Publications by authors named "Sharmin Begum"

25 Publications

  • Page 1 of 1

Autophagy is required during high MUC2 mucin biosynthesis in colonic goblet cells to contend metabolic stress.

Am J Physiol Gastrointest Liver Physiol 2021 Nov 8;321(5):G489-G499. Epub 2021 Sep 8.

Department of Microbiology, Immunology and Infectious Diseases, University of Calgary Health Sciences Centre, Calgary, Alberta, Canada.

Goblet cells are specialized for the production and secretion of MUC2 glycoproteins that forms a thick layer covering the mucosal epithelium as a protective barrier against noxious substances and invading microbes. High MUC2 mucin biosynthesis induces endoplasmic reticulum (ER) stress and apoptosis in goblet cells during inflammatory and infectious diseases. Autophagy is an intracellular degradation process required for maintenance of intestinal homeostasis. In this study, we hypothesized that autophagy was triggered during high MUC2 mucin biosynthesis from colonic goblet cells to cope with metabolic stress. To interrogate this, we analyzed the autophagy process in high MUC2-producing human HT29-H and a clone HT29-L silenced for MUC2 expression by lentivirus-mediated shRNA, and WT and CRISPR/Cas9 MUC2 KO LS174T cells. Autophagy was constitutively increased in high MUC2-producing cells characterized by elevated pULK1S555 expression and increased numbers of autophagosomes as compared with MUC2 silenced or gene edited cells. Similarly, colonoids from but not littermates differentiated into goblet cells showed increased autophagy. IL-22 treatment corrected misfolded MUC2 protein and alleviated the autophagy process in LS174T cells. This study highlights that autophagy plays an essential role in goblet cells to survive during high mucin biosynthesis by regulating cellular homeostasis. It is unclear how colonic goblet cells survive by producing high output MUC2 mucin that triggers endoplasmic stress by misfolded MUC2 proteins. To cope with metabolic stress, we interrogated if autophagy played an essential role in regulating cellular homeostasis. Indeed, high MUC2 mucin biosynthesis dysregulated autophagy processes that was regulated by IL-22 to maintain gut barrier innate host defenses.
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http://dx.doi.org/10.1152/ajpgi.00221.2021DOI Listing
November 2021

Entamoeba histolytica exploits the autophagy pathway in macrophages to trigger inflammation in disease pathogenesis.

Mucosal Immunol 2021 09 7;14(5):1038-1054. Epub 2021 May 7.

Departments of Microbiology, Immunology and Infectious Diseases, Calgary, AB, Canada.

The mechanism whereby Entamoeba histolytica (Eh) binding with macrophages at the intercellular junction triggers aggressive pro-inflammatory responses in disease pathogenesis is not well understood. The host intracellular protein degradation process autophagy and its regulatory proteins are involved in maintenance of cellular homeostasis and excessive inflammatory responses. In this study we unraveled how Eh hijacks the autophagy process in macrophages to dysregulate pro-inflammatory responses. Direct contact of live Eh with macrophages activated caspase-6 that induced rapid proteolytic degradation of the autophagy ATG16L1 protein complex independent of NLRP3 inflammasome and caspase-3/8 activation. Crohn's disease susceptible ATG16L1 T300A variant was highly susceptible to Eh-mediated degradation that augmented pro-inflammatory cytokines in mice. Quantitative proteomics revealed downregulation of autophagy and vesicle-mediated transport and upregulation of cysteine-type endopeptidase pathways in response to Eh. We conclude during Eh-macrophage outside-in signaling, ATG16L1 protein complex plays an overlooked regulatory role in shaping the pro-inflammatory landscape in amebiasis.
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http://dx.doi.org/10.1038/s41385-021-00408-4DOI Listing
September 2021

Entamoeba histolytica.

Trends Parasitol 2021 07 28;37(7):676-677. Epub 2021 Jan 28.

Departments of Microbiology, Immunology, and Infectious Diseases, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada. Electronic address:

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http://dx.doi.org/10.1016/j.pt.2021.01.001DOI Listing
July 2021

Role of inflammasomes in innate host defense against Entamoeba histolytica.

J Leukoc Biol 2020 09 4;108(3):801-812. Epub 2020 Jun 4.

Department of Microbiology, Immunology and Infectious Diseases, University of Calgary Health Sciences Centre, Calgary, Alberta, Canada.

Intestinal amebiasis is the disease caused by the extracellular protozoan parasite Entamoeba histolytica (Eh) that induces a dynamic and heterogeneous interaction profile with the host immune system during disease pathogenesis. In 90% of asymptomatic infection, Eh resides with indigenous microbiota in the outer mucus layer of the colon without prompting an immune response. However, for reasons that remain unclear, in a minority of the Eh-infected individuals, this fine tolerated relationship is switched to a pathogenic phenotype and advanced to an increasingly complex host-parasite interaction. Eh disease susceptibility depends on parasite virulence factors and their interactions with indigenous bacteria, disruption of the mucus bilayers, and adherence to the epithelium provoking host immune cells to evoke a robust pro-inflammatory response mediated by inflammatory caspases and inflammasome activation. To understand Eh pathogenicity and innate host immune responses, this review highlights recent advances in our understanding of how Eh induces outside-in signaling via Mϕs to activate inflammatory caspases and inflammasome to regulate pro-inflammatory responses.
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http://dx.doi.org/10.1002/JLB.3MR0420-465RDOI Listing
September 2020

Entamoeba histolytica stimulates the alarmin molecule HMGB1 from macrophages to amplify innate host defenses.

Mucosal Immunol 2020 03 26;13(2):344-356. Epub 2019 Nov 26.

Department of Microbiology, Immunology and Infectious Diseases, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, Canada.

Even though Entamoeba histolytica (Eh)-induced host pro-inflammatory responses play a critical role in disease, we know very little about the host factors that regulate this response. Direct contact between host cell and Eh signify the highest level of danger, and to eliminate this threat, the host immune system elicits an augmented immune response. To understand the mechanisms of this response, we investigated the induction and release of the endogenous alarmin molecule high-mobility group box 1 (HMGB1) that act as a pro-inflammatory cytokine and chemoattractant during Eh infection. Eh in contact with macrophage induced a dose- and time-dependent secretion of HMGB1 in the absence of cell death. Secretion of HMGB1 was facilitated by Eh surface Gal-lectin-activated phosphoinositide 3-kinase and nuclear factor-κB signaling and up-regulation of histone acetyltransferase activity to trigger acetylated HMGB1 translocation from the nucleus. Unlike lipopolysaccharide, Eh-induced HMGB1 release was independent of caspase-1-mediated inflammasome and gasdermin D pores. In vivo, Eh inoculation in specific pathogen-free but not germ-free mice was associated with high levels of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, and keratinocyte-derived chemokine, which was suppressed with HMGB1 neutralization. This study reveals that Eh-induced active secretion of the HMGB1 plays a key role in shaping the pro-inflammatory landscape critical in innate host defense against amebiasis.
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http://dx.doi.org/10.1038/s41385-019-0233-6DOI Listing
March 2020

Deterministic Evolutionary Trajectories Influence Primary Tumor Growth: TRACERx Renal.

Cell 2018 04 12;173(3):595-610.e11. Epub 2018 Apr 12.

Department of Scientific Computing, the Francis Crick Institute, London NW1 1AT, UK.

The evolutionary features of clear-cell renal cell carcinoma (ccRCC) have not been systematically studied to date. We analyzed 1,206 primary tumor regions from 101 patients recruited into the multi-center prospective study, TRACERx Renal. We observe up to 30 driver events per tumor and show that subclonal diversification is associated with known prognostic parameters. By resolving the patterns of driver event ordering, co-occurrence, and mutual exclusivity at clone level, we show the deterministic nature of clonal evolution. ccRCC can be grouped into seven evolutionary subtypes, ranging from tumors characterized by early fixation of multiple mutational and copy number drivers and rapid metastases to highly branched tumors with >10 subclonal drivers and extensive parallel evolution associated with attenuated progression. We identify genetic diversity and chromosomal complexity as determinants of patient outcome. Our insights reconcile the variable clinical behavior of ccRCC and suggest evolutionary potential as a biomarker for both intervention and surveillance.
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http://dx.doi.org/10.1016/j.cell.2018.03.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5938372PMC
April 2018

The macrophage cytoskeleton acts as a contact sensor upon interaction with Entamoeba histolytica to trigger IL-1β secretion.

PLoS Pathog 2017 Aug 24;13(8):e1006592. Epub 2017 Aug 24.

Department of Microbiology, Immunology and Infectious Diseases, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada.

Entamoeba histolytica (Eh) is the causative agent of amebiasis, one of the major causes of dysentery-related morbidity worldwide. Recent studies have underlined the importance of the intercellular junction between Eh and host cells as a determinant in the pathogenesis of amebiasis. Despite the fact that direct contact and ligation between Eh surface Gal-lectin and EhCP-A5 with macrophage α5β1 integrin are absolute requirements for NLRP3 inflammasome activation and IL-1β release, many other undefined molecular events and downstream signaling occur at the interface of Eh and macrophage. In this study, we investigated the molecular events at the intercellular junction that lead to recognition of Eh through modulation of the macrophage cytoskeleton. Upon Eh contact with macrophages key cytoskeletal-associated proteins were rapidly post-translationally modified only with live Eh but not with soluble Eh proteins or fragments. Eh ligation with macrophages rapidly activated caspase-6 dependent cleavage of the cytoskeletal proteins talin, Pyk2 and paxillin and caused robust release of the pro-inflammatory cytokine, IL-1β. Macrophage cytoskeletal cleavages were dependent on Eh cysteine proteinases EhCP-A1 and EhCP-A4 but not EhCP-A5 based on pharmacological blockade of Eh enzyme inhibitors and EhCP-A5 deficient parasites. These results unravel a model where the intercellular junction between macrophages and Eh form an area of highly interacting proteins that implicate the macrophage cytoskeleton as a sensor for Eh contact that leads downstream to subsequent inflammatory immune responses.
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http://dx.doi.org/10.1371/journal.ppat.1006592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587335PMC
August 2017

BCL9L Dysfunction Impairs Caspase-2 Expression Permitting Aneuploidy Tolerance in Colorectal Cancer.

Cancer Cell 2017 01;31(1):79-93

Translational Cancer Therapeutics Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK; Translational Cancer Therapeutics Laboratory, University College London Cancer Institute, Paul O'Gorman Building, 72 Huntley Street, London WC2E 6DD, UK. Electronic address:

Chromosomal instability (CIN) contributes to cancer evolution, intratumor heterogeneity, and drug resistance. CIN is driven by chromosome segregation errors and a tolerance phenotype that permits the propagation of aneuploid genomes. Through genomic analysis of colorectal cancers and cell lines, we find frequent loss of heterozygosity and mutations in BCL9L in aneuploid tumors. BCL9L deficiency promoted tolerance of chromosome missegregation events, propagation of aneuploidy, and genetic heterogeneity in xenograft models likely through modulation of Wnt signaling. We find that BCL9L dysfunction contributes to aneuploidy tolerance in both TP53-WT and mutant cells by reducing basal caspase-2 levels and preventing cleavage of MDM2 and BID. Efforts to exploit aneuploidy tolerance mechanisms and the BCL9L/caspase-2/BID axis may limit cancer diversity and evolution.
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http://dx.doi.org/10.1016/j.ccell.2016.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225404PMC
January 2017

Optimization of Quantitative PCR Methods for Enteropathogen Detection.

PLoS One 2016 23;11(6):e0158199. Epub 2016 Jun 23.

Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, United States of America.

Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P<0.001) but were still able to detect 80% of pathogens with a Cq < 30 in stool. Second, a simplified total nucleic acid (TNA) extraction procedure was compared to separate DNA and RNA extractions and showed 92% (318/344) sensitivity and 98% (951/968) specificity, with no difference in Cq value for the positive results (ΔCq(DNA+RNA-TNA) = -0.01 ± 1.17, P = 0.972, N = 318). Third, we devised a quantification scheme that adjusts pathogen quantity to the specimen's extraction and amplification efficiency, and show that this better estimates the quantity of spiked specimens than the raw target Cq. In sum, these methods for enteropathogen quantification, stool sample collection, and nucleic acid extraction will be useful for laboratories studying enteric disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158199PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918952PMC
July 2017

Impact of enterovirus and other enteric pathogens on oral polio and rotavirus vaccine performance in Bangladeshi infants.

Vaccine 2016 06 3;34(27):3068-3075. Epub 2016 May 3.

Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville 22908, USA.

Background: Oral polio vaccine (OPV) and rotavirus vaccine (RV) exhibit poorer performance in low-income settings compared to high-income settings. Prior studies have suggested an inhibitory effect of concurrent non-polio enterovirus (NPEV) infection, but the impact of other enteric infections has not been comprehensively evaluated.

Methods: In urban Bangladesh, we tested stools for a broad range of enteric viruses, bacteria, parasites, and fungi by quantitative PCR from infants at weeks 6 and 10 of life, coincident with the first OPV and RV administration respectively, and examined the association between enteropathogen quantity and subsequent OPV serum neutralizing titers, serum rotavirus IgA, and rotavirus diarrhea.

Results: Campylobacter and enterovirus (EV) quantity at the time of administration of the first dose of OPV was associated with lower OPV1-2 serum neutralizing titers, while enterovirus quantity was also associated with diminished rotavirus IgA (-0.08 change in log titer per tenfold increase in quantity; P=0.037), failure to seroconvert (OR 0.78, 95% CI: 0.64-0.96; P=0.022), and breakthrough rotavirus diarrhea (OR 1.34, 95% CI: 1.05-1.71; P=0.020) after adjusting for potential confounders. These associations were not observed for Sabin strain poliovirus quantity.

Conclusion: In this broad survey of enteropathogens and oral vaccine performance we find a particular association between EV carriage, particularly NPEV, and OPV immunogenicity and RV protection. Strategies to reduce EV infections may improve oral vaccine responses. ClinicalTrials.gov Identifier: NCT01375647.
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http://dx.doi.org/10.1016/j.vaccine.2016.04.080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912219PMC
June 2016

Role of the Gut Microbiota of Children in Diarrhea Due to the Protozoan Parasite Entamoeba histolytica.

J Infect Dis 2016 May 27;213(10):1579-85. Epub 2015 Dec 27.

Department of Medicine.

Background: An estimated 1 million children die each year before their fifth birthday from diarrhea. Previous population-based surveys of pediatric diarrheal diseases have identified the protozoan parasite Entamoeba histolytica, the etiological agent of amebiasis, as one of the causes of moderate-to-severe diarrhea in sub-Saharan Africa and South Asia.

Methods: We prospectively studied the natural history of E. histolytica colonization and diarrhea among infants in an urban slum of Dhaka, Bangladesh.

Results: Approximately 80% of children were infected with E. histolytica by the age of 2 years. Fecal anti-galactose/N-acetylgalactosamine lectin immunoglobulin A was associated with protection from reinfection, while a high parasite burden and expansion of the Prevotella copri level was associated with diarrhea.

Conclusions: E. histolytica infection was prevalent in this population, with most infections asymptomatic and diarrhea associated with both the amount of parasite and the composition of the microbiota.
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http://dx.doi.org/10.1093/infdis/jiv772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4837909PMC
May 2016

Immune Evasion Mechanisms of Entamoeba histolytica: Progression to Disease.

Front Microbiol 2015 15;6:1394. Epub 2015 Dec 15.

Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, Snyder Institute for Chronic Diseases, University of Calgary Calgary, AB, Canada.

Entamoeba histolytica (Eh) is a protozoan parasite that infects 10% of the world's population and results in 100,000 deaths/year from amebic dysentery and/or liver abscess. In most cases, this extracellular parasite colonizes the colon by high affinity binding to MUC2 mucin without disease symptoms, whereas in some cases, Eh triggers an aggressive inflammatory response upon invasion of the colonic mucosa. The specific host-parasite factors critical for disease pathogenesis are still not well characterized. From the parasite, the signature events that lead to disease progression are cysteine protease cleavage of the C-terminus of MUC2 that dissolves the mucus layer followed by Eh binding and cytotoxicity of the mucosal epithelium. The host mounts an ineffective excessive host pro-inflammatory response following contact with host cells that causes tissue damage and participates in disease pathogenesis as Eh escapes host immune clearance by mechanisms that are not completely understood. Ameba can modulate or destroy effector immune cells by inducing neutrophil apoptosis and suppressing respiratory burst or nitric oxide (NO) production from macrophages. Eh adherence to the host cells also induce multiple cytotoxic effects that can promote cell death through phagocytosis, apoptosis or by trogocytosis (ingestion of living cells) that might play critical roles in immune evasion. This review focuses on the immune evasion mechanisms that Eh uses to survive and induce disease manifestation in the host.
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http://dx.doi.org/10.3389/fmicb.2015.01394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4678226PMC
December 2015

Tracking the genomic evolution of esophageal adenocarcinoma through neoadjuvant chemotherapy.

Cancer Discov 2015 Aug 23;5(8):821-831. Epub 2015 May 23.

The Francis Crick Institute, 44 Lincoln's Inn Fields, London, UK.

Unlabelled: Esophageal adenocarcinomas are associated with a dismal prognosis. Deciphering the evolutionary history of this disease may shed light on therapeutically tractable targets and reveal dynamic mutational processes during the disease course and following neoadjuvant chemotherapy (NAC). We exome sequenced 40 tumor regions from 8 patients with operable esophageal adenocarcinomas, before and after platinum-containing NAC. This revealed the evolutionary genomic landscape of esophageal adenocarcinomas with the presence of heterogeneous driver mutations, parallel evolution, early genome-doubling events, and an association between high intratumor heterogeneity and poor response to NAC. Multiregion sequencing demonstrated a significant reduction in thymine to guanine mutations within a CpTpT context when comparing early and late mutational processes and the presence of a platinum signature with enrichment of cytosine to adenine mutations within a CpC context following NAC. Esophageal adenocarcinomas are characterized by early chromosomal instability leading to amplifications containing targetable oncogenes persisting through chemotherapy, providing a rationale for future therapeutic approaches.

Significance: This work illustrates dynamic mutational processes occurring during esophageal adenocarcinoma evolution and following selective pressures of platinum exposure, emphasizing the iatrogenic impact of therapy on cancer evolution. Identification of amplifications encoding targetable oncogenes maintained through NAC suggests the presence of stable vulnerabilities, unimpeded by cytotoxics, suitable for therapeutic intervention.
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http://dx.doi.org/10.1158/2159-8290.CD-15-0412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4529488PMC
August 2015

Recurrent chromosomal gains and heterogeneous driver mutations characterise papillary renal cancer evolution.

Nat Commun 2015 Mar 19;6:6336. Epub 2015 Mar 19.

Genomic analysis of tumour development, Instituto de Biomedicina y Biotecnología de Cantabria (CSIC-UC-Sodercan), Departamento de Biología Molecular, Universidad de Cantabria, 39011 Santander, Spain.

Papillary renal cell carcinoma (pRCC) is an important subtype of kidney cancer with a problematic pathological classification and highly variable clinical behaviour. Here we sequence the genomes or exomes of 31 pRCCs, and in four tumours, multi-region sequencing is undertaken. We identify BAP1, SETD2, ARID2 and Nrf2 pathway genes (KEAP1, NHE2L2 and CUL3) as probable drivers, together with at least eight other possible drivers. However, only ~10% of tumours harbour detectable pathogenic changes in any one driver gene, and where present, the mutations are often predicted to be present within cancer sub-clones. We specifically detect parallel evolution of multiple SETD2 mutations within different sub-regions of the same tumour. By contrast, large copy number gains of chromosomes 7, 12, 16 and 17 are usually early, monoclonal changes in pRCC evolution. The predominance of large copy number variants as the major drivers for pRCC highlights an unusual mode of tumorigenesis that may challenge precision medicine approaches.
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http://dx.doi.org/10.1038/ncomms7336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383019PMC
March 2015

Molecular genotyping and quantitation assay for rotavirus surveillance.

J Virol Methods 2015 Mar 17;213:157-63. Epub 2014 Dec 17.

Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA, United States.

Rotavirus genotyping is useful for surveillance purposes especially in areas where rotavirus vaccination has been or will be implemented. RT-PCR based molecular methods have been applied widely, but quantitative assays targeting a broad spectrum of genotypes have not been developed. Three real time RT-PCR panels were designed to identify G1, G2, G9, G12 (panel GI), G3, G4, G8, G10 (panel GII), and P[4], P[6], P[8], P[10], P[11] (panel P), respectively. An assay targeting NSP3 was included in both G panels as an internal control. The cognate assays were also formulated as one RT-PCR-Luminex panel for simultaneous detection of all the genotypes listed above plus P[9]. The assays were evaluated with various rotavirus isolates and 89 clinical samples from Virginia, Bangladesh and Tanzania, and exhibited 95% (81/85) sensitivity compared with the conventional RT-PCR-Gel-electrophoresis method, and 100% concordance with sequencing. Real time assays identified a significantly higher rate of mixed genotypes in Bangladeshi samples than the conventional gel-electrophoresis-based RT-PCR assay (32.5% versus 12.5%, P<0.05). In these mixed infections, the relative abundance of the rotavirus types could be estimated by Cq values. These typing assays detect and discriminate a broad range of G/P types circulating in different geographic regions with high sensitivity and specificity and can be used for rotavirus surveillance.
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http://dx.doi.org/10.1016/j.jviromet.2014.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4417650PMC
March 2015

Kinetics of poliovirus shedding following oral vaccination as measured by quantitative reverse transcription-PCR versus culture.

J Clin Microbiol 2015 Jan 5;53(1):206-11. Epub 2014 Nov 5.

Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia, USA.

Amid polio eradication efforts, detection of oral polio vaccine (OPV) virus in stool samples can provide information about rates of mucosal immunity and allow estimation of the poliovirus reservoir. We developed a multiplex one-step quantitative reverse transcription-PCR (qRT-PCR) assay for detection of OPV Sabin strains 1, 2, and 3 directly in stool samples with an external control to normalize samples for viral quantity and compared its performance with that of viral culture. We applied the assay to samples from infants in Dhaka, Bangladesh, after the administration of trivalent OPV (tOPV) at weeks 14 and 52 of life (on days 0 [pre-OPV], +4, +11, +18, and +25 relative to vaccination). When 1,350 stool samples were tested, the sensitivity and specificity of the quantitative PCR (qPCR) assay were 89 and 91% compared with culture. A quantitative relationship between culture(+)/qPCR(+) and culture(-)/qPCR(+) stool samples was observed. The kinetics of shedding revealed by qPCR and culture were similar. qPCR quantitative cutoffs based on the day +11 or +18 stool samples could be used to identify the culture-positive shedders, as well as the long-duration or high-frequency shedders. Interestingly, qPCR revealed that a small minority (7%) of infants contributed the vast majority (93 to 100%) of the total estimated viral excretion across all subtypes at each time point. This qPCR assay for OPV can simply and quantitatively detect all three Sabin strains directly in stool samples to approximate shedding both qualitatively and quantitatively.
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http://dx.doi.org/10.1128/JCM.02406-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4290924PMC
January 2015

Spatial and temporal diversity in genomic instability processes defines lung cancer evolution.

Science 2014 Oct;346(6206):251-6

Thermo Fisher Scientific, Carlsbad, CA 92008, USA.

Spatial and temporal dissection of the genomic changes occurring during the evolution of human non-small cell lung cancer (NSCLC) may help elucidate the basis for its dismal prognosis. We sequenced 25 spatially distinct regions from seven operable NSCLCs and found evidence of branched evolution, with driver mutations arising before and after subclonal diversification. There was pronounced intratumor heterogeneity in copy number alterations, translocations, and mutations associated with APOBEC cytidine deaminase activity. Despite maintained carcinogen exposure, tumors from smokers showed a relative decrease in smoking-related mutations over time, accompanied by an increase in APOBEC-associated mutations. In tumors from former smokers, genome-doubling occurred within a smoking-signature context before subclonal diversification, which suggested that a long period of tumor latency had preceded clinical detection. The regionally separated driver mutations, coupled with the relentless and heterogeneous nature of the genome instability processes, are likely to confound treatment success in NSCLC.
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http://dx.doi.org/10.1126/science.1253462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4636050PMC
October 2014

Development of synchronous VHL syndrome tumors reveals contingencies and constraints to tumor evolution.

Genome Biol 2014 Aug 27;15(8):433. Epub 2014 Aug 27.

Background: Genomic analysis of multi-focal renal cell carcinomas from an individual with a germline VHL mutation offers a unique opportunity to study tumor evolution.

Results: We perform whole exome sequencing on four clear cell renal cell carcinomas removed from both kidneys of a patient with a germline VHL mutation. We report that tumors arising in this context are clonally independent and harbour distinct secondary events exemplified by loss of chromosome 3p, despite an identical genetic background and tissue microenvironment. We propose that divergent mutational and copy number anomalies are contingent upon the nature of 3p loss of heterozygosity occurring early in tumorigenesis. However, despite distinct 3p events, genomic, proteomic and immunohistochemical analyses reveal evidence for convergence upon the PI3K-AKT-mTOR signaling pathway. Four germline tumors in this young patient, and in a second, older patient with VHL syndrome demonstrate minimal intra-tumor heterogeneity and mutational burden, and evaluable tumors appear to follow a linear evolutionary route, compared to tumors from patients with sporadic clear cell renal cell carcinoma.

Conclusions: In tumors developing from a germline VHL mutation, the evolutionary principles of contingency and convergence in tumor development are complementary. In this small set of patients with early stage VHL-associated tumors, there is reduced mutation burden and limited evidence of intra-tumor heterogeneity.
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http://dx.doi.org/10.1186/s13059-014-0433-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4166471PMC
August 2014

Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study.

Lancet Infect Dis 2014 Aug 9;14(8):716-724. Epub 2014 Jul 9.

International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Dhaka, Bangladesh.

Background: Childhood diarrhoea can be caused by many pathogens that are difficult to assay in the laboratory. Molecular diagnostic techniques provide a uniform method to detect and quantify candidate enteropathogens. We aimed to develop and assess molecular tests for identification of enteropathogens and their association with disease.

Methods: We developed and assessed molecular diagnostic tests for 15 enteropathogens across three platforms-PCR-Luminex, multiplex real-time PCR, and TaqMan array card-at five laboratories worldwide. We judged the analytical and clinical performance of these molecular techniques against comparator methods (bacterial culture, ELISA, and PCR) using 867 diarrhoeal and 619 non-diarrhoeal stool specimens. We also measured molecular quantities of pathogens to predict the association with diarrhoea, by univariate logistic regression analysis.

Findings: The molecular tests showed very good analytical and clinical performance at all five laboratories. Comparator methods had limited sensitivity compared with the molecular techniques (20-85% depending on the target) but good specificity (median 97·3%, IQR 96·5-98·9; mean 95·2%, SD 9·1). Positive samples by comparator methods usually had higher molecular quantities of pathogens than did negative samples, across almost all platforms and for most pathogens (p<0·05). The odds ratio for diarrhoea at a given quantity (measured by quantification cycle, Cq) showed that for most pathogens associated with diarrhoea-including Campylobacter jejuni and Campylobacter coli, Cryptosporidium spp, enteropathogenic Escherichia coli, heat-stable enterotoxigenic E coli, rotavirus, Shigella spp and enteroinvasive E coli, and Vibrio cholerae-the strength of association with diarrhoea increased at higher pathogen loads. For example, Shigella spp at a Cq range of 15-20 had an odds ratio of 8·0 (p<0·0001), but at a Cq range of 25-30 the odds ratio fell to 1·7 (p=0·043).

Interpretation: Molecular diagnostic tests can be implemented successfully and with fidelity across laboratories around the world. In the case of diarrhoea, these techniques can detect pathogens with high sensitivity and ascribe diarrhoeal associations based on quantification, including in mixed infections, providing rich and unprecedented measurements of infectious causes.

Funding: Bill & Melinda Gates Foundation Next Generation Molecular Diagnostics Project.
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http://dx.doi.org/10.1016/S1473-3099(14)70808-4DOI Listing
August 2014

Identification of an LGP2-associated MDA5 agonist in picornavirus-infected cells.

Elife 2014 Feb 18;3:e01535. Epub 2014 Feb 18.

Immunobiology Laboratory, Cancer Research UK, London Research Institute, London, United Kingdom.

The RIG-I-like receptors RIG-I, LGP2, and MDA5 initiate an antiviral response that includes production of type I interferons (IFNs). The nature of the RNAs that trigger MDA5 activation in infected cells remains unclear. Here, we purify and characterise LGP2/RNA complexes from cells infected with encephalomyocarditis virus (EMCV), a picornavirus detected by MDA5 and LGP2 but not RIG-I. We show that those complexes contain RNA that is highly enriched for MDA5-stimulatory activity and for a specific sequence corresponding to the L region of the EMCV antisense RNA. Synthesis of this sequence by in vitro transcription is sufficient to generate an MDA5 stimulatory RNA. Conversely, genomic deletion of the L region in EMCV generates viruses that are less potent at stimulating MDA5-dependent IFN production. Thus, the L region antisense RNA of EMCV is a key determinant of innate immunity to the virus and represents an RNA that activates MDA5 in virally-infected cells. DOI: http://dx.doi.org/10.7554/eLife.01535.001.
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http://dx.doi.org/10.7554/eLife.01535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967861PMC
February 2014

Genomic architecture and evolution of clear cell renal cell carcinomas defined by multiregion sequencing.

Nat Genet 2014 Mar 2;46(3):225-233. Epub 2014 Feb 2.

Translational Cancer Therapeutics Laboratory, Cancer Research UK London Research Institute, London, UK.

Clear cell renal carcinomas (ccRCCs) can display intratumor heterogeneity (ITH). We applied multiregion exome sequencing (M-seq) to resolve the genetic architecture and evolutionary histories of ten ccRCCs. Ultra-deep sequencing identified ITH in all cases. We found that 73-75% of identified ccRCC driver aberrations were subclonal, confounding estimates of driver mutation prevalence. ITH increased with the number of biopsies analyzed, without evidence of saturation in most tumors. Chromosome 3p loss and VHL aberrations were the only ubiquitous events. The proportion of C>T transitions at CpG sites increased during tumor progression. M-seq permits the temporal resolution of ccRCC evolution and refines mutational signatures occurring during tumor development.
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http://dx.doi.org/10.1038/ng.2891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4636053PMC
March 2014

Detection of Campylobacter in stool and determination of significance by culture, enzyme immunoassay, and PCR in developing countries.

J Clin Microbiol 2014 Apr 22;52(4):1074-80. Epub 2014 Jan 22.

Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, USA.

Campylobacter is a common bacterial enteropathogen that can be detected in stool by culture, enzyme immunoassay (EIA), or PCR. We compared culture for C. jejuni/C. coli, EIA (ProSpecT), and duplex PCR to distinguish Campylobacter jejuni/C. coli and non-jejuni/coli Campylobacter on 432 diarrheal and matched control stool samples from infants in a multisite longitudinal study of enteric infections in Tanzania, Bangladesh, and Peru. The sensitivity and specificity of culture were 8.5% and 97.6%, respectively, compared with the results of EIA and 8.7% and 98.0%, respectively, compared with the results of PCR for C. jejuni/C. coli. Most (71.6%) EIA-positive samples were positive by PCR for C. jejuni/C. coli, but 27.6% were positive for non-jejuni/coli Campylobacter species. Sequencing of 16S rRNA from 53 of these non-jejuni/coli Campylobacter samples showed that it most closely matched the 16S rRNA of C. hyointestinalis subsp. lawsonii (56%), C. troglodytis (33%), C. upsaliensis (7.7%), and C. jejuni/C. coli (2.6%). Campylobacter-negative stool spiked with each of the above-mentioned Campylobacter species revealed reactivity with EIA. PCR detection of Campylobacter species was strongly associated with diarrhea in Peru (odds ratio [OR] = 3.66, P < 0.001) but not in Tanzania (OR = 1.56, P = 0.24) or Bangladesh (OR = 1.13, P = 0.75). According to PCR, Campylobacter jejuni/C. coli infections represented less than half of all infections with Campylobacter species. In sum, in infants in developing country settings, the ProSpecT EIA and PCR for Campylobacter reveal extremely high rates of positivity. We propose the use of PCR because it retains high sensitivity, can ascertain burden, and can distinguish between Campylobacter infections at the species level.
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http://dx.doi.org/10.1128/JCM.02935-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993515PMC
April 2014

Etiology of diarrhea in Bangladeshi infants in the first year of life analyzed using molecular methods.

J Infect Dis 2013 Dec 16;208(11):1794-802. Epub 2013 Sep 16.

Division of Infectious Diseases and International Health, Department of Medicine.

Background: Diarrhea causes enormous morbidity and mortality in developing countries, yet the relative importance of multiple potential enteropathogens has been difficult to ascertain.

Methods: We performed a longitudinal cohort study from birth to 1 year of age in 147 infants in Dhaka, Bangladesh. Using multiplex polymerase chain reaction, we analyzed 420 episodes of diarrhea and 1385 monthly surveillance stool specimens for 32 enteropathogen gene targets. For each infant we examined enteropathogen quantities over time to ascribe each positive target as a probable or less-likely contributor to diarrhea.

Results: Multiple enteropathogens were detected by the first month of life. Diarrhea was associated with a state of overall pathogen excess (mean number of enteropathogen gene targets (± SE), 5.6 ± 0.1 vs 4.3 ± 0.1 in surveillance stool specimens; P < .05). After a longitudinal, quantitative approach was applied to filter out less-likely contributors, each diarrheal episode still had an average of 3.3 probable or dominant targets. Enteroaggregative Escherichia coli, Campylobacter, enteropathogenic E. coli, rotavirus, and Entamoeba histolytica were the most frequent probable contributors to diarrhea. Rotavirus was enriched in moderate to severe diarrheal episodes.

Conclusions: In this community-based study diarrhea seemed to be a multipathogen event and a state of enteropathogen excess above a high carriage baseline.
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http://dx.doi.org/10.1093/infdis/jit507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814844PMC
December 2013

The zebrafish reference genome sequence and its relationship to the human genome.

Nature 2013 Apr 17;496(7446):498-503. Epub 2013 Apr 17.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.
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http://dx.doi.org/10.1038/nature12111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3703927PMC
April 2013

Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.

N Engl J Med 2012 Mar;366(10):883-892

Cancer Research UK London Research Institute (M. Gerlinger, A.J.R., S.H., D.E., E.G., P.M., N.M., A.S., B.P., S.B., N.Q.M., C.R.S., B.S.-D., G.C., G.S., J.D., C.S.), Royal Marsden Hospital Department of Medicine (J.L., M.N., L.P., G.S., M. Gore), Wellcome Trust Sanger Institute (P.T., I.V., A.B., D.J., K.R., C.L., P.A.F.), Barts Cancer Institute at the Barts and the London School of Medicine and Dentistry (M. Gerlinger), and the University College London Cancer Institute (C.S.) - all in London; the Technical University of Denmark, Lyngby (A.C.E., Z.S.); and Harvard Medical School, Boston (Z.S.). Address reprint requests to Dr. Swanton at the Cancer Research UK London Research Institute, Translational Cancer Therapeutics Laboratory, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom, or at

Background: Intratumor heterogeneity may foster tumor evolution and adaptation and hinder personalized-medicine strategies that depend on results from single tumor-biopsy samples.

Methods: To examine intratumor heterogeneity, we performed exome sequencing, chromosome aberration analysis, and ploidy profiling on multiple spatially separated samples obtained from primary renal carcinomas and associated metastatic sites. We characterized the consequences of intratumor heterogeneity using immunohistochemical analysis, mutation functional analysis, and profiling of messenger RNA expression.

Results: Phylogenetic reconstruction revealed branched evolutionary tumor growth, with 63 to 69% of all somatic mutations not detectable across every tumor region. Intratumor heterogeneity was observed for a mutation within an autoinhibitory domain of the mammalian target of rapamycin (mTOR) kinase, correlating with S6 and 4EBP phosphorylation in vivo and constitutive activation of mTOR kinase activity in vitro. Mutational intratumor heterogeneity was seen for multiple tumor-suppressor genes converging on loss of function; SETD2, PTEN, and KDM5C underwent multiple distinct and spatially separated inactivating mutations within a single tumor, suggesting convergent phenotypic evolution. Gene-expression signatures of good and poor prognosis were detected in different regions of the same tumor. Allelic composition and ploidy profiling analysis revealed extensive intratumor heterogeneity, with 26 of 30 tumor samples from four tumors harboring divergent allelic-imbalance profiles and with ploidy heterogeneity in two of four tumors.

Conclusions: Intratumor heterogeneity can lead to underestimation of the tumor genomics landscape portrayed from single tumor-biopsy samples and may present major challenges to personalized-medicine and biomarker development. Intratumor heterogeneity, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure through Darwinian selection. (Funded by the Medical Research Council and others.).
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http://dx.doi.org/10.1056/NEJMoa1113205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4878653PMC
March 2012
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