Biology subjects

Johnson, A.

Publications and source records attributed to Johnson, A..

At least 91 records · Page 5Linked to original sources

bioRxiv2020

TMTPro Complementary Ion Quantification Increases Plexing and Sensitivity for Accurate Multiplexed Proteomics at the MS2 Level

Multiplexed proteomics is a powerful tool to assay cell states in health and disease, but accurate quantification of relative protein changes is impaired by interference from co-isolated peptides. Interference can be reduced by using MS3-based quantification, but this reduces sensitivity and requires specialized instrumentation. An alternative approach is quantification by complementary ions, which allows accurate and precise multiplexed quantification at the MS2 level and is compatible with the most widely distributed instruments. However, complementary ions of the popular TMT tag form inefficiently and multiplexing is limited to five channels. Here, we evaluate and optimize complementary ion quantification for the recently released TMTPro tag, which increases plexing capacity to eight channels (TMTProC). We find that the beneficial fragmentation properties of TMTPro increase quantification signal five-fold compared to TMT. This increased sensitivity results in ~65% more proteins quantified compared to TMTPro-MS3 and even slightly outperforms TMTPro-MS2. Furthermore, TMTProC quantification is more accurate than TMTPro-MS2 and even superior to TMTPro-MS3. To demonstrate the power of TMTProC, we analyzed a human and yeast interference sample and were able to quantify 13,290 proteins in 24 fractions. Thus, TMTProC advances multiplexed proteomics data quality and widens access to accurate multiplexed proteomics beyond laboratories with MS3-capable instrumentation.

systems biology↗

bioRxiv2020

Modulation of root growth by nutrient-defined fine-tuning of polar auxin transport

Nitrogen is an essential macronutrient and its availability in soil plays a critical role in plant growth, development and impacts agricultural productivity. Plants have evolved different strategies to sense and respond to heterogeneous nitrogen distribution. Modulating root system architecture, including primary root growth and branching, is among the most essential plant adaptions to ensure adequate nitrogen acquisition. However, the immediate molecular pathways coordinating the adjustment of root growth in response to varying nitrogen sources are poorly understood. Here, using a combination of physiological, live in vivo high- and super resolution imaging, we describe a novel adaptation strategy of root growth on available nitrogen source. We show that growth, i.e. tissue-specific cell division and elongation rates are fine-tuned by modulating auxin flux within and between tissues. Changes in auxin redistribution are achieved by nitrogen source dependent post-translational modification of PIN2, a major auxin efflux carrier, at an uncharacterized, evolutionary conserved phosphosite. Further, we generate a computer model based on our results which successfully recapitulate our experimental observations and creates new predictions that could broaden our understanding of root growth mechanisms in the dynamic environment.

plant biology↗

bioRxiv2020

Development of mPing-based Activation Tags for Crop Insertional Mutagenesis

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing-based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

plant biology↗

bioRxiv2020

Bats Possess Unique Variants of the Antiviral Restriction Factor Tetherin

Bats are recognised as important reservoirs of viruses deadly to other mammals, including humans. These infections are typically non-pathogenic in bats raising questions about host response differences that might exist between bats and other mammals. Tetherin is a restriction factor which inhibits the release of a diverse range of viruses from host cells, including retroviruses, coronaviruses, filoviruses, and paramyxoviruses, some of which are deadly to humans and transmitted by bats. Here we characterise the tetherin genes from 27 species of bats, revealing that they have evolved under strong selective pressure, and that fruit bats and vesper bats express unique structural variants of the tetherin protein. Tetherin was widely and variably expressed across fruit bat tissue-types and upregulated in spleen tissue when stimulated with Toll-like receptor agonists. The expression of two computationally predicted splice isoforms of fruit bat tetherin was verified. We identified an additional third unique splice isoform which includes a C-terminal region that is not homologous to known mammalian tetherin variants but was functionally capable of restricting the release of filoviral virus-like particles. We also report that vesper bats possess and express at least five tetherin genes, including structural variants, a greater number than any other mammal reported to date. These findings support the hypothesis of differential antiviral gene evolution in bats relative to other mammals.

evolutionary biology↗

bioRxiv2020

High temporal resolution reveals simultaneous plasma membrane recruitment of the TPLATE complex subunits.

The TPLATE complex (TPC) is a key endocytic adaptor protein complex in plants. TPC contains six evolutionary conserved subunits and two plant specific subunits, AtEH1/Pan1 and AtEH2/Pan1, which are not associated with the hexameric subcomplex in the cytoplasm. To investigate the dynamic assembly of the octameric TPC at the plasma membrane (PM), we performed state-of-the-art dual-color live cell imaging at physiological and a lowered temperature. Our data show that lowering the temperature slows down endocytosis and thereby enhances the temporal resolution of the differential recruitment of endocytic components. Under both normal and lowered temperature conditions, the core TPC subunit TPLATE, and the AtEH/Pan1 proteins, exhibited simultaneous recruitment at the PM. These results, together with our co-localization analysis of different TPC subunits, allow us to conclude that in plant cells, TPC is not recruited to the PM sequentially but as an octameric complex. One sentence summaryLowering the temperature increases spatiotemporal resolution of protein recruitment at the plasma membrane.

cell biology↗

bioRxiv2020

Bud23 promotes the progression of the Small Subunit Processome to the pre-40S ribosome in Saccharomyces cerevisiae

The first metastable assembly intermediate of the eukaryotic ribosomal small subunit (SSU) is the SSU Processome, a large complex of RNA and protein factors that is thought to represent an early checkpoint in the assembly pathway. Transition of the SSU Processome towards continued maturation requires the removal of the U3 snoRNA and biogenesis factors as well as ribosomal RNA processing. While the factors that drive these events are largely known, how they do so is not well understood. The methyltransferase Bud23 has a role during this transition, but its function, beyond the nonessential methylation of 18S rRNA, is not characterized. Here, we have carried out a comprehensive genetic screen to understand Bud23 function. We identified 67 unique extragenic bud23{Delta}-suppressing mutations that mapped to genes encoding the SSU Processome factors DHR1, IMP4, UTP2 (NOP14), BMS1 and the SSU protein RPS28A. These factors form a physical interaction network that links the binding site of Bud23 to the U3 snoRNA and many of the suppressing mutations weaken protein-protein and protein-RNA interactions. Importantly, this network links Bud23 to the GTPase Bms1 and the RNA helicase Dhr1. Bms1 is thought to drive conformational changes to promote rRNA cleavage, and we previously showed that Dhr1 is required for unwinding the U3 snoRNA. Moreover, particles isolated from cells lacking Bud23 accumulated late SSU Processome factors and pre-rRNAs not cleaved at sites A1 and A2. We propose a model in which Bud23 dissociates factors surrounding its binding site to promote SSU Processome progression. Author summaryRibosomes are the molecular machines that synthesize proteins and are composed of a large and a small subunit which carry out the essential functions of polypeptide synthesis and mRNA decoding, respectively. Ribosome production is tightly linked to cellular growth as cells must produce enough ribosomes to meet their protein needs. However, ribosome assembly is a metabolically expensive pathway that must be balanced with other cellular energy needs and regulated accordingly. In eukaryotes, the small subunit (SSU) Processome is a metastable intermediate that ultimately progresses towards a mature SSU through the release of biogenesis factors. The decision to progress the SSU Processome is thought to be an early checkpoint in the SSU assembly pathway, but what drives this checkpoint is unknown. Previous studies suggest that Bud23 plays an uncharacterized role during SSU Processome progression. Here, we used a genetic approach to understand its function and found that Bud23 is connected to a network of factors that stabilize the particle. Interestingly, two of these factors are enzymes that facilitate structural rearrangements needed for progression. We conclude that Bud23 promotes the release of factors surrounding its binding site to drive rearrangements during the progression of the SSU Processome.

molecular biology↗

bioRxiv2020

Global reference mapping and dynamics of human transcription factor footprints

Combinatorial binding of transcription factors to regulatory DNA underpins gene regulation in all organisms. Genetic variation in regulatory regions has been connected with diseases and diverse phenotypic traits1, yet it remains challenging to distinguish variants that impact regulatory function2. Genomic DNase I footprinting enables quantitative, nucleotide-resolution delineation of sites of transcription factor occupancy within native chromatin3-5. However, to date only a small fraction of such sites have been precisely resolved on the human genome sequence5. To enable comprehensive mapping of transcription factor footprints, we produced high-density DNase I cleavage maps from 243 human cell and tissue types and states and integrated these data to delineate at nucleotide resolution ~4.5 million compact genomic elements encoding transcription factor occupancy. We map the fine-scale structure of ~1.6 million DHS and show that the overwhelming majority is populated by well-spaced sites of single transcription factor:DNA interaction. Cell context-dependent cis-regulation is chiefly executed by wholesale actuation of accessibility at regulatory DNA versus by differential transcription factor occupancy within accessible elements. We show further that the well-described enrichment of disease- and phenotypic trait-associated genetic variants in regulatory regions1,6 is almost entirely attributable to variants localizing within footprints, and that functional variants impacting transcription factor occupancy are nearly evenly partitioned between loss- and gain-of-function alleles. Unexpectedly, we find that the global density of human genetic variation is markedly increased within transcription factor footprints, revealing an unappreciated driver of cis-regulatory evolution. Our results provide a new framework for both global and nucleotide-precision analyses of gene regulatory mechanisms and functional genetic variation.

genomics↗

bioRxiv2019

Emergence of a novel Salmonella enterica serotype Reading clone is linked to its expansion in commercial turkey production, resulting in unanticipated human illness in North America

Concurrent separate human outbreaks of Salmonella enterica serotype Reading occurred in 2017-2019 in the United States and Canada, which were both linked to the consumption of raw turkey products. In this study, a comprehensive genomic investigation was conducted to reconstruct the evolutionary history of S. Reading from turkeys, and to determine the genomic context of outbreaks involving this rarely isolated Salmonella serotype. A total of 988 isolates of U.S. origin were examined using whole genome-based approaches, including current and historical isolates from humans, meat, and live food animals. Broadly, isolates clustered into three major clades, with one apparently highly adapted turkey clade. Within the turkey clade isolates clustered into three subclades, including an "emergent" clade that only contained isolates dated 2016 or later, including many of the isolates from these outbreaks. Genomic differences were identified between emergent and other turkey subclades suggesting that the apparent success of currently circulating subclades clade is, in part, attributable to plasmid acquisitions conferring antimicrobial resistance, gain of phage-like sequences with cargo virulence factors, and mutations in systems that may be involved in beta-glucuronidase activity and resistance towards colicins. U.S. and Canadian outbreak isolates were found interspersed throughout the emergent subclade and the other circulating subclade. The emergence of a novel S. Reading turkey subclade, coinciding temporally with expansion in commercial turkey production and with U.S. and Canadian human outbreaks, indicates that emergent strains with higher potential for niche success were likely vertically transferred and rapidly disseminated from a common source. ImportanceIncreasingly, outbreak investigations involving foodborne pathogens are confounded by the inter-connectedness of food animal production and distribution, necessitating high-resolution genomic investigations to determine their basis. Fortunately, surveillance and whole genome sequencing, combined with the public availability of these data, enable comprehensive queries to determine underlying causes of such outbreaks. Utilizing this pipeline, it was determined that a novel clone of Salmonella Reading has emerged that coincides with increased abundance in raw turkey products and two outbreaks of human illness in North America. The rapid dissemination of this highly adapted and conserved clone indicates that it was likely obtained from a common source and rapidly disseminated across turkey production. Key genomic changes may have contributed to its apparent continued success in the barn environment, and ability to cause illness in humans.

microbiology↗

bioRxiv2019

Reproducible and Transparent Research Practices in Published Neurology Research

BackgroundThe objective of this study was to evaluate the nature and extent of reproducible and transparent research practices in neurology research.\n\nMethodsThe NLM catalog was used to identify MEDLINE-indexed neurology journals. A PubMed search of these journals was conducted to retrieve publications over a 5-year period from 2014 to 2018. A random sample of publications was extracted. Two authors conducted data extraction in a blinded, duplicate fashion using a pilot-tested Google form. This form prompted data extractors to determine whether publications provided access to items such as study materials, raw data, analysis scripts, and protocols. In addition, we determined if the publication was included in a replication study or systematic review, was preregistered, had a conflict of interest declaration, specified funding sources, and was open access.\n\nResultsOur search identified 223,932 publications meeting the inclusion criteria, from which 300 were randomly sampled. Only 290 articles were accessible, yielding 202 publications with empirical data for analysis. Our results indicate that 8.99% provided access to materials, 9.41% provided access to raw data, 0.50% provided access to the analysis scripts, 0.99% linked the protocol, and 3.47% were preregistered. A third of sampled publications lacked funding or conflict of interest statements. No publications from our sample were included in replication studies, but a fifth were cited in a systematic review or meta-analysis.\n\nConclusionsCurrent research in the field of neurology does not consistently provide information needed for reproducibility. The implications of poor research reporting can both affect patient care and increase research waste. Collaborative intervention by authors, peer reviewers, journals, and funding sources is needed to mitigate this problem.

scientific communication and education↗

bioRxiv2019

Evolutionary unique mechanistic framework of clathrin-mediated endocytosis in plants

In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME follows the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the surface but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes.

plant biology↗

bioRxiv2019

Cell-surface receptors enable perception of extracellular cytokinins

Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience 1,2. Although cytokinin receptors are substantially localised to the endoplasmic reticulum 3-5, the cellular sites of cytokinin perception continue to be debated 1,6,7. Several cytokinin types display bioactivity 8,9 and recently a cell-specific cytokinin gradient was reported in roots 10. Yet, the importance of spatially heterogeneous cytokinin distribution and the specific cytokinin(s) that account for the different responses remain unclear. Here we show that cytokinin perception by plasma membrane receptors is an effective path for cytokinin response in root cells. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP;11) is closely matched to intracellular cytokinin content, yet a proportion of bioactive cytokinins are detected in the extracellular fluid. Using cytokinins covalently linked to beads that could not pass the plasma membrane, we demonstrate that strong TCSn activation still occurs and that this response is greatly diminished in cytokinin receptor mutants. Although intracellular receptors play significant roles, we argue for a revision of concepts of cytokinin perception to include the spatial dimensions. In particular, selective ligand-receptor affinities, cellular localisation and tissue distribution of bioactive cytokinins, their receptors, transporters and inactivation enzymes appear all to be components of the signalling regulatory mechanisms.

plant biology↗

bioRxiv2019

FGF signals direct myotube guidance by regulating Rho/Rac activity

Nascent myotubes undergo a dramatic morphological transformation during myogenesis in which the myotubes elongate over several cell diameters and choose the correct muscle attachment sites. Although this process of myotube guidance is essential to pattern the musculoskeletal system, the mechanisms that control myotube guidance remain poorly understood. Using transcriptomics, we found that components of the Fibroblast Growth Factor (FGF) signaling pathway were enriched in nascent myotubes in Drosophila embryos. Null mutations in the FGF receptor heartless (htl), or its ligands, caused significant myotube guidance defects. Mechanistically, paracrine FGF signals to Htl in the mesoderm regulate the activity of Rho/Rac GTPases in nascent myotubes to effect changes in the actin cytoskeleton. FGF signals are thus essential regulators of myotube guidance that act through cytoskeletal regulatory proteins to pattern the musculoskeletal system.

developmental biology↗

bioRxiv2019

Movement patterns of urban free-roaming dogs: implications for rabies control in Arequipa, Peru

In 2015, a case of canine rabies in Arequipa, Peru indicated the re-emergence of rabies virus in the city. Despite mass dog vaccination campaigns across the city and reactive ring vaccination and other control activities around positive cases (e.g. elimination of unowned dogs), the outbreak has spread. Here we explore how the urban landscape of Arequipa affects the movement patterns of free-roaming dogs, the main reservoirs of the rabies virus in the area. We tracked 23 free-roaming dogs using Global Positioning System (GPS) collars. We analyzed the spatio-temporal GPS data using the time- local convex hull method. Dog movement patterns varied across local environments. We found that water channels, an urban feature of Arequipa that are dry most of the year, promote movement. Dogs that used the water channels move further, faster and more directionally than dogs that do not. Our findings suggest that water channels can be used by dogs as highways to transverse the city and have the potential to spread disease far beyond the radius of control practices. Control efforts should focus on a robust vaccination campaign attuned to the geography of the city, and not limited to small-scale rings surrounding cases.

ecology↗

bioRxiv2019

The L1 stalk is required for efficient export of nascent large ribosomal subunits in yeast

The ribosomal protein Rpl1 (uL1 in universal nomenclature) is essential in yeast and constitutes part of the L1 stalk which interacts with E site ligands on the ribosome. Structural studies of nascent pre-60S complexes in yeast have shown that a domain of the Crm1-dependent nuclear export adapter Nmd3, binds in the E site and interacts with Rpl1, inducing closure of the L1 stalk. Based on this observation, we decided to reinvestigate the role of the L1 stalk in nuclear export of pre-60S subunits despite previous work showing that Rpl1-deficient ribosomes are exported from the nucleus and engage in translation. Large cargoes, such as ribosomal subunits, require multiple export factors to facilitate their transport through the nuclear pore complex. Here, we show that pre-60S subunits lacking Rpl1 or truncated for the RNA of the L1 stalk are exported inefficiently. Surprisingly, this is not due to a measurable defect in recruitment of Nmd3 but appears to result from inefficient recruitment of the Mex67-Mtr2 heterodimer.

molecular biology↗

bioRxiv2018

Cryptic Promoter Activation Drives POU5F1 (OCT4) Expression in Renal Cell Carcinoma

Transcriptional dysregulation drives cancer formation but the underlying mechanisms are still poorly understood. As a model system, we used renal cell carcinoma (RCC), the most common malignant kidney tumor which canonically activates the hypoxia-inducible transcription factor (HIF) pathway. We performed genome-wide chromatin accessibility and transcriptome profiling on paired tumor/normal samples and found that numerous transcription factors with a RCC-selective expression pattern also demonstrated evidence of HIF binding in the vicinity of their gene body. Some of these transcription factors influenced the tumors regulatory landscape, notably the stem cell transcription factor POU5F1 (OCT4). Unexpectedly, we discovered a HIF-pathway-responsive cryptic promoter embedded within a human-specific retroviral repeat element that drives POU5F1 expression in RCC via a novel transcript. Elevat POU5F1 expression levels were correlated with advanced tumor stage and poorer overall survival in RCC patients. Thus, integrated transcriptomic and epigenomic analysis of even a small number of primary patient samples revealed remarkably convergent shared regulatory landscapes and a novel mechanism for dysregulated expression of POU5F1 in RCC.

cancer biology↗

bioRxiv2018

How small-molecule inhibitors of dengue-virus infection interfere with viral membrane fusion

Dengue virus (DV) is a compact, icoshedrally symmetric, enveloped particle, covered by 90 dimers of envelope protein (E), which mediates viral attachment and membrane fusion. Fusion requires a dimer-to-trimer transition and membrane engagement of hydrophobic \"fusion loops\". We previously characterized the steps in membrane fusion for the related West Nile virus (WNV), using recombinant, WNV virus-like particles (VLPs) for single-particle experiments. Trimerization and membrane engagement are rate-limiting; fusion requires at least two adjacent trimers; availability of competent monomers within the contact zone between virus and target membrane creates a trimerization bottleneck. We have extended that work to dengue VLPs, from all four DV serotypes, finding an essentially similar mechanism. Small-molecule inhibitors of DV infection that target E block its fusion-inducing conformation change. We show that [~]15 bound molecules per particle ([~]8.5 % occupancy) completely prevent fusion, in accord with the proposed mechanism and the likely inhibitor binding site on E.\n\nImpact statementSingle-particle studies of dengue-virus membrane fusion and the effect of small-molecule inhibitors of infection clarify the viral fusion mechanism.

biophysics↗

bioRxiv2018

Utp14 interaction with the Small Subunit Processome

The SSU Processome (sometimes referred to as 90S) is an early stabile intermediate in the small ribosomal subunit biogenesis pathway of eukaryotes. Progression of the SSU Processome to a pre-40S particle requires a large-scale compaction of the RNA and release of many biogenesis factors. The U3 snoRNA is a primary component of the SSU Processome and hybridizes to the rRNA at multiple locations to organize the structure of the SSU Processome. Thus, release of U3 is prerequisite for the transition to pre-40S. Our lab proposed that the RNA helicase Dhr1 plays a crucial role in the transition by unwinding U3 and that this activity is controlled by the SSU Processome protein Utp14. How Utp14 times the activation of Dhr1 is an open question. Despite being highly conserved, Utp14 contains no recognizable domains, and how Utp14 interacts with the SSU Processome is not well characterized. Here, we used UV crosslinking and analysis of cDNA and yeast two-hybrid interaction to characterize how Utp14 interacts with the pre-ribosome. Moreover, proteomic analysis of SSU particles lacking Utp14 revealed that Utp14 is needed for efficient recruitment of the RNA exosome. Our analysis positions Utp14 to be uniquely poised to communicate the status of assembly of the SSU Processome to Dhr1 and possibly the exosome as well.

molecular biology↗

bioRxiv2017

Data-driven brain-types and their cognitive consequences

The canonical approach to exploring brain-behaviour relationships is to group individuals according to a phenotype of interest, and then explore the neural correlates of this grouping. A limitation of this approach is that multiple aetiological pathways could result in a similar phenotype, so the role of any one brain mechanism may be substantially underestimated. Building on advances in network analysis, we used a data-driven community-clustering algorithm to identify robust subgroups based on white-matter microstructure in childhood and adolescence (total N=313, mean age: 11.24 years). The algorithm indicated the presence of two equal-size groups that show a critical difference in FA of the left and right cingulum. These different brain types had profoundly different cognitive abilities with higher performance in the higher FA group. Further, a connectomics analysis indicated reduced structural connectivity in the low FA subgroup that was strongly related to reduced functional activation of the default mode network.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC=\"FIGDIR/small/237859_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (34K):\norg.highwire.dtl.DTLVardef@135e807org.highwire.dtl.DTLVardef@145673org.highwire.dtl.DTLVardef@137cd67org.highwire.dtl.DTLVardef@8e2cff_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗