Soil-transmitted helminth and Schistosoma mansoni infections do not evoke cross-reactive antibodies to the Onchocerca volvulus peptide epitopes OvMP-1 and OvMP-23.

The Ov16 IgG4 ELISA or lateral flow test is considered the reference method for Onchocerca volvulusepidemiological mapping. Recently, two linear epitopes encoded in OvMP-1 and OvMP-23 peptides were introduced as serological markers, but the observed antibody cross-reactivity in samples originating from O. volvulusnon-endemic areas required further investigation. In this study, we evaluated these markers in an Onchocercameso-endemic setting in Jimma, Ethiopia. For all individuals(n = 303), the infection status with soil-transmitted helminths and Schistosoma mansoniwas known. In total, 11 (3.6%) individuals were positive for anti-Ov16 IgG4 antibodies, while 34 (11.2%) and 15 (5.0%) individuals had antibody responses to OvMP-1 andOvMP-23, respectively. Out of the 34 OvMP-1 positive samples, 33 were negative on the Ov16 IgG4 ELISA. Similarly, out of the 15 OvMP-23 positive samples, 14 scored negative on this reference method. Upon further analysis of the “false positive samples” for infection with non-Onchocercahelminth infections, they were not significantly correlated to soil-transmitted helminth (p> 0.05) or S. mansoniinfections (p> 0.05). This suggests that these individuals are either infected with O.volvulus and were not picked up by the Ov16 IgG4 ELISA, or that they have an immune response against other agents that cause cross-reactivity. For OvMP-1, there appeared to be a significant trend towards increased seroprevalence in older individuals. The results of this work demonstrate that both OvMP-1 and OvMP23 do not cross-react with soil-transmitted helminth or S. mansoniinfections. The discordancy with the Ov16 test requires further investigation in Onchocercaendemic populations

Patent infections with soil-transmitted helminths and Schistosoma mansoni are not associated with increased prevalence of antibodies to the Onchocerca volvulus peptide epitopes OvMP-1 and OvMP-23

Background: Ov16 serology is considered a reference method for Onchocerca volvulus epidemiological mapping. Given the suboptimal sensitivity of this test and the fact that seroconversion takes more than a year after infection, additional serological tests might be needed to guide onchocerciasis elimination programmes. Recently, two linear epitopes encoded in OvMP-1 and OvMP-23 peptides were introduced as serological markers, but the observed antibody cross-reactivity in samples originating from Onchocerca volvulus non-endemic areas required further investigation. Methods: We evaluated both peptide markers in an O. volvulus hypo-endemic setting in Jimma Town, Ethiopia using peptide ELISA. For all individuals (n = 303), the infection status with soil-transmitted helminths and Schistosoma mansoni was known. Results: We found that 11 (3.6%) individuals were positive for anti-Ov16 IgG4 antibodies, while 34 (11.2%) and 15 (5.0%) individuals were positive for OvMP-1 and OvMP-23, respectively. Out of the 34 OvMP-1 positive samples, 33 were negative on the Ov16 IgG4 ELISA. Similarly, out of the 15 OvMP-23 positive samples, 14 scored negative on this reference method. No difference in seroprevalence for all three markers could be observed between uninfected individuals and individuals infected with different soil-transmitted helminths or S. mansoni. Moreover, the intensity of the response to OvMP-1, OvMP-23 or Ov16 was not significantly stronger in individuals carrying patent STH or S. mansoni infections, nor was there any correlation between the intensities of the responses to the three different antigens. Conclusions: This study demonstrates that a patent infection with either soil-transmitted helminths or S. mansoni does not lead to increased antibody recognition of both OvMP-1 and OvMP23

Detection of Ascaris lumbricoides infection by ABA-1 coproantigen ELISA

Intestinal worms, or soil-transmitted helminths (STHs), affect hundreds of millions of people in all tropical and subtropical regions of the world. The most prevalent STH isAscaris lumbricoides. Through large-scale deworming programs, World Health Organization aims to reduce morbidity, caused by moderate-to-heavy intensity infections, below 2%. In order to monitor these control programs, stool samples are examined microscopically for the presence of worm eggs. This procedure requires well-trained personnel and is known to show variability between different operators interpreting the slides. We have investigated whether ABA-1, one of the excretory-secretory products ofA.lumbricoidescan be used as a coproantigen marker for infection with this parasite. Polyclonal antibodies were generated and a coproantigen ELISA was developed. Using this ELISA, it was found that ABA-1 in stool detectedAscarisinfection with a sensitivity of 91.5% and a specificity of 95.3%. Our results also demonstrate that there is a correlation between ABA-1 levels in stool andA.lumbricoidesDNA detected in stool. Using a threshold of 18.2 ng/g stool the ABA-1 ELISA correctly assigned 68.4% of infected individuals to the moderate-to-heavy intensity infection group, with a specificity of 97.1%. Furthermore, the levels of ABA-1 in stool were shown to rapidly and strongly decrease upon administration of a standard anthelminthic treatment (single oral dose of 400 mg albendazole). In anAscaris suuminfection model in pigs, it was found that ABA-1 remained undetectable until day 28 and was detected at day 42 or 56, concurrent with the appearance of worm eggs in the stool. This report demonstrates that ABA-1 can be considered anAscaris-specific coproantigen marker that can be used to monitor infection intensity. It also opens the path for development of point-of-care immunoassay-based tests to determineA.lumbricoidesinfection in stool at the sample collection site. Author summary Intestinal worms are one of the most common infections in tropical and subtropical parts of the world. The roundwormAscaris lumbricoidesis the most prevalent and efforts are ongoing to use preventive chemotherapy to reduce both prevalence and intensity of this infection. To monitor these programs, stool-based microscopy is currently used. We have investigated the possibility of using ABA-1, an abundantly secreted protein from the worm, as a biomarker in stool of infected individuals. We have developed an ELISA and using this assay determined that ABA-1 as stool biomarker had a sensitivity of 91.5% and a specificity of 95.3% to detect infection withA.lumbricoides. We also showed that ABA-1 in stool rapidly and strongly decreased upon administration of a standard anthelminthic treatment. The main asset of this novel stool biomarker is its potential to be used in of point-of-care immunoassay-based tests to determineA.lumbricoidesinfection in stool at the sample collection site

2-Methyl-pentanoyl-carnitine (2-MPC) : a urine biomarker for patent Ascaris lumbricoides infection

Infections with intestinal worms, such as Ascaris lumbricoides, affect hundreds of millions of people in all tropical and subtropical regions of the world. Through large-scale deworming programs, World Health Organization aims to reduce moderate-to-heavy intensity infections below 1%. Current diagnosis and monitoring of these control programs are solely based on the detection of worm eggs in stool. Here we describe how metabolome analysis was used to identify the A. lumbricoides-specific urine biomarker 2-methyl pentanoyl carnitine (2-MPC). This biomarker was found to be 85.7% accurate in determining infection and 90.5% accurate in determining a moderate-to-heavy infection. Our results also demonstrate that there is a correlation between 2-MPC levels in urine and A. lumbricoides DNA detected in stool. Furthermore, the levels of 2-MPC in urine were shown to rapidly and strongly decrease upon administration of a standard treatment (single oral dose of 400 mg albendazole). In an Ascaris suum infection model in pigs, it was found that, although 2-MPC levels were much lower compared to humans, there was a significant association between urinary 2-MPC levels and both worm counts (p=0.023) and the number of eggs per gram (epg) counts (p<0.001). This report demonstrates that urinary 2-MPC can be considered an A. lumbricoides-specific biomarker that can be used to monitor infection intensity

Amino acid sensing and signalling by Gap1 in the yeast Saccharomyces cerevisiae

For unicellular organisms, such as the yeastTABLE OF CONTENTS i LIST OF ABBREVIATIONS v LIST OF GENES vii SAMENVATTING 1 SUMMARY 3 SCOPE OF THIS THESIS 5 CHAPTER I 7 NUTRIENT SENSING PATHWAYS IN YEAST 7 1. Introduction 8 2. Pathways responding to the availability of nutrients 8 2.1. The Ras-cAMP pathway 8 2.1.1. Mechanisms controlling activity of PKA 10 2.1.2. PKA targets 12 2.1.2.1. Transcriptional control 12 2.1.2.2. Control of reserve carbohydrates 13 2.1.2.3. Other targets 14 2.2. The Fermentable Growth Medium-induced (FGM) pathway 15 2.2.1. General overview 15 2.2.2. Nutrient sensors involved in the FGM pathway 16 2.2.2.1. Gap1, an amino acid sensor 17 2.2.2.2. Mep2, an ammonium sensor 18 2.2.2.3. Pho84 and Pho87, phosphate sensors 18 2.3. The TOR pathway 19 3. Nitrogen sensing pathways 24 3.1. SPS sensing system 24 3.2. Nitrogen catabolite repression 27 3.3. General amino acid control 31 4. Regulated trafficking of the General Amino acid Permease Gap1 33 4.1. The endoplasmic reticulum: a first checkpoint 34 4.2. Early secretory pathway: getting out of the ER … 35 4.3. Early secretory pathway: … and into the Golgi 38 4.4. Late secretory pathway: from the Golgi to the plasma membrane… or vacuole 40 4.5. Endocytosis and degradation 43 4.6. Activity dependent inactivation of Gap1 47 CHAPTER II 49 ANALYSIS OF GAP1 AS AN AMINO ACID SENSOR 49 1. Abstract 50 2. Introduction 51 3. Search for Gap1 domains involved in amino acid sensing 52 3.1. Analysis of Gap1-Can1 chimeras 52 3.2. Point mutagenesis of the intracellular loops of Gap1 54 4. Discussion 57 CHAPTER III 61 ANALYSIS OF CONSTITUVELY SIGNALLING GAP1 ALLELES 61 1. Abstract 62 2. Introduction 63 3. Search for signalling pathways involved in mediating the effect of the constitutive Gap1 alleles 63 3.1. Gap1ΔC6 does not genetically interact with Tor 63 3.2. Gap1ΔC6 does not genetically interact with Sch9 66 3.3. Gap1ΔC6 does not genetically interact with the PI-3 kinase, Stt4 67 3.4. Gap1ΔC6 exerts its effect downstream of adenylate cyclase 68 4. Requirement of seg1-1 for Gap1 allele specific phenotypes 69 4.1. The overactive effect of Gap1ΔC6 is dominant only in one particular gap1Δ background 69 4.2. Effect of seg1-1 mutation on other Gap1 alleles 71 4.3. Mutations in the C-terminally localized ER exit domain of Gap1 abolish the overactive phenotype of Gap1ΔC6 74 4.4. seg1-1 affects the accumulation of Gap1 at the plasma membrane 75 4.5. Identification of the genomic location of seg1-1 76 4.6. Multi-copy suppressor analysis of the seg1-1 mutation 78 4.7. Overexpression of Gap1 does not mimic the overactivating effects of Gap1DC6 79 5. Discussion 81 CHAPTER IV 87 SEARCH FOR NOVEL COMPONENTS IN THE FGM PATHWAY 87 1. Abstract 88 2. Introduction 89 3. Buoyant density as a novel PKA target in yeast: investigation and use in library screening 91 3.1. Buoyant density is regulated by PKA 92 3.2. Buoyant density appears to be independent of trehalose or glycogen 94 3.3. Screening of the yeast deletion collection for strains deficient in amino acid induced decrease in buoyant density 95 4. Role of Kin1 and Kin2 in nitrogen sensing 97 4.1. Kin1 and Kin2 do not play a role in amino acid induced activation of the FGM pathway 98 4.2. Kin1 and Kin2 do not play a role in transition to stationary phase upon nitrogen depletion 99 5. Purification of the Gap1 protein complex 101 5.1. A TAP-tagged Gap1 is functional 102 5.2. Isolation and identification of interacting proteins 103 6. Discussion 104 CHAPTER V 107 GENERAL DISCUSSION, CONCLUSIONS AND PERSPECTIVES 107 1. Introduction 108 2. Specific domains in Gap1 involved in amino acid sensing 108 3. Characterisation of the constitutively signalling Gap1 alleles 109 4. A search for novel components involved in Gap1-mediated activation of the FGM pathway 112 CHAPTER VI 115 MATERIALS AND METHODS 115 1. Materials 116 1.1. Yeast strains 116 1.2. Bacterial strains 118 1.3. Plasmids 118 1.4. Primers used in this work 119 2. Methods 122 2.1. Molecular biology methods 122 2.1.1. Cloning techniques and sequencing 122 2.1.2. Gene deletions 122 2.1.3. TAP-tagging of Gap1 122 2.1.4. Mutagenesis of Gap1 123 2.1.5. Construction of Gap1-Can1 chimeras 124 2.1.6. Construction of pFL44LGAP1 125 2.1.7. Selection of multi-copy suppressors of seg1-1 125 2.1.8. Identification of the genomic location of seg1-1 125 2.2. Culture and starvation conditions 126 2.2.1. Bacteria 126 2.2.2. Yeast cells 126 2.3. Growth curves 127 2.4. Growth assay on plates 127 2.5. Percoll gradient centrifugation 128 2.6. Trehalase determination 128 2.7. Trehalose determination 129 2.8. Transport of L-citrulline 129 2.9. Western analysis of Gap1 130 2.10. Cell fractionation and equilibrium density centrifugation 131 2.11. Tandem affinity purification of the Gap1 complex 132 2.11.1. Preparation of the extract 132 2.11.2. Tandem affinity purification 132 2.11.3. Separation of the isolated proteins 133 References 135status: publishe

REVIEW The miRNA world of polyomaviruses

Polyomaviruses are a family of non-enveloped DNA viruses infecting several species, including humans, primates, birds, rodents, bats, horse, cattle, raccoon and sea lion. They typically cause asymptomatic infection and establish latency but can be reactivated under certain conditions causing severe diseases. MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in several cellular processes by binding to and inhibiting the translation of specific mRNA transcripts. In this review, we summarize the current knowledge of microRNAs involved in polyomavirus infection. We review in detail the different viral miRNAs that have been discovered and the role they play in controlling both host and viral protein expression. We also give an overview of the current understanding on how host miRNAs may function in controlling polyomavirus replication, immune evasion and pathogenesis

Circulating human microRNAs are not linked to JC polyomavirus serology or urinary viral load in healthy subjects

BACKGROUND: JC polyomavirus (JCPyV) is a widespread human polyomavirus that usually resides latently in its host. It can be reactivated under immunomodulating conditions and cause Progressive Multifocal Leukoencephalopathy (PML). Circulating microRNAs (miRNAs) are emerging as promising biomarkers for several pathologies. In this study, we have investigated whether circulating miRNAs exist that are differentially expressed between JCPyV seropositive and JCPyV seronegative on the one hand or between JCPyV shedders and JCPyV non-shedders on the other hand. METHODS: Human miRNA expression profiling was performed in a small set of plasma samples obtained from seronegative subjects, seropositive shedders and seropositive non-shedders. A set of 10 miRNAs was selected for further analysis in a larger group of samples. RESULTS: Based on the plasma profiling experiment of 30 samples, 6 miRNAs were selected that were possibly differentially expressed between seropositive and seronegative subjects and 4 miRNAs were selected that were possibly differentially expressed between shedders and non-shedders. Subsequently, expression of these 10 selected miRNAs was assessed in an independent set of 100 plasma samples. Results indicated that none of them were differentially expressed. CONCLUSION: This study could not identify circulating human miRNAs that were differentially expressed between plasma from JCPyV seropositive and JCPyV seronegative subjects or between JCPyV shedders and JCPyV non-shedders.status: publishe

Droplet digital PCR is an accurate method to assess methylation status on FFPE samples

Most tissue samples available for cancer research are archived as formalin-fixed paraffin-embedded (FFPE) samples. However, the fixation process and the long storage duration lead to DNA fragmentation and hinder epigenome analysis. The use of droplet digital PCR (ddPCR) to detect DNA methylation has recently emerged. In this study, we compare an optimized ddPCR assay with a conventional qPCR assay by targeting a dilution series of control DNA. In addition, we compare the ddPCR technology with results from Infinium arrays targeting two separate CpG sites on a set of colon adenoma FFPE samples. Our data demonstrate that qPCR and ddPCR assess methylation status equally well on dilution controls with a high DNA input. However, the methylation detection on low-input samples is more accurate using ddPCR. The proposed primer design (methylation-independent primers with amplification of solely the converted DNA target) will allow for methylation detection, independent of bisulfite conversion efficiency. Those data show that ddPCR can be used for methylation analysis on FFPE samples with a wide range of DNA input and that the precision of the assay depends largely on the total amount of amplifiable DNA fragments. Due to accessibility of the ddPCR technology and its accuracy on high- as well as low-DNA input samples, we propose the use of this approach for studies involving degraded FFPE samples

Reactivity in peptide ELISA can be inhibited by peptides with the same motif.

<p>Reduction in titer was determined by addition of non-biotinylated peptide to 2 different samples during incubation on the ELISA plates. Plates are coated with the biotinylated peptide as indicated in the x-axis. Titration curves were determined (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005330#pntd.0005330.s003" target="_blank">S3 Fig</a>) and titer was calculated as the dilution showing an Abs_405nm that corresponds to 10-fold reduction in absorbance of the non-inhibited sample at the start dilution (i.e. 200-fold). As a negative control, the non-relevant peptide JCV_VP2_167-15mer was included. All self-inhibition results are indicated with *.</p

Assessment of immune response against peptides with motifs using peptide ELISA.

<p>Immunoreactivity against the 12 selected peptides was determined in a set of 21 <i>O</i>. <i>volvulus</i> infected individuals (group 1), healthy controls from Southern Africa (group 2), healthy controls from Belgium (group 3) and individuals infected with HIV (group 4), HCV (group 5), Dengue (group 6), <i>P</i>. <i>falciparum</i> (group 7) and <i>W</i>. <i>bancrofti</i> (group 8) and asthma patients (group 9).</p