Read Celera Assembler v.3.2.6 (MaSuRCA). The final assembly included 4,825 scaffolds and a haploid genome size of 172 Mb. A BUSCO assessment found 88% of metazoan orthologs present in the genome. An Augustus ab initio gene prediction found 23,660 genes, of which 66% (15,635) had detectable similarity to annotated genes from the starlet sea anemone, Nematostella vectensis, or to the Uniprot database. Although the R. muelleri genome is smaller (172 Mb) than other publicly available, hexacoral genomes (256-448 Mb), the R. muelleri genome is similar to the hexacoral genomes in terms of the number of complete metazoan BUSCOs and predicted gene models.\n\nConclusionsThe R. muelleri hybrid genome provides a novel resource for researchers to investigate the evolution of genes and gene families within Octocorallia and more widely across Anthozoa. It will be a key resource for future comparative genomics with other corals and for understanding the genomic basis of coral diversity.

genomics↗

bioRxiv2018

Kinetic Model of Translational Autoregulation

We investigate dynamics of a kinetic model of inhibitory autoregulation as exemplified when a protein inhibits its own production by interfering with its messenger RNA, known in molecular biology as translational autoregulation. We first show how linear models without feedback set the stage with a nonequilibrium steady state that constitutes the target of the regulation. However, regulation in the simple linear model is far from optimal. The negative feedback mechanism whereby the protein \"jams\" the mRNA greatly enhances the effectiveness of the control, with response to perturbation that is targeted, rapid, and metabolically efficient. Understanding the full dynamics of the system phase space is essential to understanding the autoregulation process.

systems biology↗

bioRxiv2018

Integrating Vectors for Genetic Studies in the Rare Actinomycete Amycolatopsis marina

Few natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms. In this study, we sought to identify integrating vectors, derived using phage int/attP loci, that would efficiently integrate site-specifically in the rare Actinomycete, Amycolatopsis marina DSM45569. Analysis of the genome of A. marina DSM45569 indicated the presence of attB-like sequences for TG1 and R4 integrases. The TG1 and R4 attBs were active in in vitro recombination assays with their cognate purified integrases and attP loci. Integrating vectors containing either the TG1 or R4 int/attP loci yielded exconjugants in conjugation assays from E. coli to A. marina DSM45569. Site-specific recombination of the plasmids into the host TG1 or R4 attB sites was confirmed by sequencing. The presence of homologous TG1 and R4 attB sites in other species of this genus indicates that vectors based on TG1 and R4 integrases could be widely applicable.\n\nImportanceRare Actinomycetes have the same potential of natural product discovery as Streptomyces, but the potential has not been fully explored due to the lack of efficient molecular biology tools. In this study, we identified two serine integrases, TG1 and R4, which could be used in the rare Actinomycetes species, Amycolatopsis marina, as tools for genome integration. The high level of conservation between the attB sites for TG1 and R4 in a number of Amycolatopsis species suggested that plasmids with the integration systems from these phages should be widely useful in this genus.

microbiology↗

bioRxiv2018

Draft genome of the liver fluke Fasciola gigantica

Fascioliasis is a neglected food-borne disease caused by liver flukes (genus Fasciola) and affects more than 200 million people worldwide. Despite technological advances, little is known about the molecular biology and biochemistry of the fluke. We present the draft genome of Fasciola gigantica for the first time. The assembled draft genome has a size of ~1.04 Gb with an N50 of 129 kb. A total of 20,858 genes were predicted. The de novo repeats identified in the draft genome were 46.85%. In pathway analysis, all the genes of glycolysis, Krebs cycle and fatty acid metabolism were found to be present, but the key genes for fatty acid production in fatty acid biosynthesis were missing. This indicates that the fatty acid required for the survival of the fluke may be acquired from the host bile. The genomic information will provide a comprehensive resource to facilitate the development of novel interventions for fascioliasis control.

bioinformatics↗

bioRxiv2026

A Sequential Assembly Mechanism for Stable Cdc13 Dimerization on Telomeric DNA

The telomere-binding protein Cdc13 specifically binds to single-stranded telomeric DNA, playing a critical role in telomere protection and length regulation. While extensive biochemical, molecular biological, and genetic studies have shown that Cdc13 can form dimers or oligomers in solution and bind telomeric DNA with high specificity, the dynamic mechanism of its loading onto telomeres is less well characterized. Using two single-molecule methods, single-molecule fluorescence resonance energy transfer (smFRET) and colocalization single-molecule spectroscopy (CoSMoS), we demonstrate that Cdc13 initially loads onto telomeres as a monomer. This is followed by the recruitment of a second monomer, forming a stable Cdc13 dimer on a 12-nucleotide telomeric DNA segment. Although genetic studies suggest that monomeric Cdc13 binding alone is insufficient to maintain telomere length, it underscores the Cdc13 monomers regulatory importance in coordinating telomere synthesis and protection. This monomer-to-dimer transition provides a mechanistic basis for understanding the multi-tasked roles of Cdc13 in telomere replication and protection.

biophysics↗