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Results for “cell biology”

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 181 records · Page 10Linked to original sources

bioRxiv2023

Simulated microgravity during clino-rotation is disturbed by spurious fluid motion

To study processes related to weightlessness in ground-based cell biological research, a theoretically assumed microgravity environment is typically simulated using a clinostat - a small laboratory device that rotates cell culture vessels with the aim of averaging out the vector of gravitational forces. Here, we report that the rotational movement during fast clinorotation induces complex fluid motions in the cell culture vessel, which can trigger unintended cellular responses. Specifically, we demonstrate that suppression of myotube formation by 2D-clinorotation at 60 rpm is not an effect of the assumed microgravity but instead is a consequence of fluid motion. Therefore, cell biological results from fast clinorotation cannot be attributed to microgravity unless alternative explanations have been rigorously tested and ruled out. We consider two control experiments mandatory, i) a static, non-rotating control, and ii) a control for fluid motion. These control experiments are also highly recommended for other rotation speed settings and experimental conditions. Finally, we discuss strategies to minimize fluid motion in clinorotation experiments.

cell biology↗

bioRxiv2024

Impact of PIK3CA gain and PTEN loss on mantle cell lymphoma biology and sensitivity to targeted therapies

Besides many other mutations in known cancer driver genes, mantle cell lymphoma (MCL) is characterized by recurrent genetic alterations of important regulators of the phosphoinositol-3-kinase (PI3K) cascade including PIK3CA gains and PTEN losses. To evaluate the biological and functional consequences of these aberrations in MCL, we have introduced transgenic expression of PIK3CA (PIK3CA UP) and performed knockout of PTEN gene (PTEN KO) in 5 MCL cell lines. The modified cell lines were tested for associated phenotypes including dependence on upstream B-cell receptor (BCR) signaling (by an additional BCR knockout). PIK3CA overexpression decreased the dependence of the tested MCL on prosurvival signaling from BCR, decreased levels of oxidative phosphorylation, and increased resistance to 2-deoxy-glucose, a glycolysis inhibitor. Unchanged AKT phosphorylation status and unchanged sensitivity to a battery of PI3K inhibitors suggested that PIK3CA gain might impact MCL cells in AKT independent manner. PTEN KO was associated with a more distinct phenotype: AKT hyperphosphorylation and overactivation, increased resistance to multiple inhibitors (most of the tested PI3K inhibitors, BTK inhibitor ibrutinib, and BCL2 inhibitor venetoclax), increased glycolytic rates with resistance to 2-deoxy-glucose, and significantly decreased dependence on prosurvival BCR signaling. Our results suggest that the frequent aberrations of the PI3K pathway may rewire associated signaling with lower dependence on BCR signaling, better metabolic and hypoxic adaptation, and targeted therapy resistance in MCL. Key point 1PIK3CA gain and PTEN loss decrease the dependence of MCL cells on B-Cell Receptor Signaling and anti-apoptotic BCL2. Key point 2PIK3CA gain and PTEN loss lead to complex metabolic rewiring and increased survival of MCL cells under hypoxia.

cancer biology↗

bioRxiv2020

OpenAWSEM with Open3SPN2: a fast, flexible, and accessible framework for large-scale coarse-grained biomolecular simulations

We present OpenAWSEM and Open3SPN2, new cross-compatible implementations of coarse-grained models for protein (AWSEM) and DNA (3SPN2) molecular dynamics simulations within the OpenMM framework. These new implementations retain the chemical accuracy and intrinsic efficiency of the original models while adding GPU acceleration and the ease of forcefield modification provided by OpenMMs Custom Forces software framework. By utilizing GPUs, we achieve more than a 100-fold speedup in protein and protein-DNA simulations over the existing LAMMPS-based implementations running on a CPU. We showcase the benefits of OpenMMs Custom Forces framework by devising and implementing two new potentials that allow us to address important aspects of protein folding and structure prediction and by testing the ability of the combined OpenAWSEM and Open3SPN2 to model protein-DNA binding. The first potential is used to describe the changes in effective interactions that occur as a protein becomes partially buried in a membrane. We also introduced an interaction to describe proteins with multiple disulfide bonds. Using simple pairwise disulfide bonding terms results in unphysical clustering of cysteine residues, posing a problem when simulating the folding of proteins with many cysteines. We now can computationally reproduce Anfinsens early Nobel prize winning experiments [1] by using OpenMMs Custom Forces framework to introduce a multi-body disulfide bonding term that prevents unphysical clustering. Our protein-DNA simulations show that the binding landscape is funneled towards structures that are quite similar to those found using experiments. In summary, this paper provides a simulation tool for the molecular biophysics community that is both easy to use and sufficiently efficient to simulate large proteins and large protein-DNA systems that are central to many cellular processes. These codes should facilitate the interplay between molecular simulations and cellular studies, which have been hampered by the large mismatch between the time and length scales accessible to molecular simulations and those relevant to cell biology. Author summaryThe cells most important pieces of machinery are large complexes of proteins often along with nucleic acids. From the ribosome, to CRISPR-Cas9, to transcription factors and DNA-wrangling proteins like the SMC-Kleisins, these complexes allow organisms to replicate and enable cells to respond to environmental cues. Computer simulation is a key technology that can be used to connect physical theories with biological reality. Unfortunately, the time and length scales accessible to molecular simulation have not kept pace with our ambition to study the cells molecular factories. Many simulation codes also unfortunately remain effectively locked away from the user community who need to modify them as more of the underlying physics is learned. In this paper, we present OpenAWSEM and Open3SPN2, two new easy-to-use and easy to modify implementations of efficient and accurate coarse-grained protein and DNA simulation forcefields that can now be run hundreds of times faster than before, thereby making studies of large biomolecular machines more facile.

molecular biology↗

bioRxiv2023

3D environment modulates persistent lamin distribution and the biomechanical signature of the nucleus.

The interplay between cells and their surrounding microenvironment drives multiple cellular functions, including migration, proliferation, and cell fate transitions. The nucleus is a mechanosensitive organelle that adapts external mechanical and biochemical signals provided by the environment into nuclear changes with functional consequences for cell biology. However, the morphological and functional changes of the nucleus induced by 3D extracellular signals remain unclear. Here, we demonstrated that cells derived from 3D conditions conserve changes from cell confinement and show an aberrant nuclear morphology and localization of lamin B1, even in the absence of cellular confinement. We found that actin polymerization and protein kinase C (PKC) activity mediate the abnormal distribution of lamin B1 in 3D conditions-derived cells. These cells present altered chromatin compaction, gene transcription and cellular functions such as cell viability and migration. By combining biomechanical techniques and single-nucleus analysis, we have determined that the nucleus from 3D conditions-derived cells shows a different mechanical behavior and biophysical signature than the nucleus from control cells. Together, our work substantiates novel insights into how the extracellular environment alters the cell biology by promoting permanent changes in the chromatin, morphology, lamin B1 distribution, and the mechanical response of the nucleus.

cell biology↗

bioRxiv2020

Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells

The current model of the mammalian circadian oscillator is predominantly based on data from genetics and biochemistry experiments, while the cell biology of circadian clocks is still in its infancy. Here, we describe a new strategy for the efficient generation of knock-in reporter cell lines using CRISPR technology that is particularly useful for lowly or transiently expressed genes, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins, which allowed to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed ([~]5 hours), and CRY1 levels are much higher (>6 times) compared to PER2 questioning the current model of the circadian oscillator. Our knock-in strategy will allow the generation of additional single, double or triple knock-in cells for circadian clock proteins, which should greatly advance our understanding about the cell biology of circadian clocks.

cell biology↗

bioRxiv2023

Unusual actin-binding mechanism and the role of profilin in actin dynamics of trypanosomatid parasites

Diseases caused by Leishmania, and Trypanosoma parasites, such as leishmaniasis and African sleeping sickness, are a major health problem in tropical countries. Due to their complex life cycle involving both vertebrate and insect hosts, and > 1 billion years of evolutionarily distance, the cell biology of these trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is highly divergent when compared to the other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique -helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family, and is strikingly similar to the structure of WH2 domain, a small actin-binding motif found in many other cytoskeletal regulators. We demonstrate that the WH2-like motif contributes to actin monomer-binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Surprisingly, unlike other profilins characterized so far, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, as well as the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers the molecular principles by which actin dynamics are regulated by profilin in trypanosomatids. Moreover, the unusual actin-binding mechanism of profilin identified here could be applied for designing inhibitors against pathogenic trypanosomatid parasites. AUTHOR SUMMARYLeishmania and Trypanosoma parasites are a major health problem as they cause various diseases in humans and other vertebrates. Currently, there are no specific drugs to treat the diseases caused by these trypanosomatid parasites. Similar to all other eukaryotes, trypanosomatid parasites have an actin cytoskeleton, which is essential for the viability of parasites. Interestingly, both actin and actin-regulatory machineries of these parasites are highly divergent from the ones of animals, making them possible drug targets to treat diseases caused by these parasites. To uncover how the actin cytoskeleton of trypanosomatid parasites is regulated, we focused on a central actin-binding protein, profilin. Importantly, our experiments revealed that trypanosomatid profilins interact with actin through a different structural mechanism as compared to animal profilins, and have specific effects on the assembly of actin filaments. Our genetic studies demonstrate that these specific features of trypanosomatid profilin are also critical for the proper function on this protein in parasites. Our study provides new insight into the cell biology of trypanosomatid parasites. We also envision that the structural and functional differences between trypanosomatid and human profilins can be applied for developing compounds for selectively neutralizing Leishmania and Trypanosoma parasites.

biochemistry↗

bioRxiv2024

Laser Scanning Confocal printing, a new simple method for micro-device fabrication (LSCprint method).

Micro-devices and lab-on-a-chip technologies have revolutionized cell biology research, enabling a plethora of applications from single-cell sorting to organ-on-a-chip assays. However, their construction remains laborious, requiring specialized equipment and trained personnel, thereby restricting their accessibility to specialized laboratories. The conventional protocol for micro-device printing involves intricate steps, including master cast/mold production and device fabrication, leading to high costs and time consumption. Here, we present a novel, simplified method utilizing a laser scanning confocal microscope (LSCM) and commercially available photosensitive resins. By using the UV or violet excitation laser lines of an LSCM, we eliminate the need for external suppliers and complex equipment, enabling any conventional cell biology laboratory to fabricate micro-devices swiftly and inexpensively. Our method not only enhances the capabilities of standard confocal microscopes but also democratises microfluidic device fabrication, making it accessible to non-specialized laboratories. With minimal reagent consumption and high scalability, our approach offers a cost-effective solution for rapid prototyping and production of micro-devices, circumventing previous barriers to widespread adoption. Moreover, our method allows direct printing of micro-devices onto substrates, eliminating the need for molds and intermediate steps, thus facilitating greater design flexibility and accessibility for non-specialized laboratories.

bioengineering↗

bioRxiv2022

Configurable Compartmentation Enables In vitro Reconstitution of Sustained Synthetic Biology Systems

The compartmentalized and communicative nature of biological cells contributes to the complexity and endurance of living organisms. Current in vitro compartmentalization systems such as droplet emulsions reproduce the compartmentalization property of cells yet fail to recapture the configurability of cellular communication with the environment. To mimic biological cells a step further and expand the capabilities of in vitro compartmentalization, we present here a general strategy that inherits the passive transport phenomenon of biology. The strategy incorporates layered, micrometer-sized, hydrogel-based compartments featuring configurability in composition, functionality, and selective permeability of biomolecules. We demonstrated the unique advantage of our strategy in two scenarios of synthetic biology. First, a compartmentalized cell-free protein synthesis system was reconstituted that could support multiple rounds of reactions. Second, we constructed living bacteria-based biosensors in the hydrogel compartments, which could achieve long-lasting functioning with markedly enhanced fitness in complex environments. Looking forward, our strategy should be widely applicable for constructing complex, robust, and sustained in vitro synthetic molecular and cellular systems, paving the way for their practical applications.

synthetic biology↗

bioRxiv2016

Inferring Fitness Landscapes and Selection on Phenotypic States from Single-Cell Genealogical Data

Recent advances in single-cell time-lapse microscopy have revealed non-genetic heterogeneity and temporal fluctuations of cellular phenotypes. While different phenotypic traits such as abundance of growth-related proteins in single cells may have differential effects on the reproductive success of cells, rigorous experimental quantification of this process has remained elusive due to the complexity of single cell physiology within the context of a proliferating population. We introduce and apply a practical empirical method to quantify the fitness landscapes of arbitrary phenotypic traits, using genealogical data in the form of population lineage trees which can include phenotypic data of various kinds. Our inference methodology for fitness landscapes determines how reproductivity is correlated to cellular phenotypes, and provides a natural generalization of bulk growth rate measures for single-cell histories. Using this technique, we quantify the strength of selection acting on different cellular phenotypic traits within populations, which allows us to determine whether a change in population growth is caused by individual cells response, selection within a population, or by a mixture of these two processes. By applying these methods to single-cell time-lapse data of growing bacterial populations that express a resistance-conferring protein under antibiotic stress, we show how the distributions, fitness landscapes, and selection strength of single-cell phenotypes are affected by the drug. Our work provides a unified and practical framework for quantitative measurements of fitness landscapes and selection strength for any statistical quantities definable on lineages, and thus elucidates the adaptive significance of phenotypic states in time series data. The method is applicable in diverse fields, from single cell biology to stem cell differentiation and viral evolution.

Systems Biology↗

bioRxiv2020

Modular cell-free expression plasmids to accelerate biological design in cells

Industrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms--organisms that are easy to use like Escherichia coli--often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have the desired metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has emerged as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institutes DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression of enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for C. autoethanogenum expression with efficiencies ranging from 68-90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.

synthetic biology↗

bioRxiv2025

Subcellular proteomics of Paramecium tetraurelia reveals mosaic localization of glycolysis and gluconeogenesis

Ciliates are unicellular heterotrophic eukaryotes, most of which consume other microbes as prey. They exhibit nuclear dimorphism which requires reconstruction of a transcriptionally active macronucleus from the germline micronucleus after sexual recombination. This complex genomic structure has prevented the development of highly tractable genetic models leaving much of ciliate cell biology unexplored. To complicate matters further, some ciliates tend to accumulate many gene duplicates either singly or via whole genome duplications. Thus, extensive insight into the cell biology of ciliates requires the use of high-throughput tools like subcellular proteomics. Here, we use a subcellular proteomics workflow to classify over 9,000 proteins to 16 subcellular compartments in Paramecium tetraurelia. From these data, we identify a small but robust subcellular cluster containing canonical mitochondrial outer membrane proteins as well as some ER proteins, putatively at membrane contact sites. Within this cluster, we identified the important glycolytic enzyme phosphofructokinase, which contained a transmembrane domain. Further investigation revealed that several latter-acting glycolytic enzymes were localized to the mitochondrial cluster. The location of phosphoenol pyruvate carboxykinase and pyruvate carboxylase in the mitochondria but pyruvate kinase in the cytosol suggests that ciliates prefer gluconeogenesis over glycolysis. The localization of these enzymes was confirmed in a preliminary subcellular proteome of Tetrahymena thermophila. In sum, our findings suggest that mitochondrial localization of glycolytic/gluconeogenic enzymes is widespread across ciliates and that several may preferentially undergo gluconeogenesis over glycolysis using amino acids as a primary carbon source in both catabolic and anabolic metabolism. HighlightsSubcellular proteomics of Paramecium tetraurelia revealed that glycolytic and gluconeogenic enzymes are mosaically distributed between the cytosol, mitochondrial matrix, and mitochondrial outer membrane. A distinct mitochondrial outer membrane compartment was identified with 105 classified proteins, including core mitochondrial biogenesis proteins and a putative Tom70-like protein. Phosphofructokinase, a key glycolytic enzyme, was found embedded in the mitochondrial outer membrane. Localization of biochemical pathways suggest ciliates favor gluconeogenesis over glycolysis. In total, over 9000 Paramecium proteins were identified using subcellular proteomics and classified into 16 different cellular compartments.

cell biology↗

bioRxiv2022

Catch bond models explain how force amplifies TCR signaling and antigen discrimination

Central to T cell biology, the T cell receptor (TCR) integrates forces in its triggering process upon interaction with peptide-major histocompatibility complex (pMHC)1-3. Phenotypically, forces elicit TCR catch-slip bonds with strong pMHCs but slip-only bonds with weak pMHCs4-10. While such correlation is commonly observed, the quantitative bond pattern and degree of "catchiness" vary. We developed two models based on the structure, elastic properties, and force-induced conformational changes of the TCR-pMHC-I/II complexes to derive from their bond characteristics more intrinsic parameters that underlie structural mechanisms, predict T cell signaling, and discriminate antigens. Applying the models to 55 datasets of 12 {beta}TCRs and their mutants interacting with corresponding pMHCs without coreceptor engagement demonstrated the ability for structural and physical parameters to quantitatively integrate and classify a broad range of bond behaviors and biological activities. Comparing to the generic two-state model for catch-slip bond that also fits the data, our models can distinguish class I from class II MHC systems and their best-fit parameters correlate with the TCR/pMHC potency to trigger T cell activation, which the generic model cannot. The models were tested by mutagenesis using structural analysis, bond profile measurement, and functional assay of a MHC and a TCR mutated to alter conformation changes. The extensive comparisons between theory and experiment provided strong validation of the models and testable hypothesis regarding specific conformational changes that control bond profiles, thereby suggesting structural mechanisms for the inner workings of the TCR mechanosensing machinery and plausible explanation of why and how force may amplify TCR signaling and antigen discrimination.

biophysics↗

bioRxiv2023

Particle-based simulations reveal two positive feedback loops allow relocation and stabilization of the polarity site during yeast mating

Many cells adjust the direction of polarized growth or migration in response to external directional cues. The yeast Saccharomyces cerevisiae orient their cell fronts (also called polarity sites) up pheromone gradients in the course of mating. However, the initial polarity site is often not oriented towards the eventual mating partner, and cells relocate the polarity site in an indecisive manner before developing a stable orientation. During this reorientation phase, the polarity site displays erratic assembly-disassembly behavior and moves around the cell cortex. The mechanisms underlying this dynamic behavior remain poorly understood. Particle-based simulations of the core polarity circuit revealed that molecular-level fluctuations are insufficient to overcome the strong positive feedback required for polarization and generate relocating polarity sites. Surprisingly, inclusion of a second pathway that promotes polarity site orientation generated a mobile polarity site with properties similar to those observed experimentally. This pathway forms a second positive feedback loop involving the recruitment of receptors to the cell membrane and couples polarity establishment to gradient sensing. This second positive feedback loop also allows cells to stabilize their polarity site once the site is aligned with the pheromone gradient. Author summaryCells perform many complex tasks, including directed growth, migration, division and differentiation. To accomplish these tasks, the relevant molecular machinery is localized to specific cellular regions. The asymmetric distribution of cellular components is referred to as cell polarity. Polarity is established by localized activation of the protein Cdc42. Establishing mechanisms that regulate the spatiotemporal activity of Cdc42 is a fundamental area of cell biology. Mating yeast cells dynamically relocate a region of high Cdc42 activity, referred to as the polarity site, and grow toward each other after proper alignment of the sites. We investigated mechanisms that generate dynamic polarity sites by performing particle-based simulations of the biochemical reactions that regulate Cdc42 activity. The reactions contain two positive feedback loops that reinforce Cdc42 activity. The first involves autocatalytic activation of Cdc42 through recruitment of an activator. While necessary for polarity establishment, this feedback loop on its own created a stable polarity site that did not relocate. Incorporation of the second feedback loop, which couples the polarity machinery to extracellular mating signals, generated mobile polarity sites. This feedback loop also provides a mechanism for developing stable alignment of polarity sites. Our findings provide insight into how cells regulate polarity dynamics to accomplish complex tasks.

systems biology↗

bioRxiv2025

Context-dependent spatial multicellular network motifs for single-cell spatial biology

The clinical state of diseased tissue is caused by complex intercellular processes that go beyond pairwise cell-cell interactions and are difficult to infer due to the combinatorial explosion of such high-dimensionality. We present context-dependent identification of spatial motifs (CISM), a two-step method to identify local cell structures associated with a disease state in single cell spatial data. First, for each tissue, CISM enumerates structures of enriched reoccurring multicellular patterns that define modular motifs in the multicellular network. Second, discriminative motifs are selected according to the context - their presence in patients at different clinical disease states. By applying CISM, we show that modular structures composed of as little as 3-5 cells and their relative spatial arrangement can encode differences in clinical disease states in cohorts of triple-negative breast cancer (TNBC) and melanoma patients. Machine learning validation indicated that discriminative motifs outperform state-of-the-art methods for disease state prediction while enabling interpretation of which interactions in what spatial context are associated with these predictions. CISM-derived discriminative motifs may define an intermediate spatial scale of abstraction and modularity in multicellular organization and function with broad applicability in the domain of spatial single cell omics and beyond.

systems biology↗

bioRxiv2021

Multiplexed activation in mammalian cells using dFnCas12a-VPR

The adoption of CRISPR systems for the generation of synthetic transcription factors has greatly simplified the process for upregulating endogenous gene expression, with a plethora of applications in cell biology, bioproduction and cell reprogramming. In particular the recently discovered Cas12a systems offer extended potential, as Cas12a is capable of processing its own crRNA array to provide multiple individual crRNAs for subsequent targeting from a single transcript. Here we show the application of dFnCas12a-VPR in mammalian cells, with FnCas12a possessing a shorter PAM sequence than As or Lb variants, enabling denser targeting of genomic loci. We observe that synergistic activation and multiplexing can be achieved using crRNA arrays but also show that crRNAs expressed towards the 5 of 6-crRNA arrays show evidence of enhanced activity. This not only represents a more flexible tool for transcriptional modulation but further expands our understanding of the design capabilities and limitations when considering longer crRNA arrays for multiplexed targeting.

synthetic biology↗

bioRxiv2014

Conservation and structural analysis of the Xenopus laevis phospho-proteome

The African clawed frog Xenopus laevis is an important model organism for studies in developmental and cell biology, including cell-signaling. However, our knowledge of X. laevis protein post-translational modifications remains scarce. Here, we used a mass spectrometry-based approach to survey the phosphoproteome of this species, compiling a list of 3225 phosphosites. We used this resource to study the conservation between the phosphoproteomes of X. laevis and 13 other species. We found that the degree of conservation of phosphorylation across species is predictive of sites with known molecular function, kinase interactions and functionally relevant phospho-regulatory interactions. In addition, using comparative protein structure models, we find that phosphosites within structured domains tend to be located at positions with high conformational flexibility. A fraction of sites appear to occur in inaccessible positions and have the potential to regulate protein conformation.

Evolutionary Biology↗

bioRxiv2014

A curated C. difficile strain 630 metabolic network: prediction of essential targets and inhibitors

Clostridium difficile is the leading cause of hospital-borne infections occurring when the natural intestinal flora is depleted following antibiotic treatment. We present iMLTC804cdf, an extensively curated reconstructed metabolic network for the C. difficile pathogenic strain 630. iMLTC804cdf contains 804 genes, 705 metabolites and 766 metabolic, 145 exchange and 118 transport reactions. iMLTC804cdf is the most complete and accurate metabolic reconstruction of a gram-positive anaerobic bacteria to date. We validate the model with simulated growth assays in different media and carbon sources and use it to predict essential genes. We obtain 88.8% accuracy in the prediction of gene essentiality when compared to experimental data for B. subtilis homologs. We predict the existence of 83 essential genes and 68 essential gene pairs, a number of which are unique to C. difficile and have non-existing or predicted non-essential human homologs. For 19 of these potential therapeutic targets, we find 72 inhibitors of homologous proteins that could serve as starting points in the development of new antibiotics, including approved drugs with the potential for drug repositioning.\n\nSystems & Synthetic Biology Subject Category: Genome Scale & Integrative Biology\n\nMolecular & Cell Biology Subject Category: Pharmacology & Drug Discovery

Systems Biology↗

bioRxiv2014

Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI’s RNA-seq archive

Mycoplasmas are notorious contaminants of cell culture and can have profound effects on host cell biology by depriving cells of nutrients and inducing global changes in gene expression. Because they are small, they can escape filtration in culture media. Because they lack cell walls, they are resistant to commonly used antibiotics. Over the last two decades, sentinel testing has revealed wide-ranging contamination rates in mammalian culture. To obtain an unbiased assessment from hundreds of labs, we analyzed sequence data from 9395 rodent and primate samples from 884 series (or projects) in the NCBI Sequence Read Archive. We found 11% of these series were contaminated (defined as [≥] 100 reads/million mapping to mycoplasma in one or more samples). Ninety percent of mycoplasma-mapped reads aligned to ribosomal RNA. Interestingly, series using poly(A)-selection, which should bias against mycoplasma detection, had comparable contamination rates as non-poly(A)-selected series. We also examined the relationship between mycoplasma contamination and host gene expression in a single cell RNA-seq dataset and found 61 host genes (P < 0.001) were significantly associated with mycoplasma-mapped read counts. Lastly, to estimate the potential economic cost of this widespread contamination, we queried NIH RePORTER to find grants with the terms \"cell culture\" or \"cell lines\". Funding for these totaled over $3 billion, suggesting hundreds of millions of dollars in research are potentially affected. In all, this study suggests mycoplasma contamination is still prevalent today and poses substantial risk to research quality, with considerable financial consequences.

Genomics↗