Biology subjects

Smith, L.

Publications and source records attributed to Smith, L..

68 records · Page 4Linked to original sources

bioRxiv2020

A commercial autogenous injection vaccine protects ballan wrasse (Labrus bergylta, Ascanius) against Aeromonas salmonicida vapA type V

Atypical Aeromonas salmonicida (aAs) and Vibrionaceae related species are bacteria routinely recovered from diseased ballan wrasse used as cleaner fish in Atlantic salmon farming. Autogenous multivalent vaccines formulated from these microorganisms are widely used by the industry to protect farmed wrasse despite limited experimental proof that they are primary pathogens. In this study, the components of a commercial multivalent injection wrasse vaccine were tested for infectivity, pathogenicity and virulence via intra peritoneal injection at pre-deployment size (25-50g) and the efficacy of the vaccine for protection against aAs assessed. Injection with 3.5x109, 8x109 1.8x109 and 5x109 cfu/fish of Vibrio splendidus, V. ichthyoenteri, Aliivibrio logeii and A. salmonicida, respectively, did not cause significant mortalities, lesions or clinical signs after a period of 14 days. IP injection with both aAs and Photobacterium indicum successfully reproduced the clinical signs and internal lesions observed during natural outbreaks of the disease. Differences in virulence (LD50 at day 8-post infection of 3.6x106 cfu/fish and 1.6x107 cfu/fish) were observed for two aAs vapA type V isolates. In addition, the LD50 for Photobacterium indicum was 2.2x107 cfu/fish. The autogenous vaccine was highly protective against the two aAs vapA type V isolates after 700-degree days of immunisation. The RPSFINAL values for the first isolate were 95 and 91% at 1x106 cfu/fish and 1x107 cfu/fish, respectively, and 79% at 1x107 cfu/fish for the second isolate tested. In addition, significantly higher anti aAs seral antibodies (IgM), were detected by ELISA in vaccinated fish in contrast with control (mock vaccinated) fish. These results suggest wrasse can be effectively immunised and protected against aAs infection by injection with oil adjuvanted vaccines prepared with inactivated homologous isolates. Further work should assess the efficacy of vaccination against other isolates that have proven to be pathogenic such as aAs type VI and Photobacterium indicum and explore the feasibility of immersion vaccination. In addition, a full characterisation of aAs isolates within the same vapA types should be performed as differences in virulence between vapA type V isolates were observed and partial genome analysis indicated small but potentially important genomic differences in these isolates.

pathology↗

bioRxiv2020

NAT8L mRNA oxidation is linked to neurodegeneration in multiple sclerosis

RNA oxidation has been implicated in neurodegeneration, but the underlying mechanism for such effects is unclear. Recently, we demonstrated extensive RNA oxidation within the neurons in multiple sclerosis (MS) brain. In this report we identified selectively oxidized mRNAs in neuronal cells that pertained to neuropathological pathways. N-acetyl aspartate transferase 8 like (NAT8L) mRNA is one such transcript, whose translated product enzymatically synthesizes N-acetyl aspartic acid (NAA), a neuronal metabolite important for myelin synthesis. We reasoned that impediment of translation of an oxidized NAT8L mRNA will result in reduction in its cognate protein, thus lowering NAA level. This assertion is directly supported by our studies on a model cellular system, an MS animal model and postmortem human MS brain. Reduced NAA level in the brain hampers myelin integrity making neuronal axons more susceptible to damage, which contributes in MS neurodegeneration. Overall, this work provides a framework for mechanistic understanding of the link between RNA oxidation and neurodegenerative diseases.

molecular biology↗

bioRxiv2020

De Novo Phosphoinositide Synthesis in Zebrafish Is Required for Triad Formation but Not Essential for Myogenesis

Phosphoinositides (PIPs) and their regulatory enzymes are key players in many cellular processes and are required for aspects of vertebrate development. Dysregulated PIP metabolism has been implicated in several human diseases, including a subset of skeletal myopathies that feature structural defects in the triad. The role of PIPs in skeletal muscle formation, and particularly triad biogenesis, has yet to be determined. CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) catalyzes the formation of phosphatidylinositol, which is the base of all PIP species. Loss of CDIPT should, in theory, result in the failure to produce PIPs, and thus provide a strategy for establishing the requirement for PIPs during embryogenesis. In this study, we generated cdipt mutant zebrafish and determined the impact on skeletal myogenesis. Analysis of cdipt mutant muscle revealed no apparent global effect on early muscle development. However, small but significant defects were observed in triad size, with T-tubule area, inter terminal cisternae distance and gap width being smaller in cdipt mutants. This was associated with a decrease in motor performance. Overall, these data suggest that myogenesis in zebrafish does not require de novo PIP synthesis but does implicate a role for CDIPT in triad formation.

developmental biology↗

bioRxiv2019

Suramin potently inhibits binding of the mammalian high mobility group protein AT-hook 2 to DNA

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein which plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen high throughput screening (HTS) assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we discovered that suramin, a negatively charged antiparasitic drug potently inhibits the HMGA2-DNA interaction. Our results also show that the inhibition is through suramin binding to the AT-hooks of HMGA2, therefore blocking its DNA binding capacity. Furthermore, we demonstrate that suramin can induce brain tumor stem cells differentiation into cells with neurite-like structures, a process triggered by disrupting HMGA2-DNA interactions. Since suramin has strong antitumor and anti-metastasis activities, our discovery suggests that HMGA2 and HMGA2-like proteins may be the cellular target of this century-old drug.

biochemistry↗

bioRxiv2019

A photostable monomeric superfolder GFP

The green fluorescent protein GFP from Aequorea victoria has been engineered extensively in the past to generate variants suitable for protein tagging. Early efforts produced the enhanced variant EGFP and its monomeric derivative mEGFP, which have useful photophysical properties, as well as superfolder GFP, which folds efficiently under adverse conditions. We previously generated msGFP, a monomeric superfolder derivative of EGFP. Unfortunately, compared to EGFP, msGFP and other superfolder GFP variants show faster photobleaching. We now describe msGFP2, which retains monomeric superfolder properties while being as photostable as EGFP. msGFP2 contains modified N- and C-terminal peptides that are expected to reduce nonspecific interactions. Compared to EGFP and mEGFP, msGFP2 is less prone to disturbing the functions of certain partner proteins. For general-purpose protein tagging, msGFP2 may be the best available derivative of A. victoria GFP.

cell biology↗

bioRxiv2019

Reciprocal monoallelic expression of ASAR lncRNA genes controls replication timing of human chromosome 6.

AbstractDNA replication occurs on mammalian chromosomes in a cell-type distinctive temporal order known as the replication timing program. We previously found that disruption of the noncanonical lncRNA genes ASAR6 and ASAR15 results in delayed replication timing and delayed mitotic chromosome condensation of human chromosome 6 and 15, respectively. ASAR6 and ASAR15 display random monoallelic expression, and display asynchronous replication between alleles that is coordinated with other random monoallelic genes on their respective chromosomes. Disruption of the expressed allele, but not the silent allele, of ASAR6 leads to delayed replication, activation of the previously silent alleles of linked monoallelic genes, and structural instability of human chromosome 6. In this report, we describe a second lncRNA gene (ASAR6-141) on human chromosome 6 that when disrupted results in delayed replication timing in cis. ASAR6-141 is subject to random monoallelic expression and asynchronous replication, and is expressed from the opposite chromosome 6 homolog as ASAR6. ASAR6-141 RNA, like ASAR6 and ASAR15 RNAs, contains a high L1 content and remains associated with the chromosome territory where it is transcribed. Three classes of cis-acting elements control proper chromosome function in mammals: origins of replication, centromeres; and telomeres, which are responsible for replication, segregation and stability of all chromosomes. Our work supports a fourth type of essential chromosomal element, \"Inactivation/Stability Centers\", which express ASAR lncRNAs responsible for proper replication timing, monoallelic expression, and structural stability of each chromosome.\n\nAuthor summaryMammalian cells replicate their chromosomes during a highly ordered and cell type-specific program. Genetic studies have identified two long non-coding RNA genes, ASAR6 and ASAR15, as critical regulators of the replication timing program of human chromosomes 6 and 15, respectively. There are several unusual characteristics of the ASAR6 and ASAR15 RNAs that distinguish them from other long non-coding RNAs, including: being very long (>200 kb), lacking splicing of the transcripts, lacking polyadenylation, and being retained in the nucleus on the chromosomes where they are made. ASAR6 and ASAR15 also have the unusual property of being expressed from only one copy of the two genes located on homologous chromosome pairs. Using these unusual characteristics shared between ASAR6 and ASAR15, we have identified a second ASAR lncRNA gene located on human chromosome 6, which we have named ASAR6-141. ASAR6-141 is expressed from the opposite chromosome 6 homolog as ASAR6, and disruption of the expressed allele results in delayed replication of chromosome 6. ASAR6-141 RNA had previously been annotated as vlinc273. The very long intergenic non-coding (vlinc)RNAs represent a recently annotated class of RNAs that are long (>50 kb), non-spliced, and non-polyadenlyated nuclear RNAs. There are currently >2,700 vlincRNAs expressed from every chromosome, are encoded by >15% of the human genome, and with a few exceptions have no known function. Our results suggest the intriguing possibility that the vlinc class of RNAs may be functioning to control the replication timing program of all human chromosomes.

genetics↗

bioRxiv2018

Micro computed tomography as an accessible imaging platform for exploring organism development and human disease modeling

Understanding how events at the molecular and cellular scales contribute to tissue form and function is key to uncovering mechanisms driving animal development, physiology and disease. Elucidating these mechanisms has been enhanced through the study of model organisms and the use of sophisticated genetic, biochemical and imaging tools. Here we present an optimized method for non-invasive imaging of Drosophila melanogaster at high resolution using micro computed tomography (-CT). Our method allows for rapid processing of intact animals at any developmental stage, provides precise quantitative assessment of tissue size and morphology, and permits analysis of inter-organ relationships. We then use the power of -CT imaging to model human diseases through the characterization of microcephaly in the fly. Our work demonstrates that -CT is a versatile and accessible tool that complements standard imaging techniques, capable of uncovering novel biological mechanisms that have remained undocumented due to limitations of current methods.

developmental biology↗

bioRxiv2018

Occidiofungin, an actin binding antifungal with in vivo efficacy in a vulvovaginal candidiasis infection

Current antifungal treatment options are plagued with rapidly increasing occurrence of resistance, high degree of toxicity and a limited spectrum of activity. The need to develop a novel antifungal with a unique target, wider spectrum of activity, and reduced toxicity to the host, is urgent. We have identified and characterized one such compound named occidiofungin that is produced by the soil bacterium Burkholderia contaminans MS14. This study identifies the primary cellular target of the antifungal, which was determined to be actin. Actin binding metabolites are generally characterized by their ability to inhibit polymerization or depolymerization of actin filaments, which presumably accounts for their severe toxicity. Occidiofungin, instead, has a subtler effect on actin dynamics that triggers apoptotic cell death. We were able to demonstrate the effectiveness of the antifungal in treating a vulvovaginal yeast infection in a murine model. This discovery puts occidiofungin in a unique class of actin-binding antifungal compounds with minimal reported toxicity to the host. The results of this study are important for the development of a novel class of antifungals that could fill the existing gap in treatment options for fungal infections.\n\nAuthor summaryWidespread resistance to antifungal compounds currently in use has been alarming. Identification and development of a new class of antifungals with a novel cellular target is desperately needed. This study describes the assays carried out to determine the molecular target and evaluate efficacy of one such novel antifungal compound called occidiofungin. Occidiofungin modified with a functional alkyne group enabled affinity purification assays and localization studies in yeast. These studies led to the identification of the actin binding property of occidiofungin. Actin-binding by secondary metabolites often exhibit severe host toxicity, but this does not appear to be the case for occidiofungin. We have previously been able to administer occidiofungin to mice at concentrations in the range of 5 mg/kg without any serious complications. We were able to demonstrate the effectiveness of the antifungal in treating a vaginal fungal infection in a murine model. The results outlined in this manuscript establish that occidiofungin is an efficacious compound with a novel molecular target, putting it in a completely new class of antifungals.

microbiology↗

bioRxiv2018

Effects of Parkinson’s disease and dopamine on digit span measures of working memory

RationaleParkinsons disease (PD) impairs working memory (WM) - the ability to maintain items in memory for short periods of time and manipulate them. There is conflicting evidence on the nature of the deficits caused by the disease, and the potential beneficial and detrimental effects of dopaminergic medication on different WM processes.\n\nObjectivesWe hypothesised that PD impairs both maintenance and manipulation of items in WM and dopaminergic medications improve this in PD patients but impair it in healthy older adults.\n\nMethodsWe tested 68 PD patients ON and OFF their dopaminergic medication, 83 healthy age-matched controls, and 30 healthy older adults after placebo and levodopa administration. We used the digit span, a WM test with three components (forwards, backwards and sequence recall) that differ in the amount of manipulation required. We analysed the maximum spans and the percentage of lists correctly recalled, which probe capacity of WM and the accuracy of the memory processes within this capacity, respectively.\n\nResultsPD patients had lower WM capacity across all three digit span components, but only showed reduced percentage accuracy on the components requiring manipulation (backwards and sequence spans). Dopaminergic medication did not affect performance in PD patients. In healthy older adults, levodopa did not affect capacity, but did impair accuracy on one of the manipulation components (sequence), without affecting the other (backwards).\n\nConclusionsThis suggests a non-dopaminergic deficit of maintenance capacity and manipulation accuracy in PD patients, and a potential \"overdosing\" of intact manipulation mechanisms in healthy older adults by levodopa.

neuroscience↗

bioRxiv2018

Embryo timelapses can be compiled and quantified to understand canonical histone dynamics across multiple cell cycles.

In the last decade, computational analysis of big datasets has facilitated the processing of unprecedented quantities of collected biological data. Thus, automations and big data analyses have been revolutionary in detecting and quantifying subtle phenotypes in cell biological contexts. Analyzing similar quantities of data in larger and more complicated biological systems such as live embryos has been more challenging due to experimental necessities impeding both compilations of data collection and informative analysis. Here we present a streamlined workflow that can quantify cell cycle dynamics in early developing embryos using fluorescently labeled proteins. We benchmark this pipeline using Caenorhabditis elegans (nematode) embryonic development and a fluorescently labeled histone. Using our pipeline, we find that histone proteins are broadly stable in early embryonic development. In sum, we have utilized the large biological and experimental variation associated with quantification of fluorescent proteins in embryonic systems, to quantify nuclear accumulation rate, chromatin incorporation, and turnover/stability of canonical histones during early development.

developmental biology↗

bioRxiv2018

Discrimination Exposure and DNA Methylation of Stress-Related Genes in Latina Mothers

Latina mothers, who have the highest fertility rate among all ethnic groups in the US, are often exposed to discrimination. The biological impacts of this discrimination are unknown. This study is the first to explore the relationship between discrimination and DNA methylation of stress regulatory genes in Latinas. Our sample was Latina women (n = 147) with a mean age of 27.6 years who were assessed at 24-32 weeks gestation (T1) and 4-6 weeks postpartum (T2) and reside in the U.S. Blood was collected at T1, and the Everyday Discrimination Scale (EDS) was administered at T1 and T2. DNA Methylation at candidate gene regions was determined by bisulphite pyrosequencing. Associations between EDS and DNA methylation were assessed via zero-inflated Poisson models, adjusting for covariates and multiple-test comparisons. Discrimination was negatively associated with methylation at CpG sites within the glucocorticoid receptor (NR3C1) and brain-derived neurotrophic factor (BDNF) genes that were consistent over time. In addition, discrimination was negatively associated with methylation of a CpG in the glucocorticoid binding protein (FKBP5) at T1 but not at T2. This study underscores the complex biological pathways between discrimination and epigenetic modification in Latina women that warrant further investigation to better understand the genetic and psychopathological impact of discrimination on Latino mothers and their families.

genomics↗

bioRxiv2017

Tellurium Notebooks - An Environment for Dynamical Model Development, Reproducibility, and Reuse

The considerable difficulty encountered in reproducing the results of published dynamical models limits validation, exploration and reuse of this increasingly large biomedical research resource. To address this problem, we have developed Tellurium Notebook, a software system that facilitates building reproducible dynamical models and reusing models by 1) supporting the COMBINE archive format during model development for capturing model information in an exchangeable format and 2) enabling users to easily simulate and edit public COMBINE-compliant models from public repositories to facilitate studying model dynamics, variants and test cases. Tellurium Notebook, a Python-based Jupyter-like environment, is designed to seamlessly inter-operate with these community standards by automating conversion between COMBINE standards formulations and corresponding in-line, human-readable representations. Thus, Tellurium brings to systems biology the strategy used by other literate notebook systems such as Mathematica. These capabilities allow users to edit every aspect of the standards-compliant models and simulations, run the simulations in-line, and re-export to standard formats. We provide several use cases illustrating the advantages of our approach and how it allows development and reuse of models without requiring technical knowledge of standards. Adoption of Tellurium should accelerate model development, reproducibility and reuse.\n\nAuthor summaryThere is considerable value to systems and synthetic biology in creating reproducible models. An essential element of reproducibility is the use of community standards, an often challenging undertaking for modelers. This article describes Tellurium Notebook, a tool for developing dynamical models that provides an intuitive approach to building and reusing models built with community standards. Tellurium automates embedding human-readable representations of COMBINE archives in literate coding notebooks, bringing to systems biology this strategy central to other literate notebook systems such as Mathematica. We show that the ability to easily edit this human-readable representation enables users to test models under a variety of conditions, thereby providing a way to create, reuse, and modify standard-encoded models and simulations, regardless of the users level of technical knowledge of said standards.

systems biology↗

bioRxiv2017

The NSIGHT1 Randomized Controlled Trial: Rapid Whole Genome Sequencing for Accelerated Etiologic Diagnosis in Critically Ill Infants

ImportanceGenetic disorders, including congenital anomalies, are a leading cause of morbidity and mortality in infants, especially in neonatal and pediatric intensive care units (NICU and PICU). While genomic sequencing is useful for diagnosis of genetic diseases, results are usually reported too late to guide inpatient management.\n\nObjectiveTo test the hypothesis that rapid whole genome sequencing (rWGS) increases the proportion of infants in NICUs and PICUs receiving a genetic diagnosis within 28 days.\n\nDesignAn investigator-initiated, partially blinded, pragmatic, randomized controlled study with enrollment from October 2014 - June 2016, and follow up until December 2016.\n\nSettingA regional neonatal and pediatric intensive care unit in a tertiary referral childrens hospital.\n\nParticipantsSixty five of 129 screened families with infants aged less than four months, in neonatal and pediatric intensive care units, and with illnesses of unknown etiology, completed the study.\n\nInterventionParent and infant trio rWGS.\n\nMain Outcome and MeasureThe hypothesis and end-points were formulated a priori. The primary end-point was rate of genetic diagnosis within 28 days of enrollment or first standard test order.\n\nResultsTwenty six female proband infants, 37 male infants, and two infants of undetermined sex were randomized to receive rWGS plus standard tests (n=32, cases) or standard tests alone (n=33, controls). The study was terminated early due to loss of equipoise: 63% (21) controls received genomic sequencing as standard tests. Nevertheless, intention to treat analysis showed the rate of genetic diagnosis within 28 days to be higher in cases (31%, ten of 32) than controls (3%, one of 33; difference, 28% [95% CI, 10% to 46%]; p=0.003). Among infants enrolled in the first 25 days of life, the rate of neonatal diagnosis was higher in cases (32%, seven of 22) than controls (0%, zero of 23; difference, 32% [95% CI, 11% to 53%]; p=0.004). Age at diagnosis (median in cases 25 days, range 14-90 days vs median in controls 130 days, range 37-451) and time to diagnosis (median in cases thirteen days, range 1-84 days vs median in controls 107 days, range 21-429 days) were significantly less in cases than controls (p=0.04).\n\nCONCLUSIONSrWGS increased the proportion of infants in a regional NICU and PICU who received a timely diagnosis of a genetic disease. Additional, adequately powered studies are needed to determine whether accelerated diagnosis is associated with improved outcomes in this setting. ClinicalTrials.gov Identifier: NCT02225522.

clinical trials↗

bioRxiv2017

Limited contribution of rare, noncoding variation to autism spectrum disorder from sequencing of 2,076 genomes in quartet families

Genomic studies to date in autism spectrum disorder (ASD) have largely focused on newly arising mutations that disrupt protein coding sequence and strongly influence risk. We evaluate the contribution of noncoding regulatory variation across the size and frequency spectrum through whole genome sequencing of 519 ASD cases, their unaffected sibling controls, and parents. Cases carry a small excess of de novo (1.02-fold) noncoding variants, which is not significant after correcting for paternal age. Assessing 51,801 regulatory classes, no category is significantly associated with ASD after correction for multiple testing. The strongest signals are observed in coding regions, including structural variation not detected by previous technologies and missense variation. While rare noncoding variation likely contributes to risk in neurodevelopmental disorders, no category of variation has impact equivalent to loss-of-function mutations. Average effect sizes are likely to be smaller than that for coding variation, requiring substantially larger samples to quantify this risk.

genomics↗