Publications by authors named "Sarah J Medina"

12 Publications

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Dual RNA-Seq characterization of host and pathogen gene expression in liver cells infected with Crimean-Congo Hemorrhagic Fever Virus.

PLoS Negl Trop Dis 2020 04 6;14(4):e0008105. Epub 2020 Apr 6.

National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that can cause a hemorrhagic fever in humans, with a case fatality rate of up to 40%. Cases of CCHFV have been reported in Africa, Asia, and southern Europe; and recently, due to the expanding range of its vector, autochthonous cases have been reported in Spain. Although it was discovered over 70 years ago, our understanding of the pathogenesis of this virus remains limited. We used RNA-Seq in two human liver cell lines (HepG2 and Huh7) infected with CCHFV (strain IbAr10200), to examine kinetic changes in host expression and viral replication simultaneously at 1 and 3 days post infection. Through this, numerous host pathways were identified that were modulated by the virus including: antiviral response and endothelial cell leakage. Notably, the genes encoding DDX60, a cytosolic component of the RIG-I signalling pathway and OAS2 were both shown to be dysregulated. Interestingly, PTPRR was induced in Huh7 cells but not HepG2 cells. This has been associated with the TLR9 signalling cascade, and polymorphisms in TLR9 have been associated with poor outcomes in patients. Additionally, we performed whole-genome sequencing on CCHFV to assess viral diversity over time, and its relationship to the host response. As a result, we have demonstrated that through next-generation mRNA deep-sequencing it is possible to not only examine mRNA gene expression, but also to examine viral quasispecies and typing of the infecting strain. This demonstrates a proof-of-principle that CCHFV specimens can be analyzed to identify both the virus and host biomarkers that may have implications for prognosis.
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http://dx.doi.org/10.1371/journal.pntd.0008105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162549PMC
April 2020

Downregulation of circulating miR 802-5p and miR 194-5p and upregulation of brain MEF2C along breast cancer brain metastasization.

Mol Oncol 2020 03 5;14(3):520-538. Epub 2020 Feb 5.

Faculdade de Farmácia, Research Institute for Medicines (iMed.ULisboa), Universidade de Lisboa, Portugal.

Breast cancer brain metastases (BCBMs) have been underinvestigated despite their high incidence and poor outcome. MicroRNAs (miRNAs), and particularly circulating miRNAs, regulate multiple cellular functions, and their deregulation has been reported in different types of cancer and metastasis. However, their signature in plasma along brain metastasis development and their relevant targets remain undetermined. Here, we used a mouse model of BCBM and next-generation sequencing (NGS) to establish the alterations in circulating miRNAs during brain metastasis formation and development. We further performed bioinformatics analysis to identify their targets with relevance in the metastatic process. We additionally analyzed human resected brain metastasis samples of breast cancer patients for target expression validation. Breast cancer cells were injected in the carotid artery of mice to preferentially induce metastasis in the brain, and samples were collected at different timepoints (5 h, 3, 7, and 10 days) to follow metastasis development in the brain and in peripheral organs. Metastases were detected from 7 days onwards, mainly in the brain. NGS revealed a deregulation of circulating miRNA profile during BCBM progression, rising from 18% at 3 days to 30% at 10 days following malignant cells' injection. Work was focused on those altered prior to metastasis detection, among which were miR-802-5p and miR-194-5p, whose downregulation was validated by qPCR. Using targetscan and diana tools, the transcription factor myocyte enhancer factor 2C (MEF2C) was identified as a target for both miRNAs, and its expression was increasingly observed in malignant cells along brain metastasis development. Its upregulation was also observed in peritumoral astrocytes pointing to a role of MEF2C in the crosstalk between tumor cells and astrocytes. MEF2C expression was also observed in human BCBM, validating the observation in mouse. Collectively, downregulation of circulating miR-802-5p and miR-194-5p appears as a precocious event in BCBM and MEF2C emerges as a new player in brain metastasis development.
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http://dx.doi.org/10.1002/1878-0261.12632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053247PMC
March 2020

Bacteriophage lambda overcomes a perturbation in its host-viral genetic network through mutualism and evolution of life history traits.

Evolution 2020 04 9;74(4):764-774. Epub 2020 Jan 9.

Division of Physics, University of California San Diego, La Jolla, California.

An important driver of evolution in viruses is natural selection to optimize the use of their hosts' genetic network. To learn how viruses respond to this pressure, we disrupted the genetic network of Escherichia coli to inhibit replication of its virus, bacteriophage lambda, and then observed how λ evolved to compensate. We deleted E. coli's dnaJ gene, which lambda uses to initiate DNA replication. Lambda partially restored its ability to reproduce with just two adaptive mutations associated with genes J and S. The location of the mutations was unexpected because they were not in genes that directly interact with DnaJ, rather they affected seemingly unrelated life history traits. A nonsynonymous J mutation increased lambda's adsorption rate and an S regulatory mutation delayed lysis timing. Lambda also recovered some of its reproductive potential through intracellular mutualism. This study offers two important lessons: first, viruses can rapidly adapt to disruptive changes in their host's genetic network. Second, organisms can employ mechanisms thought to operate at the population scale, such as evolution of life history traits and social interactions, in order to overcome hurdles at the molecular level. As life science research progresses and new fields become increasingly specialized, these results remind us of the importance of multiscale and interdisciplinary approaches to understand adaptation.
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http://dx.doi.org/10.1111/evo.13920DOI Listing
April 2020

Identification of circulating microRNA signatures as potential biomarkers in the serum of elk infected with chronic wasting disease.

Sci Rep 2019 12 23;9(1):19705. Epub 2019 Dec 23.

Zoonotic Diseases & Special Pathogens, Public Health Agency of Canada, National Microbiology Laboratory, 1015 Arlington St., Winnipeg, MB, R3E 3R2, Canada.

Chronic wasting disease (CWD) is an emerging infectious prion disorder that is spreading rapidly in wild populations of cervids in North America. The risk of zoonotic transmission of CWD is as yet unclear but a high priority must be to minimize further spread of the disease. No simple diagnostic tests are available to detect CWD quickly or in live animals; therefore, easily accessible biomarkers may be useful in identifying infected animals. MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules that circulate in blood and are promising biomarkers for several infectious diseases. In this study we used next-generation sequencing to characterize the serum miRNA profiles of 35 naturally infected elk that tested positive for CWD in addition to 35 elk that tested negative for CWD. A total of 21 miRNAs that are highly conserved amongst mammals were altered in abundance in sera, irrespective of hemolysis in the samples. A number of these miRNAs have previously been associated with prion diseases. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the discriminative potential of these miRNAs as biomarkers for the diagnosis of CWD. We also determined that a subgroup of 6 of these miRNAs were consistently altered in abundance in serum from hamsters experimentally infected with scrapie. This suggests that common miRNA candidate biomarkers could be selected for prion diseases in multiple species. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses pointed to a strong correlation for 3 of these miRNAs, miR-148a-3p, miR-186-5p, miR-30e-3p, with prion disease.
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http://dx.doi.org/10.1038/s41598-019-56249-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928025PMC
December 2019

The cell type resolved mouse transcriptome in neuron-enriched brain tissues from the hippocampus and cerebellum during prion disease.

Sci Rep 2019 01 31;9(1):1099. Epub 2019 Jan 31.

Zoonotic Diseases and Special Pathogens, National Microbiology Laboratory, Canadian Science Centre for Human and Animal Health, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.

Multiple cell types and complex connection networks are an intrinsic feature of brain tissue. In this study we used expression profiling of specific microscopic regions of heterogeneous tissue sections isolated by laser capture microdissection (LCM) to determine insights into the molecular basis of brain pathology in prion disease. Temporal profiles in two mouse models of prion disease, bovine spongiform encephalopathy (BSE) and a mouse-adapted strain of scrapie (RML) were performed in microdissected regions of the CA1 hippocampus and granular layer of the cerebellum which are both enriched in neuronal cell bodies. We noted that during clinical disease the number of activated microglia and astrocytes that occur in these areas are increased, thereby likely diluting the neuronal gene expression signature. We performed a comparative analysis with gene expression profiles determined from isolated populations of neurons, microglia and astrocytes to identify transcripts that are enriched in each of these cell types. Although the incubation periods of these two models are quite different, over 300 days for BSE and ~160 days for RML scrapie, these regional microdissections revealed broadly similar profiles. Microglial and astrocyte-enriched genes contributed a profound inflammatory profile consisting of inflammatory cytokines, genes related to phagocytosis, proteolysis and genes coding for extracellular matrix proteins. CA1 pyramidal neurons displayed a net upregulation of transcription factors and stress induced genes at pre-clinical stages of disease while all tissues showed profound decrease of overlapping genes related to neuronal function, in particular transcripts related to neuronal communication including glutamate receptors, phosphatase subunits and numerous synapse-related markers. Of note, we found a small number of genes expressed in neurons that were upregulated during clinical disease including, COX6A2, FZD9, RXRG and SOX11, that may be biomarkers of neurodegeneration.
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http://dx.doi.org/10.1038/s41598-018-37715-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355796PMC
January 2019

A Novel Triple-Mutant AAV6 Capsid Induces Rapid and Potent Transgene Expression in the Muscle and Respiratory Tract of Mice.

Mol Ther Methods Clin Dev 2018 Jun 14;9:323-329. Epub 2018 Apr 14.

Department of Pathobiology, University of Guelph, Guelph, ON N1G 2W1, Canada.

Gene therapy for the treatment of genetic disorders has demonstrated considerable therapeutic success in clinical trials. Among the most effective and commonly used gene delivery vectors are those based on adeno-associated virus (AAV). Despite these advances in clinical gene therapy, further improvements in AAV vector properties such as rapid intracellular processing and transgene expression, targeted transduction of therapeutically relevant cell types, and longevity of transgene expression, will render extension of such successes to many other human diseases. Engineering of AAV capsids continues to evolve the specificity and efficiency of AAV-mediated gene transfer. Here, we describe a triple AAV6 mutant, termed AAV6.2FF, containing F129L, Y445F, and Y731F mutations. AAV6.2FF yielded 10-fold greater transgene expression in lung than AAV6 after 21 days. Additionally, this novel capsid demonstrated 101-fold and 49-fold increased transgene expression in the muscle and lungs, respectively, 24 hr post vector delivery when compared with the parental AAV6. Furthermore, AAV6.2FF retains heparin sulfate binding capacity and displays a 10-fold increase in resistance to pooled immunoglobulin neutralization . The rapid and potent expression mediated by AAV6.2FF is ideally suited to applications such as vectored immunoprophylaxis, in which rapid transgene expression is vital for use during an outbreak response scenario.
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http://dx.doi.org/10.1016/j.omtm.2018.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054702PMC
June 2018

Destabilizing mutations encode nongenetic variation that drives evolutionary innovation.

Science 2018 Mar;359(6383):1542-1545

Division of Biological Sciences, University of California (UC) San Diego, La Jolla, CA 92093, USA.

Evolutionary innovations are often achieved by repurposing existing genes to perform new functions; however, the mechanisms enabling the transition from old to new remain controversial. We identified mutations in bacteriophage λ's host-recognition gene that confer enhanced adsorption to λ's native receptor, LamB, and the ability to access a new receptor, OmpF. The mutations destabilize λ particles and cause conformational bistability of J, which yields progeny of multiple phenotypic forms, each proficient at different receptors. This work provides an example of how nongenetic protein variation can catalyze an evolutionary innovation. We propose that cases where a single genotype can manifest as multiple phenotypes may be more common than previously expected and offer a general mechanism for evolutionary innovation.
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http://dx.doi.org/10.1126/science.aar1954DOI Listing
March 2018

MicroRNA and mRNA Dysregulation in Astrocytes Infected with Zika Virus.

Viruses 2017 10 14;9(10). Epub 2017 Oct 14.

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

The Zika virus (ZIKV) epidemic is an ongoing public health concern. ZIKV is a flavivirus reported to be associated with microcephaly, and recent work in animal models demonstrates the ability of the virus to cross the placenta and affect fetal brain development. Recent findings suggest that the virus preferentially infects neural stem cells and thereby deregulates gene expression, cell cycle progression, and increases cell death. However, neuronal stem cells are not the only brain cells that are susceptible to ZIKV and infection of other brain cells may contribute to disease progression. Herein, we characterized ZIKV replication in astrocytes, and profiled temporal changes in host microRNAs (miRNAs) and transcriptomes during infection. We observed the deregulation of numerous processes known to be involved in flavivirus infection, including genes involved in the unfolded protein response pathway. Moreover, a number of miRNAs were upregulated, including miR-30e-3p, miR-30e-5p, and, miR-17-5p, which have been associated with other flavivirus infections. This study highlights potential miRNAs that may be of importance in ZIKV pathogenesis.
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http://dx.doi.org/10.3390/v9100297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691648PMC
October 2017

Ecological speciation of bacteriophage lambda in allopatry and sympatry.

Science 2016 Dec 24;354(6317):1301-1304. Epub 2016 Nov 24.

Department of Microbiology and Molecular Genetics and BEACON Center for the Study of Evolution in Action, Michigan State University, East Lansing, MI 48824, USA.

Understanding the conditions that allow speciation to occur is difficult because most research has focused on either long-lived organisms or asexual microorganisms. We propagated bacteriophage λ, a virus with rapid generations and frequent recombination, on two Escherichia coli host genotypes that expressed either the LamB or OmpF receptor. When supplied with either single host (allopatry), phage λ improved its binding to the available receptor while losing its ability to use the alternative. When evolving on both hosts together (sympatry), the viruses split into two lineages with divergent receptor preferences. Although the level of divergence varied among replicates, some lineages evolved reproductive isolation via genetic incompatibilities. This outcome indicates that, under suitable conditions, allopatric and sympatric speciation can occur with similar ease.
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http://dx.doi.org/10.1126/science.aai8446DOI Listing
December 2016

A functional SNP catalog of overlapping miRNA-binding sites in genes implicated in prion disease and other neurodegenerative disorders.

Hum Mutat 2014 Oct 18;35(10):1233-48. Epub 2014 Aug 18.

Molecular PathoBiology, Public Health Agency of Canada, National Microbiology Laboratory, Winnipeg, Manitoba, R3E 3R2, Canada.

The involvement of SNPs in miRNA target sites remains poorly investigated in neurodegenerative disease. In addition to associations with disease risk, such genetic variations can also provide novel insight into mechanistic pathways that may be responsible for disease etiology and/or pathobiology. To identify SNPs associated specifically with degenerating neurons, we restricted our analysis to genes that are dysregulated in CA1 hippocampal neurons of mice during early, preclinical phase of Prion disease. The 125 genes chosen are also implicated in other numerous degenerative and neurological diseases and disorders and are therefore likely to be of fundamental importance. We predicted those SNPs that could increase, decrease, or have neutral effects on miRNA binding. This group of genes was more likely to possess DNA variants than were genes chosen at random. Furthermore, many of the SNPs are common within the human population, and could contribute to the growing awareness that miRNAs and associated SNPs could account for detrimental neurological states. Interestingly, SNPs that overlapped miRNA-binding sites in the 3'-UTR of GABA-receptor subunit coding genes were particularly enriched. Moreover, we demonstrated that SNP rs9291296 would strengthen miR-26a-5p binding to a highly conserved site in the 3'-UTR of gamma-aminobutyric acid receptor subunit alpha-4.
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http://dx.doi.org/10.1002/humu.22627DOI Listing
October 2014

Early mechanisms of pathobiology are revealed by transcriptional temporal dynamics in hippocampal CA1 neurons of prion infected mice.

PLoS Pathog 2012 8;8(11):e1003002. Epub 2012 Nov 8.

Molecular PathoBiology, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.

Prion diseases typically have long pre-clinical incubation periods during which time the infectious prion particle and infectivity steadily propagate in the brain. Abnormal neuritic sprouting and synaptic deficits are apparent during pre-clinical disease, however, gross neuronal loss is not detected until the onset of the clinical phase. The molecular events that accompany early neuronal damage and ultimately conclude with neuronal death remain obscure. In this study, we used laser capture microdissection to isolate hippocampal CA1 neurons and determined their pre-clinical transcriptional response during infection. We found that gene expression within these neurons is dynamic and characterized by distinct phases of activity. We found that a major cluster of genes is altered during pre-clinical disease after which expression either returns to basal levels, or alternatively undergoes a direct reversal during clinical disease. Strikingly, we show that this cluster contains a signature highly reminiscent of synaptic N-methyl-D-aspartic acid (NMDA) receptor signaling and the activation of neuroprotective pathways. Additionally, genes involved in neuronal projection and dendrite development were also altered throughout the disease, culminating in a general decline of gene expression for synaptic proteins. Similarly, deregulated miRNAs such as miR-132-3p, miR-124a-3p, miR-16-5p, miR-26a-5p, miR-29a-3p and miR-140-5p follow concomitant patterns of expression. This is the first in depth genomic study describing the pre-clinical response of hippocampal neurons to early prion replication. Our findings suggest that prion replication results in the persistent stimulation of a programmed response that is mediated, at least in part, by synaptic NMDA receptor activity that initially promotes cell survival and neurite remodelling. However, this response is terminated prior to the onset of clinical symptoms in the infected hippocampus, seemingly pointing to a critical juncture in the disease. Manipulation of these early neuroprotective pathways may redress the balance between degeneration and survival, providing a potential inroad for treatment.
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http://dx.doi.org/10.1371/journal.ppat.1003002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493483PMC
April 2013

Enhancement of in vitro and in vivo microdialysis recovery of SB-265123 using Intralipid and Encapsin as perfusates.

Biopharm Drug Dispos 2003 Jan;24(1):17-25

Preclinical Drug Discovery, Cardiovascular & Urogenitary Centre of Excellence in Drug Discovery, GlaxoSmithKline, King of Prussia, PA 19406, USA.

This study was conducted to compare the ability of two potential microdialysis perfusates to enhance the recovery of SB-265123, a lipophilic, highly protein-bound compound, both in vitro and in vivo. Initial in vitro experiments established that the recovery of SB-265123 by microdialysis using normal saline as a perfusate was poor (1.7%). Different concentrations of Intralipid and Encapsin also were evaluated in an identical in vitro setting, to determine enhancement of recovery. In vitro recovery was enhanced to approximately 24 and 65% with 5 and 20% Intralipid, and to approximately 59 and 62% with 5 and 20% Encapsin, respectively. A rat in vivo study was conducted with 20% Encapsin to confirm the in vitro observations. In the in vivo study, 75-80% recovery of free SB-265123 was achieved using 20% Encapsin as a perfusate. The results from this study indicate that for SB-265123, a lipophilic, highly protein-bound molecule, Encapsin is an efficient recovery enhancer in vitro. The results from this investigation further demonstrate that a recovery enhancer may be useful for in vivo applications, even with a compound that is highly bound to plasma protein.
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http://dx.doi.org/10.1002/bdd.332DOI Listing
January 2003