Biology subjects

Johnson, S.

Publications and source records attributed to Johnson, S..

12 recordsLinked to original sources

bioRxiv2018

Cyclodextrin triggers MCOLN1-dependent endo-lysosome secretion in Niemann-Pick type C cells

In specialized cell types, lysosome-related organelles support regulated secretory pathways, while in non-specialized cells, lysosomes can undergo fusion with the plasma membrane in response to a transient rise in cytosolic calcium. Recent evidence also indicates that lysosome secretion can be controlled transcriptionally and promote clearance in lysosome storage diseases. In addition, evidence is also accumulating that low concentrations of cyclodextrins reduce the cholesterol storage phenotype in cells and animals with the cholesterol storage disease Niemann-Pick type C, via an unknown mechanism. Here, we report that cyclodextrin triggers the secretion of the endo/lysosomal content in non-specialized cells, and that this mechanism is responsible for the decreased cholesterol overload in Niemann-Pick type C cells. We also find that that the secretion of the endo/lysosome content occurs via a mechanism dependent on the endosomal calcium channel MCOLN1, as well as FYCO1, the AP1 adaptor and its partner Gadkin. We conclude that endolysosomes in non-specialized cells can acquire secretory functions elicited by cyclodextrin, and that this pathway is responsible for the decrease in cholesterol storage in Niemann-Pick C cells.

cell biology↗

bioRxiv2018

Oral iron exacerbates colitis and influences the intestinal microbiome

Inflammatory bowel disease (IBD) is associated with anaemia and oral iron replacement to correct this can be problematic, intensifying inflammation and tissue damage. The intestinal microbiota also plays a key role in the pathogenesis of IBD, and iron supplementation likely influences gut bacterial diversity in patients with IBD. Here, we assessed the impact of dietary iron, using chow diets containing either 100, 200 or 400 ppm, fed ad libitum to adult female C57BL/6 mice in the presence or absence of colitis induced using dextran sulfate sodium (DSS), on (i) clinical and histological severity of acute DSS-induced colitis, and (ii) faecal microbial diversity, as assessed by sequencing the V4 region of 16S rRNA. Increasing or decreasing dietary iron concentration from the standard 200 ppm exacerbated both clinical and histological severity of DSS-induced colitis. DSS-treated mice provided only half the standard levels of iron ad libitum (i.e. chow containing 100 ppm iron) lost more body weight than those receiving double the amount of standard iron (i.e. 400 ppm); p<0.01. Faecal calprotectin levels were significantly increased in the presence of colitis in those consuming 100 ppm iron at day 8 (5.94-fold) versus day-10 group (4.14-fold) (p<0.05), and for the 400 ppm day-8 group (8.17-fold) versus day-10 group (4.44-fold) (p<0.001). In the presence of colitis, dietary iron at 400 ppm resulted in a significant reduction in faecal abundance of Firmicutes and Bacteroidetes, and increase of Proteobacteria, changes which were not observed with lower dietary intake of iron at 100 ppm. Overall, altering dietary iron intake exacerbated DSS-induced colitis; increasing the iron content of the diet also led to changes in intestinal bacteria diversity and composition after colitis was induced with DSS.

physiology↗

bioRxiv2018

RepB C-terminus mutation of an ori pRi vector affects plasmid copy number in Agrobacterium and transgene copy number in plants

A native repABC replication origin, ori pRi, was previously reported as a single copy plasmid in Agrobacterium tumefaciens and can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors for Agrobacterium-mediated transformation. A high copy ori pRi variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the literature. Sequencing the high copy pTF::Ri repABC operon revealed the presence of two mutations: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type oriRi binary vector showed that Agrobacterium cells with the RepBY299H mutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to investigate the impact of the RepBY299H mutation on transformation and quality plant production, the RepBY299H mutated ori pRi binary vector was compared with the original wild-type ori pRi binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy ori pRi with the RepBY299H mutation in Agrobacterium cells lost the advantage of generating high frequency single copy, backbone-free transgenic plants compared to using the single copy wild-type ori pRi binary vector.

molecular biology↗

bioRxiv2018

Spontaneous and coordinated Ca2+ activity of cochlear sensory and non-sensory cells drives the maturation of OHC afferent innervation

Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine tuning and high sensitivity of the mammalian cochlea to acoustic stimuli. Here, by genetically manipulating spontaneous Ca2+ signalling in vivo, through a period of early postnatal development, we find that the refinement of OHC afferent innervation is regulated by complementary spontaneous Ca2+ signals originating in OHCs and non-sensory cells. OHCs fire spontaneous Ca2+ spikes during a narrow period of immature development. Simultaneously, waves of Ca2+ activity in the non-sensory greater epithelial ridge act, via ATP-induced activation of P2X receptors, to synchronize OHC firing, resulting in the refinement of their afferent innervation. In the absence of connexin channels Ca2+ waves are impaired, leading to a reduction in the number of ribbon synapses and afferent fibres on OHCs. We propose that the correct maturation of the afferent connectivity in OHCs requires experience-independent Ca2+ signals from sensory and non-sensory cells.

neuroscience↗

bioRxiv2018

Towards a bioelectronic computer: A theoretical study of a multi-layer biomolecular computing system that can process electronic inputs

DNA molecular machines have great potential for use in computing systems. Since Adleman originally introduced the concept of DNA computing through his use of DNA strands to solve a Hamiltonian path problem, a range of DNA-based computing elements have been developed, including logic gates, neural networks, finite state machines (FSMs) and non-deterministic universal Turing machines. It has also been established that DNA molecular machines can be controlled using electrical signals and that the state of DNA nanodevices can be measured using an electrochemical readout. However, to the best of our knowledge there has as yet been no demonstration of a fully integrated biomolecular computing system that has multiple levels of information processing capacity, can accept electronic inputs and is capable of independent operation. In this paper we address the question of how such a system could work. We present simulation results showing that such an integrated hybrid system could convert electrical impulses into biomolecular signals, perform logical operations and take a decision, storing its history. We also illustrate theoretically how such a system could potentially be used to perform a task such as controlling an autonomous robot navigating through a maze.

synthetic biology↗

bioRxiv2018

Bioelectric-calcineurin signaling module regulates allometric growth and size of the zebrafish fin

The establishment of relative size of organs and structures is paramount for attaining final form and function of an organism. Importantly, variation in the proportions of structures frequently underlies adaptive change in morphology in evolution and maybe a common mechanism underlying selection. However, the mechanism by which growth is integrated within tissues during development to achieve proper proportionality is poorly understood. We have shown that signaling by potassium channels mediates coordinated size regulation in zebrafish fins. Recently, calcineurin inhibitors were shown to elicit changes in zebrafish fin allometry as well. Here, we identify the potassium channel kcnk5b as a key player in integrating calcineurins growth effects, in part through regulation of the cytoplasmic C-terminus of the channel. We propose that the interaction between Kcnk5b and calcineurin acts as a signaling node to regulate allometric growth. Importantly, we find that this regulation is epistatic to inherent mechanisms instructing overall size as inhibition of calcineurin is able to bypass genetic instruction of size as seen in sof and wild-type fins, however, it is not sufficient to re-specify positional memory of size of the fin. These findings integrate classic signaling mediators such as calcineurin with ion channel function in the regulation of size and proportion during growth.

developmental biology↗

bioRxiv2018

Structure of the Core of the Type Three Secretion System ExportApparatus

Export of proteins through type three secretion systems is critical for bacterial motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP/FliQ/FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. We here present the structure of this complex at 4.2 [A] by cryo-electron microscopy. None of the subunits adopt canonical integral membrane protein topologies and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems localize the export gate as a core component of the periplasmic portion of the machinery, and cross-linking experiments confirm this observation. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.\n\nOne Sentence SummaryThe core of the T3SS export gate forms a supra-membrane helical assembly

microbiology↗

bioRxiv2017

Age-accelerated cognitive decline in asymptomatic adults with CSF β-amyloid

ObjectiveCompare cognitive and hippocampal volume (HCV) trajectories in asymptomatic middle-aged and older adults with positive cerebrospinal fluid (CSF) markers of {beta}-amyloid (A{beta}) or tau to adults without an AD-associated biomarker profile.\n\nMethod392 adults enrolled in a longitudinal cohort study (Wisconsin Registry for Alzheimers Prevention or Wisconsin Alzheimers Disease Research Center) completed a lumbar puncture and at least two biennial or annual neuropsychological evaluations. Cutoffs for A{beta}42, total tau, and phosphorylated tau were developed via receiver operating characteristic curve analyses on a sample of 78 participants (38 dementia, 40 controls). These cutoffs were applied to a separate sample of 314 cognitively healthy adults (mean age at CSF collection = 61.5) and mixed-effects regression analyses tested linear and quadratic interactions of biomarker group x age at each visit on cognitive and HCV outcomes.\n\nResults215 participants (69%) were biomarker negative (preclinical AD Stage 0), 46 (15%) were A{beta}+ only (preclinical AD Stage 1), 25 (8%) were A{beta}+ and tau+ (preclinical AD Stage 2), and 28 (9%) were tau+ only. Both Stage 1 and Stage 2 groups exhibited greater rates of linear decline on story memory and processing speed measures, and non-linear decline on list-learning and set-shifting measures compared to Stage 0. The tau+ only group did not significantly differ from Stage 0 in rates of cognitive decline.\n\nConclusionIn an asymptomatic at-risk cohort, elevated CSF A{beta} (with or without elevated tau) was associated with greater rates of cognitive decline, with the specific pattern of decline varying across cognitive measures.

neuroscience↗

bioRxiv2017

Mitochondrial genomes of the regionally extinct Nittany Lion (Puma concolor from Pennsylvania)

Mountain lions (Puma concolor) were once endemic across the United States. The Northeastern population of mountain lions has been largely nonexistent since the early 1800s and was officially declared extinct in 2011. This regionally extinct mountain lion is Pennsylvania State Universitys official mascot, where it is referred to as the Nittany Lion. Our goal in this study was to use recent methodological advances in ancient DNA and massively parallel sequencing to reconstruct complete mitochondrial DNA (mtDNA) genomes of multiple Nittany Lions by sampling from preserved skins. This effort is part of a broader Nittany Lion Genome project intended to involve undergraduates in ancient DNA and bioinformatics research and to engage the broader Penn State community in discussions about conservation biology and extinction. Complete mtDNA genome sequences were obtained from five individuals. When compared to previously published sequences, Nittany Lions are not more similar to each other than to individuals from the Western U.S. and Florida. Supporting previous findings, North American mountain lions overall were more closely related to each other than to those from South America and had lower genetic diversity. This result emphasizes the importance of continued conservation in the Western U.S. and Florida to prevent further regional extinctions.

ecology↗

bioRxiv2017

Structural Design of Chimeric Antigens for Multivalent Protein Vaccines

The development of prophylactic vaccines against pathogenic bacteria is a major objective of the World Health Organisation. However, vaccine development is often hindered by antigenic diversity and the difficulties encountered manufacturing immunogenic membrane proteins. Here, we employed structure-based design as a strategy to develop Chimeric Antigens (ChAs) for subunit vaccines. ChAs were generated against serogroup B Neisseria meningitidis (MenB), the predominant cause of meningococcal disease in the Western hemisphere. MenB ChAs exploit the lipoprotein factor H binding protein (fHbp) as a molecular scaffold to display the immunogenic VR2 epitope from the integral membrane protein PorA. Structural analyses demonstrate fHbp is correctly folded and that PorA VR2 epitope adopts an immunogenic conformation. In mice, ChAs elicit antibodies directed against fHbp and PorA, with antibody responses correlating to protection against meningococcal disease. ChAs offer a novel approach for generating multivalent subunit vaccines, containing of epitopes from integral membrane proteins, whose composition can be selected to circumvent pathogen diversity.

molecular biology↗

bioRxiv2017

Natural Colonization of Laboratory Mice with Staphylococcus aureus Primes a Systemic Immune Response

BackgroundWhether mice are an appropriate model for S. aureus infection and vaccination studies is a matter of debate, because they are not considered as natural hosts of S. aureus. Sparked by an outbreak of S. aureus infections in laboratory mice, we investigated whether laboratory mice are commonly colonized with S. aureus and whether this might impact on infection experiments.\n\nMethodsWe characterized 99 S. aureus isolates from laboratory mice (spa typing, virulence gene PCR), and quantified murine antibodies using FlexMap technology.\n\nResultsSpecific-pathogen-free mice from various vendors were frequently colonized with S. aureus (0-21%). S. aureus was readily transmitted from murine parents to offspring, which became persistently colonized. Most murine isolates belonged to the lineage CC88 (54%). Murine strains showed features of host adaptation, such as absence of hlb-converting phages and superantigen genes, as well as enhanced coagulation of murine plasma. Importantly, S. aureus colonization induced a systemic IgG response specific for numerous S. aureus proteins, including several vaccine candidates.\n\nConclusionLaboratory mice are natural hosts of S. aureus and, therefore, provide better infection models than previously assumed. Pre-exposure to S. aureus is a possible confounder in S. aureus infection and vaccination studies.

immunology↗

bioRxiv2017

Biocuration as an undergraduate training experience: Improving the annotation of the insect vector of Citrus greening disease

The Asian citrus psyllid (Diaphorina citri Kuwayama) is the insect vector of the bacterium Candidatus Liberibacter asiaticus (CLas), the pathogen associated with citrus Huanglongbing (HLB, citrus greening). HLB threatens citrus production worldwide. Suppression or reduction of the insect vector using chemical insecticides has been the primary method to inhibit the spread of citrus greening disease. Accurate structural and functional annotation of the Asian citrus psyllid genome, as well as a clear understanding of the interactions between the insect and CLas, are required for development of new molecular-based HLB control methods. A draft assembly of the D. citri genome has been generated and annotated with automated pipelines. However, knowledge transfer from well-curated reference genomes such as that of Drosophila melanogaster to newly sequenced ones is challenging due to the complexity and diversity of insect genomes. To identify and improve gene models as potential targets for pest control, we manually curated several gene families with a focus on genes that have key functional roles in D. citri biology and CLas interactions. This community effort produced 530 manually curated gene models across developmental, physiological, RNAi regulatory, and immunity-related pathways. As previously shown in the pea aphid, RNAi machinery genes putatively involved in the microRNA pathway have been specifically duplicated. A comprehensive transcriptome enabled us to identify a number of gene families that are either missing or misassembled in the draft genome. In order to develop biocuration as a training experience, we included undergraduate and graduate students from multiple institutions, as well as experienced annotators from the insect genomics research community. The resulting gene set (OGS v1.0) combines both automatically predicted and manually curated gene models. All data are available on https://citrusgreening.org/.

genomics↗