Publications by authors named "Amol C Shetty"

47 Publications

Comprehensive molecular profiling of UV-induced metastatic melanoma in Nme1/Nme2-deficient mice reveals novel markers of survival in human patients.

Oncogene 2021 Aug 25. Epub 2021 Aug 25.

Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland-Baltimore, Baltimore, MD, USA.

Hepatocyte growth factor-overexpressing mice that harbor a deletion of the Ink4a/p16 locus (HP mice) form melanomas with low metastatic potential in response to UV irradiation. Here we report that these tumors become highly metastatic following hemizygous deletion of the Nme1 and Nme2 metastasis suppressor genes (HPN mice). Whole-genome sequencing of melanomas from HPN mice revealed a striking increase in lung metastatic activity that is associated with missense mutations in eight signature genes (Arhgap35, Atp8b4, Brca1, Ift172, Kif21b, Nckap5, Pcdha2, and Zfp869). RNA-seq analysis of transcriptomes from HP and HPN primary melanomas identified a 32-gene signature (HPN lung metastasis signature) for which decreased expression is strongly associated with lung metastatic potential. Analysis of transcriptome data from The Cancer Genome Atlas revealed expression profiles of these genes that predict improved survival of patients with cutaneous or uveal melanoma. Silencing of three representative HPN lung metastasis signature genes (ARRDC3, NYNRIN, RND3) in human melanoma cells resulted in increased invasive activity, consistent with roles for these genes as mediators of the metastasis suppressor function of NME1 and NME2. In conclusion, our studies have identified a family of genes that mediate suppression of melanoma lung metastasis, and which may serve as prognostic markers and/or therapeutic targets for clinical management of metastatic melanoma.
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http://dx.doi.org/10.1038/s41388-021-01998-wDOI Listing
August 2021

Tissue Damage in Radiation-Induced Oral Mucositis Is Mitigated by IL-17 Receptor Signaling.

Front Immunol 2021 17;12:687627. Epub 2021 Jun 17.

Department of Biological Sciences, University of Toledo, Toledo, OH, United States.

Oral mucositis (OM) is a treatment-limiting adverse side effect of radiation and chemotherapy. Approximately 80% of patients undergoing radiotherapy (RT) for head and neck cancers (HNC) develop OM, representing a major unmet medical condition. Our understanding of the immunopathogenesis of OM is limited, due in part to the surprising paucity of information regarding healing mechanisms in the oral mucosa. RNAseq of oral tissue in a murine model that closely mimics human OM, showed elevated expression of IL-17 and related immune pathways in response to head and neck irradiation (HNI). Strikingly, mice lacking the IL-17 receptor (IL-17RA) exhibited markedly more severe OM. Restoration of the oral mucosa was compromised in mice and components associated with healing, including matrix metalloproteinase 3, 10 and IL-24 were diminished. IL-17 is typically associated with recruitment of neutrophils to mucosal sites following oral infections. Unexpectedly, in OM the absence of IL-17RA resulted in excessive neutrophil recruitment and immunopathology. Instead, neutrophil activation was IL-1R-driven in mice. Blockade of IL-1R and depletion of neutrophils lessened the severity of damage in these mice. Overall, we show IL-17 is protective in OM through multiple mechanisms including restoration of the damaged epithelia and control of the neutrophil response. We also present a clinically relevant murine model of human OM to improve mechanistic understanding and develop rational translational therapeutics.
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http://dx.doi.org/10.3389/fimmu.2021.687627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8248500PMC
June 2021

Best practices on the differential expression analysis of multi-species RNA-seq.

Genome Biol 2021 04 29;22(1):121. Epub 2021 Apr 29.

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.

Advances in transcriptome sequencing allow for simultaneous interrogation of differentially expressed genes from multiple species originating from a single RNA sample, termed dual or multi-species transcriptomics. Compared to single-species differential expression analysis, the design of multi-species differential expression experiments must account for the relative abundances of each organism of interest within the sample, often requiring enrichment methods and yielding differences in total read counts across samples. The analysis of multi-species transcriptomics datasets requires modifications to the alignment, quantification, and downstream analysis steps compared to the single-species analysis pipelines. We describe best practices for multi-species transcriptomics and differential gene expression.
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http://dx.doi.org/10.1186/s13059-021-02337-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082843PMC
April 2021

Determining Aspergillus fumigatus transcription factor expression and function during invasion of the mammalian lung.

PLoS Pathog 2021 03 29;17(3):e1009235. Epub 2021 Mar 29.

Division of Infectious Diseases, Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, CA, United States of America.

To gain a better understanding of the transcriptional response of Aspergillus fumigatus during invasive pulmonary infection, we used a NanoString nCounter to assess the transcript levels of 467 A. fumigatus genes during growth in the lungs of immunosuppressed mice. These genes included ones known to respond to diverse environmental conditions and those encoding most transcription factors in the A. fumigatus genome. We found that invasive growth in vivo induces a unique transcriptional profile as the organism responds to nutrient limitation and attack by host phagocytes. This in vivo transcriptional response is largely mimicked by in vitro growth in Aspergillus minimal medium that is deficient in nitrogen, iron, and/or zinc. From the transcriptional profiling data, we selected 9 transcription factor genes that were either highly expressed or strongly up-regulated during in vivo growth. Deletion mutants were constructed for each of these genes and assessed for virulence in mice. Two transcription factor genes were found to be required for maximal virulence. One was rlmA, which is required for the organism to achieve maximal fungal burden in the lung. The other was sltA, which regulates of the expression of multiple secondary metabolite gene clusters and mycotoxin genes independently of laeA. Using deletion and overexpression mutants, we determined that the attenuated virulence of the ΔsltA mutant is due in part to decreased expression aspf1, which specifies a ribotoxin, but is not mediated by reduced expression of the fumigaclavine gene cluster or the fumagillin-pseruotin supercluster. Thus, in vivo transcriptional profiling focused on transcription factors genes provides a facile approach to identifying novel virulence regulators.
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http://dx.doi.org/10.1371/journal.ppat.1009235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8031882PMC
March 2021

Therapeutic implications of mixed biofilm in a murine subcutaneous catheter model of polymicrobial infection.

Virulence 2021 12;12(1):835-851

Department of Oncology and Diagnostic Sciences, School of Dentistry, University of Maryland, Baltimore, MD, USA.

Biofilm-associated polymicrobial infections tend to be challenging to treat. and are leading pathogens due to their ability to form biofilms on medical devices. However, the therapeutic implications of their interactions in a host is largely unexplored. In this study, we used a mouse subcutaneous catheter model for -grown polymicrobial biofilms to validate our in vitro findings on -mediated enhanced S. aureus tolerance to vancomycin . Comparative assessment of recovery from catheters with single- or mixed-species infection demonstrated failure of vancomycin against in mice with co-infected catheters. To provide some mechanistic insights, RNA-seq analysis was performed on catheter biofilms to delineate transcriptional modulations during polymicrobial infections. induced the activation of the biofilm formation network down-regulation of the lrg operon, repressor of autolysis, and up-regulation of the ica operon and production of polysaccharide intercellular adhesin (PIA), indicating an increase in eDNA production, and extracellular polysaccharide matrix, respectively. Interestingly, virulence factors important for disseminated infections, and superantigen-like proteins were down-regulated during mixed-species infection, whereas capsular polysaccharide genes were up-regulated, signifying a strategy favoring survival, persistence and host immune evasion. follow-up experiments using DNA enzymatic digestion, lrg operon mutant strains, and confocal scanning microscopy confirmed the role of -mediated enhanced eDNA production in mixed-biofilms on tolerance to vancomycin. Combined, these findings provide mechanistic insights into the therapeutic implications of interspecies interactions, underscoring the need for novel strategies to overcome limitations of current therapies.
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http://dx.doi.org/10.1080/21505594.2021.1894834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946022PMC
December 2021

Sequencing of 53,831 diverse genomes from the NHLBI TOPMed Program.

Nature 2021 02 10;590(7845):290-299. Epub 2021 Feb 10.

The Broad Institute of MIT and Harvard, Cambridge, MA, USA.

The Trans-Omics for Precision Medicine (TOPMed) programme seeks to elucidate the genetic architecture and biology of heart, lung, blood and sleep disorders, with the ultimate goal of improving diagnosis, treatment and prevention of these diseases. The initial phases of the programme focused on whole-genome sequencing of individuals with rich phenotypic data and diverse backgrounds. Here we describe the TOPMed goals and design as well as the available resources and early insights obtained from the sequence data. The resources include a variant browser, a genotype imputation server, and genomic and phenotypic data that are available through dbGaP (Database of Genotypes and Phenotypes). In the first 53,831 TOPMed samples, we detected more than 400 million single-nucleotide and insertion or deletion variants after alignment with the reference genome. Additional previously undescribed variants were detected through assembly of unmapped reads and customized analysis in highly variable loci. Among the more than 400 million detected variants, 97% have frequencies of less than 1% and 46% are singletons that are present in only one individual (53% among unrelated individuals). These rare variants provide insights into mutational processes and recent human evolutionary history. The extensive catalogue of genetic variation in TOPMed studies provides unique opportunities for exploring the contributions of rare and noncoding sequence variants to phenotypic variation. Furthermore, combining TOPMed haplotypes with modern imputation methods improves the power and reach of genome-wide association studies to include variants down to a frequency of approximately 0.01%.
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http://dx.doi.org/10.1038/s41586-021-03205-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875770PMC
February 2021

FADU: a Quantification Tool for Prokaryotic Transcriptomic Analyses.

mSystems 2021 Jan 12;6(1). Epub 2021 Jan 12.

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA

Quantification tools for RNA sequencing (RNA-Seq) analyses are often designed and tested using human transcriptomics data sets, in which full-length transcript sequences are well annotated. For prokaryotic transcriptomics experiments, full-length transcript sequences are seldom known, and coding sequences must instead be used for quantification steps in RNA-Seq analyses. However, operons confound accurate quantification of coding sequences since a single transcript does not necessarily equate to a single gene. Here, we introduce FADU (Feature Aggregate Depth Utility), a quantification tool designed specifically for prokaryotic RNA-Seq analyses. FADU assigns partial count values proportional to the length of the fragment overlapping the target feature. To assess the ability of FADU to quantify genes in prokaryotic transcriptomics analyses, we compared its performance to those of eXpress, featureCounts, HTSeq, kallisto, and Salmon across three paired-end read data sets of (i) , (ii) , and (iii) the endosymbiont Bm. Across each of the three data sets, we find that FADU can more accurately quantify operonic genes by deriving proportional counts for multigene fragments within operons. FADU is available at https://github.com/IGS/FADU Most currently available quantification tools for transcriptomics analyses have been designed for human data sets, in which full-length transcript sequences, including the untranslated regions, are well annotated. In most prokaryotic systems, full-length transcript sequences have yet to be characterized, leading to prokaryotic transcriptomics analyses being performed based on only the coding sequences. In contrast to eukaryotes, prokaryotes contain polycistronic transcripts, and when genes are quantified based on coding sequences instead of transcript sequences, this leads to an increased abundance of improperly assigned ambiguous multigene fragments, specifically those mapping to multiple genes in operons. Here, we describe FADU, a quantification tool for prokaryotic RNA-Seq analyses designed to assign proportional counts with the purpose of better quantifying operonic genes while minimizing the pitfalls associated with improperly assigning fragment counts from ambiguous transcripts.
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http://dx.doi.org/10.1128/mSystems.00917-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901478PMC
January 2021

Venetoclax and pegcrisantaspase for complex karyotype acute myeloid leukemia.

Leukemia 2021 07 16;35(7):1907-1924. Epub 2020 Nov 16.

University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, MD, USA.

Complex karyotype acute myeloid leukemia (CK-AML) has a dismal outcome with current treatments, underscoring the need for new therapies. Here, we report synergistic anti-leukemic activity of the BCL-2 inhibitor venetoclax (Ven) and the asparaginase formulation Pegylated Crisantaspase (PegC) in CK-AML in vitro and in vivo. Ven-PegC combination inhibited growth of multiple AML cell lines and patient-derived primary CK-AML cells in vitro. In vivo, Ven-PegC showed potent reduction of leukemia burden and improved survival, compared with each agent alone, in a primary patient-derived CK-AML xenograft. Superiority of Ven-PegC, compared to single drugs, and, importantly, the clinically utilized Ven-azacitidine combination, was also demonstrated in vivo in CK-AML. We hypothesized that PegC-mediated plasma glutamine depletion inhibits 4EBP1 phosphorylation, decreases the expression of proteins such as MCL-1, whose translation is cap dependent, synergizing with the BCL-2 inhibitor Ven. Ven-PegC treatment decreased cellular MCL-1 protein levels in vitro by enhancing eIF4E-4EBP1 interaction on the cap-binding complex via glutamine depletion. In vivo, Ven-PegC treatment completely depleted plasma glutamine and asparagine and inhibited mRNA translation and cellular protein synthesis. Since this novel mechanistically-rationalized regimen combines two drugs already in use in acute leukemia treatment, we plan a clinical trial of the Ven-PegC combination in relapsed/refractory CK-AML.
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http://dx.doi.org/10.1038/s41375-020-01080-6DOI Listing
July 2021

Repeated sampling facilitates within- and between-subject modeling of the human sperm transcriptome to identify dynamic and stress-responsive sncRNAs.

Sci Rep 2020 10 15;10(1):17498. Epub 2020 Oct 15.

Department of Pharmacology and Center for Epigenetic Research in Child Health and Brain Development, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.

Epidemiological studies from the last century have drawn strong associations between paternal life experiences and offspring health and disease outcomes. Recent studies have demonstrated sperm small non-coding RNA (sncRNA) populations vary in response to diverse paternal insults. However, for studies in retrospective or prospective human cohorts to identify changes in paternal germ cell epigenetics in association with offspring disease risk, a framework must first be built with insight into the expected biological variation inherent in human populations. In other words, how will we know what to look for if we don't first know what is stable and what is dynamic, and what is consistent within and between men over time? From sperm samples from a 'normative' cohort of healthy human subjects collected repeatedly from each subject over 6 months, 17 healthy male participants met inclusion criteria and completed donations and psychological evaluations of perceived stress monthly. sncRNAs (including miRNA, piRNA, and tRNA) isolated from mature sperm from these samples were subjected to Illumina small RNA sequencing, aligned to subtype-specific reference transcriptomes, and quantified. The repeated measures design allowed us to define both within- and between-subject variation in the expression of 254 miRNA, 194 tRNA, and 937 piRNA in sperm over time. We developed screening criteria to identify a subset of potential environmentally responsive 'dynamic' sperm sncRNA. Implementing complex modeling of the relationships between individual dynamic sncRNA and perceived stress states in these data, we identified 5 miRNA (including let-7f-5p and miR-181a-5p) and 4 tRNA that are responsive to the dynamics of prior stress experience and fit our established mouse model. In the current study, we aligned repeated sampling of human sperm sncRNA expression data with concurrent measures of perceived stress as a novel framework that can now be applied across a range of studies focused on diverse environmental factors able to influence germ cell programming and potentially impact offspring development.
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http://dx.doi.org/10.1038/s41598-020-73867-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7562703PMC
October 2020

Tobacco Hornworm () caterpillars as a novel host model for the study of fungal virulence and drug efficacy.

Virulence 2020 12;11(1):1075-1089

Department of Biology & Biochemistry, University of Bath , Bath, UK.

The two leading yeast pathogens of humans, and , cause systemic infections in >1.4 million patients worldwide with mortality rates approaching 75%. It is thus imperative to study fungal virulence mechanisms, efficacy of antifungal drugs, and host response pathways. While this is commonly done in mammalian models, which are afflicted by ethical and practical concerns, invertebrate models, such as wax moth larvae and nematodes have been introduced over the last two decades. To complement existing invertebrate host models, we developed fifth instar caterpillars of the Tobacco Hornworm moth as a novel host model. These caterpillars can be maintained at 37°C, are suitable for injections with defined amounts of yeast cells, and are susceptible to the most threatening yeast pathogens, including , and . Importantly, fungal burden can be assessed daily throughout the course of infection in a single caterpillar's feces and hemolymph. Infected caterpillars can be rescued by treatment with antifungal drugs. Notably, these animals are large enough for weight to provide a reliable and reproducible measure of fungal disease and to facilitate host tissue-specific expression analyses. caterpillars combine a suite of parameters that make them suitable for the study of fungal virulence.
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http://dx.doi.org/10.1080/21505594.2020.1806665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549948PMC
December 2020

Monocyte Subsets With High Osteoclastogenic Potential and Their Epigenetic Regulation Orchestrated by IRF8.

J Bone Miner Res 2021 01 11;36(1):199-214. Epub 2020 Sep 11.

Division of Periodontology, University of Maryland School of Dentistry, Baltimore, MD, USA.

Osteoclasts (OCs) are bone-resorbing cells formed by the serial fusion of monocytes. In mice and humans, three distinct subsets of monocytes exist; however, it is unclear if all of them exhibit osteoclastogenic potential. Here we show that in wild-type (WT) mice, Ly6C and Ly6C monocytes are the primary source of OC formation when compared to Ly6C monocytes. Their osteoclastogenic potential is dictated by increased expression of signaling receptors and activation of preestablished transcripts, as well as de novo gain in enhancer activity and promoter changes. In the absence of interferon regulatory factor 8 (IRF8), a transcription factor important for myelopoiesis and osteoclastogenesis, all three monocyte subsets are programmed to display higher osteoclastogenic potential. Enhanced NFATc1 nuclear translocation and amplified transcriptomic and epigenetic changes initiated at early developmental stages direct the increased osteoclastogenesis in Irf8-deficient mice. Collectively, our study provides novel insights into the transcription factors and active cis-regulatory elements that regulate OC differentiation. © 2020 American Society for Bone and Mineral Research (ASBMR).
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http://dx.doi.org/10.1002/jbmr.4165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8168257PMC
January 2021

Tornadic Shear Stress Induces a Transient, Calcineurin-Dependent Hypervirulent Phenotype in Mucorales Molds.

mBio 2020 06 30;11(3). Epub 2020 Jun 30.

Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA

Trauma-related necrotizing myocutaneous mucormycosis (NMM) has a high morbidity and mortality in victims of combat-related injuries, geometeorological disasters, and severe burns. Inspired by the observation that several recent clusters of NMM have been associated with extreme mechanical forces (e.g., during tornados), we studied the impact of mechanical stress on Mucoralean biology and virulence in a infection model. In contrast to other experimental procedures to exert mechanical stress, tornadic shear challenge (TSC) by magnetic stirring induced a hypervirulent phenotype in several clinically relevant Mucorales species but not in or Whereas fungal growth rates, morphogenesis, and susceptibility to noxious environments or phagocytes were not altered by TSC, soluble factors released in the supernatant of shear-challenged spores rendered static spores hypervirulent. Consistent with a rapid decay of TSC-induced hypervirulence, minimal transcriptional changes were revealed by comparative RNA sequencing analysis of static and shear-challenged However, inhibition of the calcineurin/heat shock protein 90 (hsp90) stress response circuitry by cyclosporine and tanespimycin abrogated the increased pathogenicity of spores following TSC. Similarly, calcineurin loss-of-function mutants of displayed no increased virulence capacity in flies after undergoing TSC. Collectively, these results establish that TSC induces hypervirulence specifically in Mucorales and point out the calcineurin/hsp90 pathway as a key orchestrator of this phenotype. Our findings invite future studies of topical calcineurin inhibitor treatment of wounds as an adjunct mitigation strategy for NMM following high-energy trauma. Given the limited efficacy of current medical treatments in trauma-related necrotizing mucormycosis, there is a dire need to better understand the Mucoralean pathophysiology in order to develop novel strategies to counteract fungal tissue invasion following severe trauma. Here, we describe that tornadic shear stress challenge transiently induces a hypervirulent phenotype in various pathogenic Mucorales species but not in other molds known to cause wound infections. Pharmacological and genetic inhibition of calcineurin signaling abrogated hypervirulence in shear stress-challenged Mucorales, encouraging further evaluation of (topical) calcineurin inhibitors to improve therapeutic outcomes of NMM after combat-related blast injuries or violent storms.
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http://dx.doi.org/10.1128/mBio.01414-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327176PMC
June 2020

Oral epithelial IL-22/STAT3 signaling licenses IL-17-mediated immunity to oral mucosal candidiasis.

Sci Immunol 2020 06;5(48)

Division of Rheumatology and Clinical Immunology, University of Pittsburgh, Pittsburgh, PA, USA.

Oropharyngeal candidiasis (OPC; thrush) is an opportunistic infection caused by the commensal fungus Interleukin-17 (IL-17) and IL-22 are cytokines produced by type 17 lymphocytes. Both cytokines mediate antifungal immunity yet activate quite distinct downstream signaling pathways. While much is now understood about how IL-17 promotes immunity in OPC, the activities of IL-22 are far less well delineated. We show that, despite having similar requirements for induction from type 17 cells, IL-22 and IL-17 function nonredundantly during OPC. We find that the IL-22 and IL-17 receptors are required in anatomically distinct locations within the oral mucosa; loss of IL-22RA1 or signal transducer and activator of transcription 3 (STAT3) in the oral basal epithelial layer (BEL) causes susceptibility to OPC, whereas IL-17RA is needed in the suprabasal epithelial layer (SEL). Transcriptional profiling of the tongue linked IL-22/STAT3 not only to oral epithelial cell proliferation and survival but also, unexpectedly, to driving an IL-17-specific gene signature. We show that IL-22 mediates regenerative signals on the BEL that replenish the IL-17RA-expressing SEL, thereby restoring the ability of the oral epithelium to respond to IL-17 and thus to mediate antifungal events. Consequently, IL-22 signaling in BEL "licenses" IL-17 signaling in the oral mucosa, revealing spatially distinct yet cooperative activities of IL-22 and IL-17 in oral candidiasis.
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http://dx.doi.org/10.1126/sciimmunol.aba0570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340112PMC
June 2020

Evolutionary history of modern Samoans.

Proc Natl Acad Sci U S A 2020 04 14;117(17):9458-9465. Epub 2020 Apr 14.

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201;

Archaeological studies estimate the initial settlement of Samoa at 2,750 to 2,880 y ago and identify only limited settlement and human modification to the landscape until about 1,000 to 1,500 y ago. At this point, a complex history of migration is thought to have begun with the arrival of people sharing ancestry with Near Oceanic groups (i.e., Austronesian-speaking and Papuan-speaking groups), and was then followed by the arrival of non-Oceanic groups during European colonialism. However, the specifics of this peopling are not entirely clear from the archaeological and anthropological records, and is therefore a focus of continued debate. To shed additional light on the Samoan population history that this peopling reflects, we employ a population genetic approach to analyze 1,197 Samoan high-coverage whole genomes. We identify population splits between the major Samoan islands and detect asymmetrical gene flow to the capital city. We also find an extreme bottleneck until about 1,000 y ago, which is followed by distinct expansions across the islands and subsequent bottlenecks consistent with European colonization. These results provide for an increased understanding of Samoan population history and the dynamics that inform it, and also demonstrate how rapid demographic processes can shape modern genomes.
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http://dx.doi.org/10.1073/pnas.1913157117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196816PMC
April 2020

Detecting geospatial patterns of Plasmodium falciparum parasite migration in Cambodia using optimized estimated effective migration surfaces.

Int J Health Geogr 2020 04 10;19(1):13. Epub 2020 Apr 10.

Center for Geospatial Information Science, Department of Geographical Sciences, University of Maryland, College Park, 20742, MD, USA.

Background: Understanding the genetic structure of natural populations provides insight into the demographic and adaptive processes that have affected those populations. Such information, particularly when integrated with geospatial data, can have translational applications for a variety of fields, including public health. Estimated effective migration surfaces (EEMS) is an approach that allows visualization of the spatial patterns in genomic data to understand population structure and migration. In this study, we developed a workflow to optimize the resolution of spatial grids used to generate EEMS migration maps and applied this optimized workflow to estimate migration of Plasmodium falciparum in Cambodia and bordering regions of Thailand and Vietnam.

Methods: The optimal density of EEMS grids was determined based on a new workflow created using density clustering to define genomic clusters and the spatial distance between genomic clusters. Topological skeletons were used to capture the spatial distribution for each genomic cluster and to determine the EEMS grid density; i.e., both genomic and spatial clustering were used to guide the optimization of EEMS grids. Model accuracy for migration estimates using the optimized workflow was tested and compared to grid resolutions selected without the optimized workflow. As a test case, the optimized workflow was applied to genomic data generated from P. falciparum sampled in Cambodia and bordering regions, and migration maps were compared to estimates of malaria endemicity, as well as geographic properties of the study area, as a means of validating observed migration patterns.

Results: Optimized grids displayed both high model accuracy and reduced computing time compared to grid densities selected in an unguided manner. In addition, EEMS migration maps generated for P. falciparum using the optimized grid corresponded to estimates of malaria endemicity and geographic properties of the study region that might be expected to impact malaria parasite migration, supporting the validity of the observed migration patterns.

Conclusions: Optimized grids reduce spatial uncertainty in the EEMS contours that can result from user-defined parameters, such as the resolution of the spatial grid used in the model. This workflow will be useful to a broad range of EEMS users as it can be applied to analyses involving other organisms of interest and geographic areas.
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http://dx.doi.org/10.1186/s12942-020-00207-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149848PMC
April 2020

Cost effective, experimentally robust differential-expression analysis for human/mammalian, pathogen and dual-species transcriptomics.

Microb Genom 2020 01;6(1)

Institute for Genome Sciences, School of Medicine, University of Maryland, Baltimore, MD 21201, USA.

As sequencing read length has increased, researchers have quickly adopted longer reads for their experiments. Here, we examine 14 pathogen or host-pathogen differential gene expression data sets to assess whether using longer reads is warranted. A variety of data sets was used to assess what genomic attributes might affect the outcome of differential gene expression analysis including: gene density, operons, gene length, number of introns/exons and intron length. No genome attribute was found to influence the data in principal components analysis, hierarchical clustering with bootstrap support, or regression analyses of pairwise comparisons that were undertaken on the same reads, looking at all combinations of paired and unpaired reads trimmed to 36, 54, 72 and 101 bp. Read pairing had the greatest effect when there was little variation in the samples from different conditions or in their replicates (e.g. little differential gene expression). But overall, 54 and 72 bp reads were typically most similar. Given differences in costs and mapping percentages, we recommend 54 bp reads for organisms with no or few introns and 72 bp reads for all others. In a third of the data sets, read pairing had absolutely no effect, despite paired reads having twice as much data. Therefore, single-end reads seem robust for differential-expression analyses, but in eukaryotes paired-end reads are likely desired to analyse splice variants and should be preferred for data sets that are acquired with the intent to be community resources that might be used in secondary data analyses.
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http://dx.doi.org/10.1099/mgen.0.000320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067034PMC
January 2020

quorum-sensing molecule farnesol modulates staphyloxanthin production and activates the thiol-based oxidative-stress response in .

Virulence 2019 12;10(1):625-642

a Department of Oncology and Diagnostic Sciences, Dental School , University of Maryland , Baltimore , MD , USA.

Microbial species utilize secreted-signaling molecules to coordinate their behavior. Our previous investigations demonstrated a key role for the -secreted quorum-sensing molecule farnesol in modulating response to antimicrobials in mixed biofilms. In this study, we aimed to provide mechanistic insights into the impact of farnesol on within the context of inter-species interactions. To mimic biofilm dynamics, farnesol-sensitized cells were generated sequential farnesol exposure. The sensitized phenotype exhibited dramatic loss of the typical pigment, which we identified as staphyloxanthin, an important virulence factor synthesized by the Crt operon in . Additionally, farnesol exposure exerted oxidative-stress as indicated by transcriptional analysis demonstrating alterations in redox-sensors and major virulence regulators. Paradoxically, the activated stress-response conferred with enhanced tolerance to HO and phagocytic killing. Since expression of enzymes in the staphyloxanthin biosynthesis pathway was not impacted by farnesol, we generated a theoretical-binding model which indicated that farnesol may block staphyloxanthin biosynthesis competitive-binding to the CrtM enzyme crucial for staphyloxanthin synthesis, due to high structural similarity to the CrtM substrate. Finally, mixed growth with was found to similarly induce depigmentation, but not during growth with a farnesol-deficient strain. Collectively, the findings demonstrate that a fungal molecule acts as a redox-cycler eliciting a bacterial stress response activation of the thiol-based redox system under the control of global regulators. Therefore, farnesol-induced transcriptional modulations of key regulatory networks in may modulate the pathogenesis of co-infections.
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http://dx.doi.org/10.1080/21505594.2019.1635418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629188PMC
December 2019

Genomic structure and diversity of Plasmodium falciparum in Southeast Asia reveal recent parasite migration patterns.

Nat Commun 2019 06 17;10(1):2665. Epub 2019 Jun 17.

Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.

Estimates of Plasmodium falciparum migration may inform strategies for malaria elimination. Here we elucidate fine-scale parasite population structure and infer recent migration across Southeast Asia using identity-by-descent (IBD) approaches based on genome-wide single nucleotide polymorphisms called in 1722 samples from 54 districts. IBD estimates are consistent with isolation-by-distance. We observe greater sharing of larger IBD segments between artemisinin-resistant parasites versus sensitive parasites, which is consistent with the recent spread of drug resistance. Our IBD analyses reveal actionable patterns, including isolated parasite populations, which may be prioritized for malaria elimination, as well as asymmetrical migration identifying potential sources and sinks of migrating parasites.
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http://dx.doi.org/10.1038/s41467-019-10121-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6572796PMC
June 2019

Whole blood transcriptomic profiles can differentiate vulnerability to chronic low back pain.

PLoS One 2019 16;14(5):e0216539. Epub 2019 May 16.

Center for Advancement in Managing Pain, University of Connecticut School of Nursing, Storrs, Connecticut, United States of America.

The mechanisms underlying the transition from acute to chronic pain remain unclear. Here, we sought to characterize the transcriptome associated with chronic low back pain as well as the transcriptome of the transition from acute to chronic low back pain. For the analysis, we compared the whole blood transcriptome of: (a) patients at the onset of low back pain who no longer had pain within 6 weeks after onset (acute) with patients who developed chronic low back pain at 6 months (chronic T5); and, (b) patients at the onset of low back pain (chronic T1) who developed chronic pain at 6 months with healthy pain-free (normal) controls. The majority of differentially expressed genes were protein coding. We illustrate a unique chronic low back pain transcriptome characterized by significant enrichment for known pain genes, extracellular matrix genes, and genes from the extended major histocompatibility complex (MHC) genomic locus. The transcriptome of the transition from acute to chronic low back pain was characterized by significant upregulation of antigen presentation pathway (MHC class I and II) genes and downregulation of mitochondrial genes associated with oxidative phosphorylation, suggesting a unique genomic signature of vulnerability to low back pain chronicity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0216539PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522025PMC
January 2020

Author Correction: Iron restriction inside macrophages regulates pulmonary host defense against Rhizopus species.

Nat Commun 2018 11 22;9(1):5015. Epub 2018 Nov 22.

Department of Medicine, University of Crete, Foundation for Research and Technology, 71300, Heraklion, Crete, Greece.

The original version of this Article contained an error in the spelling of the author Emilien Etienne, which was incorrectly given as Emilien Ettiene. These errors have now been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-018-07301-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6250689PMC
November 2018

Iron restriction inside macrophages regulates pulmonary host defense against Rhizopus species.

Nat Commun 2018 08 20;9(1):3333. Epub 2018 Aug 20.

Department of Medicine, University of Crete, Foundation for Research and Technology, 71300, Heraklion, Crete, Greece.

Mucormycosis is a life-threatening respiratory fungal infection predominantly caused by Rhizopus species. Mucormycosis has incompletely understood pathogenesis, particularly how abnormalities in iron metabolism compromise immune responses. Here we show how, as opposed to other filamentous fungi, Rhizopus spp. establish intracellular persistence inside alveolar macrophages (AMs). Mechanistically, lack of intracellular swelling of Rhizopus conidia results in surface retention of melanin, which induces phagosome maturation arrest through inhibition of LC3-associated phagocytosis. Intracellular inhibition of Rhizopus is an important effector mechanism, as infection of immunocompetent mice with swollen conidia, which evade phagocytosis, results in acute lethality. Concordantly, AM depletion markedly increases susceptibility to mucormycosis. Host and pathogen transcriptomics, iron supplementation studies, and genetic manipulation of iron assimilation of fungal pathways demonstrate that iron restriction inside macrophages regulates immunity against Rhizopus. Our findings shed light on the pathogenetic mechanisms of mucormycosis and reveal the role of macrophage-mediated nutritional immunity against filamentous fungi.
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http://dx.doi.org/10.1038/s41467-018-05820-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102248PMC
August 2018

Inhibition of EGFR Signaling Protects from Mucormycosis.

mBio 2018 08 14;9(4). Epub 2018 Aug 14.

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA

Mucormycosis is a life-threatening, invasive fungal infection that is caused by various species belonging to the order Mucorales. species are the most common cause of the disease, responsible for approximately 70% of all cases of mucormycosis. During pulmonary mucormycosis, inhaled spores must adhere to and invade airway epithelial cells in order to establish infection. The molecular mechanisms that govern this interaction are poorly understood. We performed an unbiased survey of the host transcriptional response during early stages of var. () infection in a murine model of pulmonary mucormycosis using transcriptome sequencing (RNA-seq). Network analysis revealed activation of the host's epidermal growth factor receptor (EGFR) signaling. Consistent with the RNA-seq results, EGFR became phosphorylated upon infection of human alveolar epithelial cells with several members of the Mucorales, and this phosphorylated, activated form of EGFR colocalized with spores. Inhibition of EGFR signaling with cetuximab or gefitinib, specific FDA-approved inhibitors of EGFR, significantly reduced the ability of to invade and damage airway epithelial cells. Furthermore, gefitinib treatment significantly prolonged survival of mice with pulmonary mucormycosis, reduced tissue fungal burden, and attenuated the activation of EGFR in response to pulmonary mucormycosis. These results indicate EGFR represents a novel host target to block invasion of alveolar epithelial cells by , and inhibition of EGFR signaling provides a novel approach for treating mucormycosis by repurposing an FDA-approved drug. Mucormycosis is an increasingly common, highly lethal fungal infection with very limited treatment options. Using a combination of animal models, transcriptomics, cell biology, and pharmacological approaches, we have demonstrated that Mucorales fungi activate EGFR signaling to induce fungal uptake into airway epithelial cells. Inhibition of EGFR signaling with existing FDA-approved drugs significantly increased survival following var. infection in mice. This study enhances our understanding of how Mucorales fungi invade host cells during the establishment of pulmonary mucormycosis and provides a proof-of-concept for the repurposing of FDA-approved drugs that target EGFR function.
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http://dx.doi.org/10.1128/mBio.01384-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094478PMC
August 2018

Evolutionary genomic dynamics of Peruvians before, during, and after the Inca Empire.

Proc Natl Acad Sci U S A 2018 07 26;115(28):E6526-E6535. Epub 2018 Jun 26.

Laboratorio de Biotecnología y Biología Molecular, Instituto Nacional de Salud, Lima 11, Perú;

Native Americans from the Amazon, Andes, and coastal geographic regions of South America have a rich cultural heritage but are genetically understudied, therefore leading to gaps in our knowledge of their genomic architecture and demographic history. In this study, we sequence 150 genomes to high coverage combined with an additional 130 genotype array samples from Native American and mestizo populations in Peru. The majority of our samples possess greater than 90% Native American ancestry, which makes this the most extensive Native American sequencing project to date. Demographic modeling reveals that the peopling of Peru began ∼12,000 y ago, consistent with the hypothesis of the rapid peopling of the Americas and Peruvian archeological data. We find that the Native American populations possess distinct ancestral divisions, whereas the mestizo groups were admixtures of multiple Native American communities that occurred before and during the Inca Empire and Spanish rule. In addition, the mestizo communities also show Spanish introgression largely following Peruvian Independence, nearly 300 y after Spain conquered Peru. Further, we estimate migration events between Peruvian populations from all three geographic regions with the majority of between-region migration moving from the high Andes to the low-altitude Amazon and coast. As such, we present a detailed model of the evolutionary dynamics which impacted the genomes of modern-day Peruvians and a Native American ancestry dataset that will serve as a beneficial resource to addressing the underrepresentation of Native American ancestry in sequencing studies.
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http://dx.doi.org/10.1073/pnas.1720798115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048481PMC
July 2018

Alternative Splicing of Differentiated Myeloid Cell Transcripts after Infection by Impacts a Selective Group of Cellular Programs.

Front Cell Infect Microbiol 2018 2;8:14. Epub 2018 Feb 2.

Institute for Genome Sciences, University of Maryland, Baltimore, Baltimore, MD, United States.

Eukaryotic proteome diversity exceeds that encoded within individual genes, and results in part from alternative splicing events of pre-messenger RNA. The diversity of these splicing events can shape the outcome in development and differentiation of normal tissues, and is important in pathogenic circumstances such as cancer and some heritable conditions. A role for alternative splicing of eukaryotic genes in response to viral and intracellular bacterial infections has only recently been recognized, and plays an important role in providing fitness for microbial survival, while potentially enhancing pathogenicity. survives within mammalian neutrophils by reshaping transcriptional programs that govern cellular functions. We applied next generation RNAseq to ATRA-differentiated HL-60 cells established to possess transcriptional and functional responses similar to -infected human neutrophils. This demonstrated an increase in transcripts with infection and high proportion of alternatively spliced transcript events (ASEs) for which predicted gene ontology processes were in part distinct from those identified by evaluation of single transcripts or gene-level analyses alone. The alternative isoforms are not on average shorter, and no alternative splicing in genes encoding spliceosome components is noted. Although not evident at gene-level analyses, individual spliceosome transcripts that impact nearly all spliceosome components were significantly upregulated. How the distinct GO processes predicted by ASEs are regulated by infection and whether they are relevant to fitness or pathogenicity of should be addressed in more detailed studies.
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http://dx.doi.org/10.3389/fcimb.2018.00014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5801399PMC
January 2019

Cross-species transcriptional analysis reveals conserved and host-specific neoplastic processes in mammalian glioma.

Sci Rep 2018 01 19;8(1):1180. Epub 2018 Jan 19.

Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, Maryland, USA.

Glioma is a unique neoplastic disease that develops exclusively in the central nervous system (CNS) and rarely metastasizes to other tissues. This feature strongly implicates the tumor-host CNS microenvironment in gliomagenesis and tumor progression. We investigated the differences and similarities in glioma biology as conveyed by transcriptomic patterns across four mammalian hosts: rats, mice, dogs, and humans. Given the inherent intra-tumoral molecular heterogeneity of human glioma, we focused this study on tumors with upregulation of the platelet-derived growth factor signaling axis, a common and early alteration in human gliomagenesis. The results reveal core neoplastic alterations in mammalian glioma, as well as unique contributions of the tumor host to neoplastic processes. Notable differences were observed in gene expression patterns as well as related biological pathways and cell populations known to mediate key elements of glioma biology, including angiogenesis, immune evasion, and brain invasion. These data provide new insights regarding mammalian models of human glioma, and how these insights and models relate to our current understanding of the human disease.
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http://dx.doi.org/10.1038/s41598-018-19451-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775420PMC
January 2018

Aligner optimization increases accuracy and decreases compute times in multi-species sequence data.

Microb Genom 2017 09 8;3(9):e000122. Epub 2017 Jul 8.

2Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA.

As sequencing technologies have evolved, the tools to analyze these sequences have made similar advances. However, for multi-species samples, we observed important and adverse differences in alignment specificity and computation time for bwa- mem (Burrows-Wheeler aligner-maximum exact matches) relative to bwa-aln. Therefore, we sought to optimize bwa-mem for alignment of data from multi-species samples in order to reduce alignment time and increase the specificity of alignments. In the multi-species cases examined, there was one majority member (i.e. or ) and one minority member (i.e. human or the endosymbiont Bm) of the sequence data. Increasing bwa-mem seed length from the default value reduced the number of read pairs from the majority sequence member that incorrectly aligned to the reference genome of the minority sequence member. Combining both source genomes into a single reference genome increased the specificity of mapping, while also reducing the central processing unit (CPU) time. In , at a seed length of 18 nt, 24.1 % of reads mapped to the human genome using 1.7±0.1 CPU hours, while 83.6 % of reads mapped to the genome using 0.2±0.0 CPU hours (total: 107.7 % reads mapping; in 1.9±0.1 CPU hours). In contrast, 97.1 % of the reads mapped to a combined human reference in only 0.7±0.0 CPU hours. Overall, the results suggest that combining all references into a single reference database and using a 23 nt seed length reduces the computational time, while maximizing specificity. Similar results were found for simulated sequence reads from a mock metagenomic data set. We found similar improvements to computation time in a publicly available human-only data set.
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http://dx.doi.org/10.1099/mgen.0.000122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643015PMC
September 2017

Identification of a gene expression signature associated with the metastasis suppressor function of NME1: prognostic value in human melanoma.

Lab Invest 2018 03 23;98(3):327-338. Epub 2017 Oct 23.

Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland-Baltimore, Baltimore, MD, USA.

Although NME1 is well known for its ability to suppress metastasis of melanoma, the molecular mechanisms underlying this activity are not completely understood. Herein, we utilized a bioinformatics approach to systematically identify genes whose expression is correlated with the metastasis suppressor function of NME1. This was accomplished through a search for genes that were regulated by NME1, but not by NME1 variants lacking metastasis suppressor activity. This approach identified a number of novel genes, such as ALDOC, CXCL11, LRP1b, and XAGE1 as well as known targets such as NETO2, which were collectively designated as an NME1-Regulated Metastasis Suppressor Signature (MSS). The MSS was associated with prolonged overall survival in a large cohort of melanoma patients in The Cancer Genome Atlas (TCGA). The median overall survival of melanoma patients with elevated expression of the MSS genes was >5.6 years longer compared with that of patients with lower expression of the MSS genes. These data demonstrate that NMEl represents a powerful tool for identifying genes whose expression is associated with metastasis and survival of melanoma patients, suggesting their potential applications as prognostic markers and therapeutic targets in advanced forms of this lethal cancer.
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http://dx.doi.org/10.1038/labinvest.2017.108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839922PMC
March 2018

Association of a Novel Mutation in the Plasmodium falciparum Chloroquine Resistance Transporter With Decreased Piperaquine Sensitivity.

J Infect Dis 2017 08;216(4):468-476

Division of Malaria Research, Institute for Global Health.

Background: Amplified copy number in the plasmepsin II/III genes within Plasmodium falciparum has been associated with decreased sensitivity to piperaquine. To examine this association and test whether additional loci might also contribute, we performed a genome-wide association study of ex vivo P. falciparum susceptibility to piperaquine.

Methods: Plasmodium falciparum DNA from 183 samples collected primarily from Cambodia was genotyped at 33716 genome-wide single nucleotide polymorphisms (SNPs). Linear mixed models and random forests were used to estimate associations between parasite genotypes and piperaquine susceptibility. Candidate polymorphisms were evaluated for their association with dihydroartemisinin-piperaquine treatment outcomes in an independent dataset.

Results: Single nucleotide polymorphisms on multiple chromosomes were associated with piperaquine 90% inhibitory concentrations (IC90) in a genome-wide analysis. Fine-mapping of genomic regions implicated in genome-wide analyses identified multiple SNPs in linkage disequilibrium with each other that were significantly associated with piperaquine IC90, including a novel mutation within the gene encoding the P. falciparum chloroquine resistance transporter, PfCRT. This mutation (F145I) was associated with dihydroartemisinin-piperaquine treatment failure after adjusting for the presence of amplified plasmepsin II/III, which was also associated with decreased piperaquine sensitivity.

Conclusions: Our data suggest that, in addition to plasmepsin II/III copy number, other loci, including pfcrt, may also be involved in piperaquine resistance.
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http://dx.doi.org/10.1093/infdis/jix334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5853219PMC
August 2017

Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing.

PLoS Pathog 2017 Aug 25;13(8):e1006582. Epub 2017 Aug 25.

Department of Microbiology, The University of Alabama at Birmingham, Birmingham, AL, United States of America.

For over 130 years, invasive pneumococcal disease has been associated with the presence of extracellular planktonic pneumococci, i.e. diplococci or short chains in affected tissues. Herein, we show that Streptococcus pneumoniae that invade the myocardium instead replicate within cellular vesicles and transition into non-purulent biofilms. Pneumococci within mature cardiac microlesions exhibited salient biofilm features including intrinsic resistance to antibiotic killing and the presence of an extracellular matrix. Dual RNA-seq and subsequent principal component analyses of heart- and blood-isolated pneumococci confirmed the biofilm phenotype in vivo and revealed stark anatomical site-specific differences in virulence gene expression; the latter having major implications on future vaccine antigen selection. Our RNA-seq approach also identified three genomic islands as exclusively expressed in vivo. Deletion of one such island, Region of Diversity 12, resulted in a biofilm-deficient and highly inflammogenic phenotype within the heart; indicating a possible link between the biofilm phenotype and a dampened host-response. We subsequently determined that biofilm pneumococci released greater amounts of the toxin pneumolysin than did planktonic or RD12 deficient pneumococci. This allowed heart-invaded wildtype pneumococci to kill resident cardiac macrophages and subsequently subvert cytokine/chemokine production and neutrophil infiltration into the myocardium. This is the first report for pneumococcal biofilm formation in an invasive disease setting. We show that biofilm pneumococci actively suppress the host response through pneumolysin-mediated immune cell killing. As such, our findings contradict the emerging notion that biofilm pneumococci are passively immunoquiescent.
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http://dx.doi.org/10.1371/journal.ppat.1006582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589263PMC
August 2017

Transcriptional Variation of Diverse Enteropathogenic Isolates under Virulence-Inducing Conditions.

mSystems 2017 Jul-Aug;2(4). Epub 2017 Jul 25.

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA.

Enteropathogenic (EPEC) bacteria are a diverse group of pathogens that cause moderate to severe diarrhea in young children in developing countries. EPEC isolates can be further subclassified as typical EPEC (tEPEC) isolates that contain the bundle-forming pilus (BFP) or as atypical EPEC (aEPEC) isolates that do not contain BFP. Comparative genomics studies have recently highlighted the considerable genomic diversity among EPEC isolates. In the current study, we used RNA sequencing (RNA-Seq) to characterize the global transcriptomes of eight tEPEC isolates representing the identified genomic diversity, as well as one aEPEC isolate. The global transcriptomes were determined for the EPEC isolates under conditions of laboratory growth that are known to induce expression of virulence-associated genes. The findings demonstrate that unique genes of EPEC isolates from diverse phylogenomic lineages contribute to variation in their global transcriptomes. There were also phylogroup-specific differences in the global transcriptomes, including genes involved in iron acquisition, which had significant differential expression in the EPEC isolates belonging to phylogroup B2. Also, three EPEC isolates from the same phylogenomic lineage (EPEC8) had greater levels of similarity in their genomic content and exhibited greater similarities in their global transcriptomes than EPEC from other lineages; however, even among closely related isolates there were isolate-specific differences among their transcriptomes. These findings highlight the transcriptional variability that correlates with the previously unappreciated genomic diversity of EPEC. Recent studies have demonstrated that there is considerable genomic diversity among EPEC isolates; however, it is unknown if this genomic diversity leads to differences in their global transcription. This study used RNA-Seq to compare the global transcriptomes of EPEC isolates from diverse phylogenomic lineages. We demonstrate that there are lineage- and isolate-specific differences in the transcriptomes of genomically diverse EPEC isolates during growth under virulence-inducing conditions. This study addressed biological variation among isolates of a single pathovar in an effort to demonstrate that while each of these isolates is considered an EPEC isolate, there is significant transcriptional diversity among members of this pathovar. Future studies should consider whether this previously undescribed transcriptional variation may play a significant role in isolate-specific variability of EPEC clinical presentations.
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http://dx.doi.org/10.1128/mSystems.00024-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527300PMC
July 2017
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