Publications by authors named "Thomas Otto"

205 Publications

Essential Genes of the Parasitic Apicomplexa.

Trends Parasitol 2021 Jan 5. Epub 2021 Jan 5.

Center for Global Health and Infectious Diseases and USF Genomics Program, College of Public Health, University of South Florida, 3720 Spectrum Boulevard, Suite 404, Tampa, FL 33612, USA. Electronic address:

Genome-scale mutagenesis screens for genes essential for apicomplexan parasite survival have been completed in three species: Plasmodium falciparum, the major human malaria parasite, Plasmodium berghei, a model rodent malaria parasite, and the more distantly related Toxoplasma gondii, the causative agent of toxoplasmosis. These three species share 2606 single-copy orthologs, 1500 of which have essentiality data in all three screens. In this review, we explore the overlap between these datasets to define the core essential genes of the phylum Apicomplexa. We further discuss the implications of these groundbreaking studies for understanding apicomplexan parasite biology, and we identify promising areas of focus for developing new pan-apicomplexan parasite interventions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pt.2020.11.007DOI Listing
January 2021

Evaluating the Utility of Combined Bladder Cancer Biomarkers, the Molecular Prognostication of Tumor Subtypes, or What Else Is Needed to Illuminate Our Vision?

Int J Mol Sci 2020 Dec 18;21(24). Epub 2020 Dec 18.

International Agency for Research on Cancer (IARC), F-69372 Lyon, France.

In the last few years, we published two special issues devoted to highlighting important scientific results in the field of bladder cancer research and clinical implications [...].
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21249657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766346PMC
December 2020

Machine learning approaches classify clinical malaria outcomes based on haematological parameters.

BMC Med 2020 11 30;18(1):375. Epub 2020 Nov 30.

Institute of Infection, Immunity & Inflammation, MVLS, University of Glasgow, Glasgow, UK.

Background: Malaria is still a major global health burden, with more than 3.2 billion people in 91 countries remaining at risk of the disease. Accurately distinguishing malaria from other diseases, especially uncomplicated malaria (UM) from non-malarial infections (nMI), remains a challenge. Furthermore, the success of rapid diagnostic tests (RDTs) is threatened by Pfhrp2/3 deletions and decreased sensitivity at low parasitaemia. Analysis of haematological indices can be used to support the identification of possible malaria cases for further diagnosis, especially in travellers returning from endemic areas. As a new application for precision medicine, we aimed to evaluate machine learning (ML) approaches that can accurately classify nMI, UM, and severe malaria (SM) using haematological parameters.

Methods: We obtained haematological data from 2,207 participants collected in Ghana: nMI (n = 978), SM (n = 526), and UM (n = 703). Six different ML approaches were tested, to select the best approach. An artificial neural network (ANN) with three hidden layers was used for multi-classification of UM, SM, and uMI. Binary classifiers were developed to further identify the parameters that can distinguish UM or SM from nMI. Local interpretable model-agnostic explanations (LIME) were used to explain the binary classifiers.

Results: The multi-classification model had greater than 85% training and testing accuracy to distinguish clinical malaria from nMI. To distinguish UM from nMI, our approach identified platelet counts, red blood cell (RBC) counts, lymphocyte counts, and percentages as the top classifiers of UM with 0.801 test accuracy (AUC = 0.866 and F1 score = 0.747). To distinguish SM from nMI, the classifier had a test accuracy of 0.96 (AUC = 0.983 and F1 score = 0.944) with mean platelet volume and mean cell volume being the unique classifiers of SM. Random forest was used to confirm the classifications, and it showed that platelet and RBC counts were the major classifiers of UM, regardless of possible confounders such as patient age and sampling location.

Conclusion: The study provides proof of concept methods that classify UM and SM from nMI, showing that the ML approach is a feasible tool for clinical decision support. In the future, ML approaches could be incorporated into clinical decision-support algorithms for the diagnosis of acute febrile illness and monitoring response to acute SM treatment particularly in endemic settings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12916-020-01823-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702702PMC
November 2020

2D Scanning Micromirror with Large Scan Angle and Monolithically Integrated Angle Sensors Based on Piezoelectric Thin Film Aluminum Nitride.

Sensors (Basel) 2020 Nov 18;20(22). Epub 2020 Nov 18.

Center for Microtechnologies, Chemnitz University of Technology, 09111 Chemnitz, Germany.

A 2D scanning micromirror with piezoelectric thin film aluminum nitride (AlN), separately used as actuator and sensor material, is presented. For endoscopic applications, such as fluorescence microscopy, the devices have a mirror plate diameter of 0.7 mm with a 4 mm chip footprint. After an initial design optimization procedure, two micromirror designs were realized. Different spring parameters for x- and y-tilt were chosen to generate spiral (Design 1) or Lissajous (Design 2) scan patterns. An additional layout, with integrated tilt angle sensors, was introduced (Design 1-S) to enable a closed-loop control. The micromirror devices were monolithically fabricated in 150 mm silicon-on-insulator (SOI) technology. Si (111) was used as the device silicon layer to support a high C-axis oriented growth of AlN. The fabricated micromirror devices were characterized in terms of their scanning and sensor characteristics in air. A scan angle of 91.2° was reached for Design 1 at 13 834 Hz and 50 V. For Design 2 a scan angle of 92.4° at 12 060 Hz, and 123.9° at 13 145 Hz, was reached at 50 V for the x- and y-axis, respectively. The desired 2D scan patterns were successfully generated. A sensor angle sensitivity of 1.9 pC/° was achieved.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/s20226599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698969PMC
November 2020

Conversion coefficients from total air kerma to the newly proposed ICRU/ICRP operational quantities for radiation protection for photon reference radiation qualities.

J Radiol Prot 2020 Nov 6. Epub 2020 Nov 6.

Technology Department, CERN, Geneve, Genève, SWITZERLAND.

The International Commission on Radiation Units and Measurements (ICRU) has recently proposed a set of new operational quantities for radiation protection. ICRU supplied conversion coefficients for mono-energetic photons but not for photon reference radiation qualities defined by the International Organization for Standardization (ISO) in ISO 4037 and by the International Electrotechnical Commission (IEC) in IEC 61267. Therefore, in this work, conversion coefficients from total air kerma to the newly proposed operational quantities are averaged for photon reference radiation qualities. Also, parameters necessary to determine the influence of the air density on the conversion coefficients are determined. Finally, the impact of the newly proposed quantities upon the response of dosemeters is investigated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1088/1361-6498/abc860DOI Listing
November 2020

Increased circulation time of Plasmodium falciparum underlies persistent asymptomatic infection in the dry season.

Nat Med 2020 12 26;26(12):1929-1940. Epub 2020 Oct 26.

Center for Infectious Diseases, Parasitology, Heidelberg University Hospital, Heidelberg, Germany.

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquito vectors to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the human host or compromising host survival, is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of individuals with febrile malaria in the transmission season, coinciding with longer circulation within each replicative cycle of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication but rather to increased splenic clearance of longer-circulating infected erythrocytes, which likely maintain parasitemias below clinical and immunological radar. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-020-1084-0DOI Listing
December 2020

Tolerance induction in memory CD4 T cells is partial and reversible.

Immunology 2021 Jan 27;162(1):68-83. Epub 2020 Oct 27.

Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, UK.

Memory T cells respond rapidly in part because they are less reliant on a heightened levels of costimulatory molecules. This enables rapid control of secondary infecting pathogens but presents challenges to efforts to control or silence memory CD4 T cells, for example in antigen-specific tolerance strategies for autoimmunity. We have examined the transcriptional and functional consequences of reactivating memory CD4 T cells in the absence of an adjuvant. We find that memory CD4 T cells generated by infection or immunisation survive secondary activation with antigen delivered without adjuvant, regardless of their location in secondary lymphoid organs or peripheral tissues. These cells were, however, functionally altered following a tertiary immunisation with antigen and adjuvant, proliferating poorly but maintaining their ability to produce inflammatory cytokines. Transcriptional and cell cycle analysis of these memory CD4 T cells suggests they are unable to commit fully to cell division potentially because of low expression of DNA repair enzymes. In contrast, these memory CD4 T cells could proliferate following tertiary reactivation by viral re-infection. These data indicate that antigen-specific tolerogenic strategies must examine multiple parameters of Tcell function, and provide insight into the molecular mechanisms that may lead to deletional tolerance of memory CD4 T cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/imm.13263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730012PMC
January 2021

Green Precursors and Soft Templating for Printing Porous Carbon-Based Micro-supercapacitors.

Chemistry 2021 Jan 7;27(4):1356-1363. Epub 2020 Dec 7.

Chemistry and Food Chemistry, Inorganic Chemistry I, Bergstraße 66, 01159, Dresden, Germany.

A combination of soft lithographic printing and soft templating has been used to fabricate high-resolution interdigitated micro-supercapacitors (MSC). Surfactant-assisted self-assembly produces high surface area ordered mesoporous carbons (490 m  g ). For the first time, such precursors have been printed by nano-imprint lithography as microdevices with a line width of only 250 nm and a spacing of only 1 μm. The devices are crack-free with low specific resistance (1.2×10  Ωm) and show good device capacitance up to 0.21 F cm .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chem.202003124DOI Listing
January 2021

β2 Integrins differentially regulate γδ T cell subset thymic development and peripheral maintenance.

Proc Natl Acad Sci U S A 2020 09 26;117(36):22367-22377. Epub 2020 Aug 26.

Institute of Infection, Immunity & Inflammation, University of Glasgow, G12 8TA Glasgow, United Kingdom;

The γδ T cells reside predominantly at barrier sites and play essential roles in immune protection against infection and cancer. Despite recent advances in the development of γδ T cell immunotherapy, our understanding of the basic biology of these cells, including how their numbers are regulated in vivo, remains poor. This is particularly true for tissue-resident γδ T cells. We have identified the β family of integrins as regulators of γδ T cells. β-integrin-deficient mice displayed a striking increase in numbers of IL-17-producing Vγ6Vδ1 γδ T cells in the lungs, uterus, and circulation. Thymic development of this population was normal. However, single-cell RNA sequencing revealed the enrichment of genes associated with T cell survival and proliferation specifically in β-integrin-deficient IL-17 cells compared to their wild-type counterparts. Indeed, β-integrin-deficient Vγ6 cells from the lungs showed reduced apoptosis ex vivo, suggesting that increased survival contributes to the accumulation of these cells in β-integrin-deficient tissues. Furthermore, our data revealed an unexpected role for β integrins in promoting the thymic development of the IFNγ-producing CD27 Vγ4 γδ T cell subset. Together, our data reveal that β integrins are important regulators of γδ T cell homeostasis, inhibiting the survival of IL-17-producing Vγ6Vδ1 cells and promoting the thymic development of the IFNγ-producing Vγ4 subset. Our study introduces unprecedented mechanisms of control for γδ T cell subsets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1921930117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486781PMC
September 2020

Author Correction: Genomes of all known members of a Plasmodium subgenus reveal paths to virulent human malaria.

Nat Microbiol 2020 Oct;5(10):1306

Laboratoire MIVEGEC, UMR 5290-224 CNRS 5290-IRD224-UM, Montpellier, France.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-020-0787-9DOI Listing
October 2020

The Impact of Malaria Parasites on Dendritic Cell-T Cell Interaction.

Front Immunol 2020 24;11:1597. Epub 2020 Jul 24.

Institute of Infection, Immunity & Inflammation, University of Glasgow, Glasgow, United Kingdom.

Malaria is caused by apicomplexan parasites of the genus . While infection continues to pose a risk for the majority of the global population, the burden of disease mainly resides in Sub-Saharan Africa. Although immunity develops against disease, this requires years of persistent exposure and is not associated with protection against infection. Repeat infections occur due to the parasite's ability to disrupt or evade the host immune responses. However, despite many years of study, the mechanisms of this disruption remain unclear. Previous studies have demonstrated a parasite-induced failure in dendritic cell (DCs) function affecting the generation of helper T cell responses. These T cells fail to help B cell responses, reducing the production of antibodies that are necessary to control malaria infection. This review focuses on our current understanding of the effect of parasite on DC function, DC-T cell interaction, and T cell activation. A better understanding of how parasites disrupt DC-T cell interactions will lead to new targets and approaches to reinstate adaptive immune responses and enhance parasite immunity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.01597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393936PMC
July 2020

Testing the impact of a single nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor on experimental cerebral malaria in mice.

Sci Rep 2020 08 12;10(1):13630. Epub 2020 Aug 12.

Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 5625 Fishers Lane, Room 4S04, Rockville, MD, USA.

Cerebral malaria (CM) is the deadliest form of severe Plasmodium infections. Currently, we have limited understanding of the mechanisms by which Plasmodium parasites induce CM. The mouse model of CM, experimental CM (ECM), induced by infection with the rodent parasite, Plasmodium berghei ANKA (PbANKA) has been extensively used to study the pathophysiology of CM. Recent genomic analyses revealed that the coding regions of PbANKA and the closely related Plasmodium berghei NK65 (PbNK65), that does not cause ECM, differ in only 21 single nucleotide polymorphysims (SNPs). Thus, the SNP-containing genes might contribute to the pathogenesis of ECM. Although the majority of these SNPs are located in genes of unknown function, one SNP is located in the DNA binding site of a member of the Plasmodium ApiAP2 transcription factor family, that we recently showed functions as a virulence factor alternating the host's immune response to the parasite. Here, we investigated the impact of this SNP on the development of ECM. Our results using CRISPR-Cas9 engineered parasites indicate that despite its immune modulatory function, the SNP is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific differences in ECM phenotypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-70617-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424516PMC
August 2020

Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.

Nat Med 2020 08 29;26(8):1295-1306. Epub 2020 Jun 29.

Research into Inflammatory Arthritis Centre Versus Arthritis (RACE), .

Immune-regulatory mechanisms of drug-free remission in rheumatoid arthritis (RA) are unknown. We hypothesized that synovial tissue macrophages (STM), which persist in remission, contribute to joint homeostasis. We used single-cell transcriptomics to profile 32,000 STMs and identified phenotypic changes in patients with early/active RA, treatment-refractory/active RA and RA in sustained remission. Each clinical state was characterized by different frequencies of nine discrete phenotypic clusters within four distinct STM subpopulations with diverse homeostatic, regulatory and inflammatory functions. This cellular atlas, combined with deep-phenotypic, spatial and functional analyses of synovial biopsy fluorescent activated cell sorted STMs, revealed two STM subpopulations (MerTKTREM2 and MerTKLYVE1) with unique remission transcriptomic signatures enriched in negative regulators of inflammation. These STMs were potent producers of inflammation-resolving lipid mediators and induced the repair response of synovial fibroblasts in vitro. A low proportion of MerTK STMs in remission was associated with increased risk of disease flare after treatment cessation. Therapeutic modulation of MerTK STM subpopulations could therefore be a potential treatment strategy for RA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-020-0939-8DOI Listing
August 2020

Refining the transcriptome of the human malaria parasite Plasmodium falciparum using amplification-free RNA-seq.

BMC Genomics 2020 Jun 8;21(1):395. Epub 2020 Jun 8.

Department of Biochemistry & Molecular Biology and Huck Center for Malaria Research, Pennsylvania State University, University Park, PA, 16802, USA.

Background: Plasmodium parasites undergo several major developmental transitions during their complex lifecycle, which are enabled by precisely ordered gene expression programs. Transcriptomes from the 48-h blood stages of the major human malaria parasite Plasmodium falciparum have been described using cDNA microarrays and RNA-seq, but these assays have not always performed well within non-coding regions, where the AT-content is often 90-95%.

Results: We developed a directional, amplification-free RNA-seq protocol (DAFT-seq) to reduce bias against AT-rich cDNA, which we have applied to three strains of P. falciparum (3D7, HB3 and IT). While strain-specific differences were detected, overall there is strong conservation between the transcriptional profiles. For the 3D7 reference strain, transcription was detected from 89% of the genome, with over 78% of the genome transcribed into mRNAs. We also find that transcription from bidirectional promoters frequently results in non-coding, antisense transcripts. These datasets allowed us to refine the 5' and 3' untranslated regions (UTRs), which can be variable, long (> 1000 nt), and often overlap those of adjacent transcripts.

Conclusions: The approaches applied in this study allow a refined description of the transcriptional landscape of P. falciparum and demonstrate that very little of the densely packed P. falciparum genome is inactive or redundant. By capturing the 5' and 3' ends of mRNAs, we reveal both constant and dynamic use of transcriptional start sites across the intraerythrocytic developmental cycle that will be useful in guiding the definition of regulatory regions for use in future experimental gene expression studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12864-020-06787-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278070PMC
June 2020

Identification of a conserved var gene in different Plasmodium falciparum strains.

Malar J 2020 May 29;19(1):194. Epub 2020 May 29.

Institute of Tropical Medicine, University of Tuebingen, Wilhelmstr. 27, 72074, Tuebingen, Germany.

Background: The multicopy var gene family of Plasmodium falciparum is of crucial importance for pathogenesis and antigenic variation. So far only var2csa, the var gene responsible for placental malaria, was found to be highly conserved among all P. falciparum strains. Here, a new conserved 3D7 var gene (PF3D7_0617400) is identified in several field isolates.

Methods: DNA sequencing, transcriptional analysis, Cluster of Differentiation (CD) 36-receptor binding, indirect immunofluorescence with PF3D7_0617400-antibodies and quantification of surface reactivity against semi-immune sera were used to characterize an NF54 clone and a Gabonese field isolate clone (MOA C3) transcribing the gene. A population of 714 whole genome sequenced parasites was analysed to characterize the conservation of the locus in African and Asian isolates. The genetic diversity of two var2csa fragments was compared with the genetic diversity of 57 microsatellites fragments in field isolates.

Results: PFGA01_060022400 was identified in a Gabonese parasite isolate (MOA) from a chronic infection and found to be 99% identical with PF3D7_0617400 of the 3D7 genome strain. Transcriptional analysis and immunofluorescence showed expression of the gene in an NF54 and a MOA clone but CD36 binding assays and surface reactivity to semi-immune sera differed markedly in the two clones. Long-read Pacific bioscience whole genome sequencing showed that PFGA01_060022400 is located in the internal cluster of chromosome 6. The full length PFGA01_060022400 was detected in 36 of 714 P. falciparum isolates and 500 bp fragments were identified in more than 100 isolates. var2csa was in parts highly conserved (H = 0) but in other parts as variable (H = 0.86) as the 57 microsatellites markers (H = 0.8).

Conclusions: Individual var gene sequences exhibit conservation in the global parasite population suggesting that purifying selection may limit overall genetic diversity of some var genes. Notably, field and laboratory isolates expressing the same var gene exhibit markedly different phenotypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12936-020-03257-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260770PMC
May 2020

Illumination of a Vision 2020-Urinary Based Biomarkers for Bladder Cancer on the Way to Clinical Decisions-Dream or Nightmare?

Int J Mol Sci 2020 Mar 2;21(5). Epub 2020 Mar 2.

Department of Urology, Lukaskrankenhaus Neuss, D-41464 Neuss, Germany.

Bladder cancer is one of the most frequent malignancies worldwide [...].
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21051694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084301PMC
March 2020

A single-nucleotide polymorphism in a ApiAP2 transcription factor alters the development of host immunity.

Sci Adv 2020 02 5;6(6):eaaw6957. Epub 2020 Feb 5.

Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA.

The acquisition of malaria immunity is both remarkably slow and unpredictable. At present, we know little about the malaria parasite genes that influence the host's ability to mount a protective immune response. Here, we show that a single-nucleotide polymorphism (SNP) resulting in a single amino acid change (S to F) in an ApiAP2 transcription factor in the rodent malaria parasite () NK65 allowed infected mice to mount a T helper cell 1 (T1)-type immune response that controlled subsequent infections. As compared to NK65, NK65 parasites differentially expressed 46 genes, most of which are predicted to play roles in immune evasion. NK65 infections resulted in an early interferon-γ response and a later expansion of germinal centers, resulting in high levels of infected red blood cell-specific T1-type immunoglobulin G2b (IgG2b) and IgG2c antibodies. Thus, the ApiAP2 transcription factor functions as a critical parasite virulence factor in malaria infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aaw6957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002124PMC
February 2020

Evolutionary analysis of the most polymorphic gene family in malaria.

Wellcome Open Res 2019 3;4:193. Epub 2019 Dec 3.

Parasite Genetics, Wellcome Trust Sanger Institute, Hinxton, UK.

The gene family of the human malaria parasite encode proteins that are crucial determinants of both pathogenesis and immune evasion and are highly polymorphic. Here we have assembled nearly complete gene repertoires from 2398 field isolates and analysed a normalised set of 714 from across 12 countries. This therefore represents the first large scale attempt to catalogue the worldwide distribution of gene sequences We confirm the extreme polymorphism of this gene family but also demonstrate an unexpected level of sequence sharing both within and between continents. We show that this is likely due to both the remnants of selective sweeps as well as a worrying degree of recent gene flow across continents with implications for the spread of drug resistance. We also address the evolution of the repertoire with respect to the ancestral genes within the and show that diversity generated by recombination is concentrated in a number of hotspots. An analysis of the subdomain structure indicates that some existing definitions may need to be revised From the analysis of this data, we can now understand the way in which the family has evolved and how the diversity is continuously being generated. Finally, we demonstrate that because the genes are distributed across the genome, sequence sharing between genotypes acts as a useful population genetic marker.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12688/wellcomeopenres.15590.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001760PMC
December 2019

Application of Dried Human Amnion Graft to Improve Post-Prostatectomy Incontinence and Potency: A Randomized Exploration Study Protocol.

Adv Ther 2020 01 28;37(1):592-602. Epub 2019 Nov 28.

Department of Urology, Rhineland Clinic, Lukas Hospital Neuss, Preussenstr. 84, 41464, Neuss, Germany.

Introduction: Incontinence (up to 20%) and erectile dysfunction (up to 70%) occur frequently after radical prostatectomy (RP) in patients with localized prostate cancer. Human amniotic membrane (HAM) can improve tissue regeneration and functional outcome after RP owing to the growth factors and unique immune tolerance. Preliminary studies showed the potential value of HAM in the reconstruction of the urinary tract and nerve protection during RP.

Methods: A protocol is developed for a prospective, randomized, single-blind, single-surgeon, placebo-controlled exploration study of the efficacy and safety of dehydrated human amnion membrane placed around the neurovascular bundle (NVB) and vesicourethral anastomosis (VUA) during RP for the treatment of localized prostate cancer. Eligible for inclusion are patients with localized prostate cancer, requiring a surgical procedure and exclusion of preoperative incontinence and erectile dysfunction. The patients are randomized 1:1 to HAM vs. placebo and blinded during the study period. According to the T test with an alpha of 0.05 and a power of 80% and expecting a dropout of 20% of the patients, an adjusted sample size per arm of 164 patients is required.

Planned Outcomes: The primary outcome is a postoperative continence measured as 24-h pad test up to 12 months postoperatively. Secondary outcomes are potency, time of postoperative catheter removal, postoperative complications, and biochemical recurrence. The protocol for this randomized exploration study defines the conditions to assess the efficacy and safety of HAM application during RP in order to improve the postoperative functional outcome. This trial should pave the way for future studies of tissue engineering in an effort to reduce the morbidity of RP.

Trial Registration: Clinicaltrials.gov, identifier NCT03864939.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12325-019-01158-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6979451PMC
January 2020

Transcriptional and genomic parallels between the monoxenous parasite Herpetomonas muscarum and Leishmania.

PLoS Genet 2019 11 11;15(11):e1008452. Epub 2019 Nov 11.

Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

Trypanosomatid parasites are causative agents of important human and animal diseases such as sleeping sickness and leishmaniasis. Most trypanosomatids are transmitted to their mammalian hosts by insects, often belonging to Diptera (or true flies). These are called dixenous trypanosomatids since they infect two different hosts, in contrast to those that infect just insects (monoxenous). However, it is still unclear whether dixenous and monoxenous trypanosomatids interact similarly with their insect host, as fly-monoxenous trypanosomatid interaction systems are rarely reported and under-studied-despite being common in nature. Here we present the genome of monoxenous trypanosomatid Herpetomonas muscarum and discuss its transcriptome during in vitro culture and during infection of its natural insect host Drosophila melanogaster. The H. muscarum genome is broadly syntenic with that of human parasite Leishmania major. We also found strong similarities between the H. muscarum transcriptome during fruit fly infection, and those of Leishmania during sand fly infections. Overall this suggests Drosophila-Herpetomonas is a suitable model for less accessible insect-trypanosomatid host-parasite systems such as sand fly-Leishmania.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1008452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6872171PMC
November 2019

Exploring Gene Expression to Assess Host Selection Pressure on Parasites During Infancy.

Front Immunol 2019 9;10:2328. Epub 2019 Oct 9.

KEMRI Wellcome Trust Research Programme, Kilifi, Kenya.

In sub-Saharan Africa, children below 5 years bear the greatest burden of severe malaria because they lack naturally acquired immunity that develops following repeated exposure to infections by . Antibodies to the surface of infected erythrocytes (IE) play an important role in this immunity. In children under the age of 6 months, relative protection from severe malaria is observed and this is thought to be partly due to trans-placental acquired protective maternal antibodies. However, the protective effect of maternal antibodies has not been fully established, especially the role of antibodies to variant surface antigens (VSA) expressed on IE. Here, we assessed the immune pressure on parasites infecting infants using markers associated with the acquisition of naturally acquired immunity to surface antigens. We hypothesized that, if maternal antibodies to VSA imposed a selection pressure on parasites, then the expression of a relatively conserved subset of genes called group A genes in infants should change with waning maternal antibodies. To test this, we compared their expression in parasites from children between 0 and 12 months and above 12 months of age. The transcript quantity and the proportional expression of group A subgroup, including those containing domain cassette 13, were positively associated with age during the first year of life, which contrasts with above 12 months. This was accompanied by a decline in infected erythrocyte surface antibodies and an increase in parasitemia during this period. The observed increase in group A gene expression with age in the first year of life, when the maternal antibodies are waning and before acquisition of naturally acquired antibodies with repeated exposure, is consistent with the idea that maternally acquired antibodies impose a selection pressure on parasites that infect infants and may play a role in protecting these infants against severe malaria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.02328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798654PMC
October 2020

Modelling pathogen load dynamics to elucidate mechanistic determinants of host-Plasmodium falciparum interactions.

Nat Microbiol 2019 09 17;4(9):1592-1602. Epub 2019 Jun 17.

Section of Paediatrics, Imperial College, London, UK.

During infection, increasing pathogen load stimulates both protective and harmful aspects of the host response. The dynamics of this interaction are hard to quantify in humans, but doing so could improve understanding of the mechanisms of disease and protection. We sought to model the contributions of the parasite multiplication rate and host response to observed parasite load in individual subjects infected with Plasmodium falciparum malaria, using only data obtained at the time of clinical presentation, and then to identify their mechanistic correlates. We predicted higher parasite multiplication rates and lower host responsiveness in cases of severe malaria, with severe anaemia being more insidious than cerebral malaria. We predicted that parasite-growth inhibition was associated with platelet consumption, lower expression of CXCL10 and type 1 interferon-associated genes, but increased cathepsin G and matrix metallopeptidase 9 expression. We found that cathepsin G and matrix metallopeptidase 9 directly inhibit parasite invasion into erythrocytes. The parasite multiplication rate was associated with host iron availability and higher complement factor H levels, lower expression of gametocyte-associated genes but higher expression of translation-associated genes in the parasite. Our findings demonstrate the potential of using explicit modelling of pathogen load dynamics to deepen understanding of host-pathogen interactions and identify mechanistic correlates of protection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-019-0474-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6708439PMC
September 2019

Progression of the canonical reference malaria parasite genome from 2002-2019.

Wellcome Open Res 2019 28;4:58. Epub 2019 May 28.

Parasite Genomics, Wellcome Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK.

Here we describe the ways in which the sequence and annotation of the reference genome has changed since its publication in 2002. As the malaria species responsible for the most deaths worldwide, the richness of annotation and accuracy of the sequence are important resources for the research community as well as the basis for interpreting the genomes of subsequently sequenced species. At the time of publication in 2002 over 60% of predicted genes had unknown functions. As of March 2019, this number has been significantly decreased to 33%. The reduction is due to the inclusion of genes that were subsequently characterised experimentally and genes with significant similarity to others with known functions. In addition, the structural annotation of genes has been significantly refined; 27% of gene structures have been changed since 2002, comprising changes in exon-intron boundaries, addition or deletion of exons and the addition or deletion of genes. The sequence has also undergone significant improvements. In addition to the correction of a large number of single-base and insertion or deletion errors, a major miss-assembly between the subtelomeres of chromosome 7 and 8 has been corrected. As the number of sequenced isolates continues to grow rapidly, a single reference genome will not be an adequate basis for interpreting intra-species sequence diversity. We therefore describe in this publication a population reference genome of , called Pfref1. This reference will enable the community to map to regions that are not present in the current assembly. 3D7 will continue to be maintained, with ongoing curation ensuring continual improvements in annotation quality.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12688/wellcomeopenres.15194.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484455PMC
May 2019

An Experimental Human Blood-Stage Model for Studying Plasmodium malariae Infection.

J Infect Dis 2020 03;221(6):948-955

QIMR Berghofer Medical Research Institute.

Background: Plasmodium malariae is considered a minor malaria parasite, although its global disease burden is underappreciated. The aim of this study was to develop an induced blood-stage malaria (IBSM) model of P. malariae to study parasite biology, diagnostic assays, and treatment.

Methods: This clinical trial involved 2 healthy subjects who were intravenously inoculated with cryopreserved P. malariae-infected erythrocytes. Subjects were treated with artemether-lumefantrine after development of clinical symptoms. Prior to antimalarial therapy, mosquito-feeding assays were performed to investigate transmission, and blood samples were collected for rapid diagnostic testing and parasite transcription profiling. Serial blood samples were collected for biomarker analysis.

Results: Both subjects experienced symptoms and signs typical of early malaria. Parasitemia was detected 7 days after inoculation, and parasite concentrations increased until antimalarial treatment was initiated 25 and 21 days after inoculation for subjects 1 and 2 respectively (peak parasitemia levels, 174 182 and 50 291 parasites/mL, respectively). The parasite clearance half-life following artemether-lumefantrine treatment was 6.7 hours. Mosquito transmission was observed for 1 subject, while in vivo parasite transcription and biomarkers were successfully profiled.

Conclusions: An IBSM model of P. malariae has been successfully developed and may be used to study the biology of, diagnostic testing for, and treatment of this neglected malaria species.

Clinical Trials Registration: ACTRN12617000048381.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/infdis/jiz102DOI Listing
March 2020

A comparative analysis of response times shows that multisensory benefits and interactions are not equivalent.

Sci Rep 2019 02 27;9(1):2921. Epub 2019 Feb 27.

School of Psychology & Neuroscience, St. Mary's Quad, South Street, St. Andrews, KY16 9JP, United Kingdom.

Multisensory signals allow faster responses than the unisensory components. While this redundant signals effect (RSE) has been studied widely with diverse signals, no modelling approach explored the RSE systematically across studies. For a comparative analysis, here, we propose three steps: The first quantifies the RSE compared to a simple, parameter-free race model. The second quantifies processing interactions beyond the race mechanism: history effects and so-called violations of Miller's bound. The third models the RSE on the level of response time distributions using a context-variant race model with two free parameters that account for the interactions. Mimicking the diversity of studies, we tested different audio-visual signals that target the interactions using a 2 × 2 design. We show that the simple race model provides overall a strong prediction of the RSE. Regarding interactions, we found that history effects do not depend on low-level feature repetition. Furthermore, violations of Miller's bound seem linked to transient signal onsets. Critically, the latter dissociates from the RSE, demonstrating that multisensory interactions and multisensory benefits are not equivalent. Overall, we argue that our approach, as a blueprint, provides both a general framework and the precision needed to understand the RSE when studied across diverse signals and participant groups.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-39924-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393672PMC
February 2019

Integrated transcriptomic and proteomic analysis of pathogenic mycobacteria and their esx-1 mutants reveal secretion-dependent regulation of ESX-1 substrates and WhiB6 as a transcriptional regulator.

PLoS One 2019 23;14(1):e0211003. Epub 2019 Jan 23.

Pathogen Genomics Laboratory, BESE Division, King Abdullah University of Science and Technology (KAUST), Thuwal-Jeddah, Kingdom of Saudi Arabia.

The mycobacterial type VII secretion system ESX-1 is responsible for the secretion of a number of proteins that play important roles during host infection. The regulation of the expression of secreted proteins is often essential to establish successful infection. Using transcriptome sequencing, we found that the abrogation of ESX-1 function in Mycobacterium marinum leads to a pronounced increase in gene expression levels of the espA operon during the infection of macrophages. In addition, the disruption of ESX-1-mediated protein secretion also leads to a specific down-regulation of the ESX-1 substrates, but not of the structural components of this system, during growth in culture medium. This effect is observed in both M. marinum and M. tuberculosis. We established that down-regulation of ESX-1 substrates is the result of a regulatory process that is influenced by the putative transcriptional regulator whib6, which is located adjacent to the esx-1 locus. In addition, the overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in a significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Taken together, these data show that WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates and that ESX-1 substrates are regulated independently from the structural components, both during infection and as a result of active secretion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0211003PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343904PMC
October 2019

Low sulfated heparins target multiple proteins for central nervous system repair.

Glia 2019 04 26;67(4):668-687. Epub 2018 Dec 26.

Institute of Infection, Immunity, and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.

The lack of endogenous repair following spinal cord injury (SCI) accounts for the frequent permanent deficits for which effective treatments are absent. Previously, we demonstrated that low sulfated modified heparin mimetics (LS-mHeps) attenuate astrocytosis, suggesting they may represent a novel therapeutic approach. mHeps are glycomolecules with structural similarities to resident heparan sulfates (HS), which modulate cell signaling by both sequestering ligands, and acting as cofactors in the formation of ligand-receptor complexes. To explore whether mHeps can affect the myelination and neurite outgrowth necessary for repair after SCI, we created lesioned or demyelinated neural cell co-cultures and exposed them with a panel of mHeps with varying degrees and positions of their sulfate moieties. LS-mHep7 enhanced neurite outgrowth and myelination, whereas highly sulfated mHeps (HS-mHeps) had attenuating effects. LS-mHeps had no effects on myelination or neurite extension in developing, uninjured myelinating cultures, suggesting they might exert their proregenerating effects by modulating or sequestering inhibitory factors secreted after injury. To investigate this, we examined conditioned media from cultures using chemokine arrays and conducted an unbiased proteomics approach by applying TMT-LC/MS to mHep7 affinity purified conditioned media from these cultures. Multiple protein factors reported to play a role in damage or repair mechanisms were identified, including amyloid betaA4. Amyloid beta peptide (1-42) was validated as an important candidate by treating myelination cultures and shown to inhibit myelination. Thus, we propose that LS-mHeps exert multiple beneficial effects on mechanisms supporting enhanced repair, and represent novel candidates as therapeutics for CNS damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/glia.23562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492281PMC
April 2019

Schizont transcriptome variation among clinical isolates and laboratory-adapted clones of the malaria parasite Plasmodium falciparum.

BMC Genomics 2018 Dec 10;19(1):894. Epub 2018 Dec 10.

Pathogen Molecular Biology Department, London School of Hygiene and Tropical Medicine, London, UK.

Background: Malaria parasites are genetically polymorphic and phenotypically plastic. In studying transcriptome variation among parasites from different infections, it is challenging to overcome potentially confounding technical and biological variation between samples. We investigate variation in the major human parasite Plasmodium falciparum, generating RNA-seq data on multiple independent replicate sample preparations of merozoite-containing intra-erythrocytic schizonts from a panel of clinical isolates and from long-term laboratory-adapted clones, with a goal of robustly identifying differentially expressed genes.

Results: Analysis of biological sample replicates shows that increased numbers improve the true discovery rate of differentially expressed genes, and that six independent replicates of each parasite line allowed identification of most differences that could be detected with larger numbers. For highly expressed genes, focusing on the top quartile at schizont stages, there was more power to detect differences. Comparing cultured clinical isolates and laboratory-adapted clones, genes more highly expressed in the laboratory-adapted clones include those encoding an AP2 transcription factor (PF3D7_0420300), a ubiquitin-binding protein and two putative methyl transferases. In contrast, higher expression in clinical isolates was seen for the merozoite surface protein gene dblmsp2, proposed to be a marker of schizonts forming merozoites committed to sexual differentiation. Variable expression was extremely strongly, but not exclusively, associated with genes known to be targeted by Heterochromatin Protein 1. Clinical isolates show variable expression of several known merozoite invasion ligands, as well as other genes for which new RT-qPCR assays validate the quantitation and allow characterisation in samples with more limited material. Expression levels of these genes vary among schizont preparations of different clinical isolates in the first ex vivo cycle in patient erythrocytes, but mean levels are similar to those in continuously cultured clinical isolates.

Conclusions: Analysis of multiple biological sample replicates greatly improves identification of genes variably expressed between different cultured parasite lines. Clinical isolates recently established in culture show differences from long-term adapted clones in transcript levels of particular genes, and are suitable for analyses requiring biological replicates to understand parasite phenotypes and variable expression likely to be relevant in nature.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12864-018-5257-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6288915PMC
December 2018

UBC Test-A Urinary Point-of-Care (POC) Assay for Diagnosis of Bladder Cancer with a focus on Non-Muscle Invasive High-Grade Tumors: Results of a Multicenter-Study.

Int J Mol Sci 2018 Dec 2;19(12). Epub 2018 Dec 2.

University Hospital for Urology, Klinikum Oldenburg, School of Medicine and Health Sciences Carl von Ossietzky University Oldenburg, Oldenburg D-26133, Germany.

Objectives: UBC Test measures soluble fragments of cytokeratins 8 and 18 in urine. We present results of a multicenter study using an updated version of UBC Test in bladder cancer patients, patients with urinary bladder cancer positive history, and healthy controls.

Material And Methods: In total 530 urine samples have been included in this study. Clinical urine samples were used from 242 patients with tumors of the urinary bladder (134 non-muscle-invasive low-grade tumors (NMI-LG), 48 non-muscle-invasive high-grade tumors (NMI-HG), and 60 muscle-invasive high-grade tumors (MI-HG)), 62 patients with non-evidence of disease (NED), and 226 healthy controls. Urine samples were analyzed by the UBC Rapid point-of-care (POC) assay and evaluated by Concile Omega 100 POC Reader. All statistical analyses have been performed using R version 3.2.3.

Results: Elevated levels of UBC Rapid Test in urine are higher in patients with bladder cancer in comparison to the control group ( < 0.001). The sensitivity for the whole bladder cancer cohort was 53.3% (positive predictive value (PPV) 90.2%, negative predictive value (NPV) 65.2%) and was 38.8% (PPV 78.8%, NPV 72.1%) for non-muscle-invasive low-grade bladder cancer; 75.0% (PPV 72.0%, NPV 94.7%) for non-muscle-invasive high-grade bladder cancer and 68.3% (PPV 74.6%, NPV 91.8%) for muscle-invasive high-grade bladder cancer. The specificity for the statistical calculations was 93.8%. The cut-off value (10 µg/L) was evaluated for the whole patient cohort. The area under the curve of the quantitative UBC Rapid Test using the optimal threshold obtained by receiver operating characteristics (ROC) analysis was 0.774. Elevated values of UBC Test in urine are higher in patients with high-grade bladder cancer in comparison to low-grade tumors and the healthy control group.

Conclusions: UBC Test has potential to be a clinically valuable urinary protein biomarker for detection of high-grade bladder cancer patients and could be added in the management of NMI-HG tumors. UBC results generated in both study centers in the present multicenter study are very similar and reproducible. Furthermore UBC Test is standardized and calibrated and thus independent of used batch of test as well as study site.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms19123841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321532PMC
December 2018

Pre-clinical evaluation of a -based whole-sporozoite malaria vaccine candidate.

NPJ Vaccines 2018 27;3:54. Epub 2018 Nov 27.

1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal.

Whole-sporozoite vaccination/immunization induces high levels of protective immunity in both rodent models of malaria and in humans. Recently, we generated a transgenic line of the rodent malaria parasite () that expresses the () circumsporozoite protein (CS), and showed that this parasite line (Vac) was capable of (1) infecting and developing in human hepatocytes but not in human erythrocytes, and (2) inducing neutralizing antibodies against the human parasite. Here, we analyzed Vac in detail and developed tools necessary for its use in clinical studies. A microbiological contaminant-free Master Cell Bank of Vac parasites was generated through a process of cyclic propagation and clonal expansion in mice and mosquitoes and was genetically characterized. A highly sensitive qRT-PCR-based method was established that enables Vac parasite detection and quantification at low parasite densities in vivo. This method was employed in a biodistribution study in a rabbit model, revealing that the parasite is only present at the site of administration and in the liver up to 48 h post infection and is no longer detectable at any site 10 days after administration. An extensive toxicology investigation carried out in rabbits further showed the absence of Vac-related toxicity. In vivo drug sensitivity assays employing rodent models of infection showed that both the liver and the blood stage forms of Vac were completely eliminated by Malarone treatment. Collectively, our pre-clinical safety assessment demonstrates that Vac possesses all characteristics necessary to advance into clinical evaluation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41541-018-0091-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258718PMC
November 2018