Epigenetics and proteomics join transcriptomics in the quest for tuberculosis biomarkers

CITATION: Esterhuyse, M. M. et al. 2015. Epigenetics and proteomics join transcriptomics in the quest for tuberculosis biomarkers. mBio, 6(5):e01187-15, doi:10.1128/mBio.01187-15.The original publication is available at http://mbio.asm.orgAn estimated one-third of the world’s population is currently latently infected with Mycobacterium tuberculosis. Latent M. tuberculosis infection (LTBI) progresses into active tuberculosis (TB) disease in ~5 to 10% of infected individuals. Diagnostic and prognostic biomarkers to monitor disease progression are urgently needed to ensure better care for TB patients and to decrease the spread of TB. Biomarker development is primarily based on transcriptomics. Our understanding of biology combined with evolving technical advances in high-throughput techniques led us to investigate the possibility of additional platforms (epigenetics and proteomics) in the quest to (i) understand the biology of the TB host response and (ii) search for multiplatform biosignatures in TB. We engaged in a pilot study to interrogate the DNA methylome, transcriptome, and proteome in selected monocytes and granulocytes from TB patients and healthy LTBI participants. Our study provides first insights into the levels and sources of diversity in the epigenome and proteome among TB patients and LTBI controls, despite limitations due to small sample size. Functionally the differences between the infection phenotypes (LTBI versus active TB) observed in the different platforms were congruent, thereby suggesting regulation of function not only at the transcriptional level but also by DNA methylation and microRNA. Thus, our data argue for the development of a large-scale study of the DNA methylome, with particular attention to study design in accounting for variation based on gender, age, and cell type.http://mbio.asm.org/content/6/5/e01187-15.abstract?sid=fe0ea1c7-6da2-4e53-b4a4-5cd8233777c7Publisher's versio

Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage

Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use meta-genomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.Peer reviewe

Setting a baseline for global urban virome surveillance in sewage

The rapid development of megacities, and their growing connectedness across the world is becoming a distinct driver for emerging disease outbreaks. Early detection of unusual disease emergence and spread should therefore include such cities as part of risk-based surveillance. A catch-all metagenomic sequencing approach of urban sewage could potentially provide an unbiased insight into the dynamics of viral pathogens circulating in a community irrespective of access to care, a potential which already has been proven for the surveillance of poliovirus. Here, we present a detailed characterization of sewage viromes from a snapshot of 81 high density urban areas across the globe, including in-depth assessment of potential biases, as a proof of concept for catch-all viral pathogen surveillance. We show the ability to detect a wide range of viruses and geographical and seasonal differences for specific viral groups. Our findings offer a cross-sectional baseline for further research in viral surveillance from urban sewage samples and place previous studies in a global perspective

Characterization of candidate genes related to estrogenic activity in Oreochromis mossambicus

Thesis (PhD (Botany and Zoology))--Stellenbosch University, 2008.Endocrine disruption is an alteration of the chemical messaging processes in the body. The value of studies‐ and monitoring of endocrine disruption using techniques included in the field of toxicogenomics is undoubtedly supported by scientific literature over the past four decades, as is demonstrated in Chapter 1 where I review relevant literature on the topic. Clearly, well sustained bio‐monitoring will include studies both in vitro and in vivo, and very well on transcriptional and translational levels. Animals are providing good models for in vivo studies to report or monitor endocrine disruption. It is imperative though to first understand such an animal’s biology, especially its endocrine system, and characterize what is considered “normal” for a species before engaging in endocrine disrupting exposures. A multitude of studies report endocrine disruption in relation to reproductive systems, with more recent work illustrating alteration of metabolism related to thyroidogenic disruption within the last decade. It is therefore essential to consider sex determination and ‐differentiation when studying sentinel species. Apart from the obvious academic interest in the matter of sex differentiation, altered patterns of sex differentiation in certain appropriate species provide for a very convincing endpoint in monitoring estrogenic endocrine disruption. As I approach to study a potential sentinel species for the southern African subcontinent, I set forward to study aspects of endocrine disruption influencing the reproductive system in a piece‐meal manner, starting with estrogenic endocrine disruption as this is the best studied facet of the endocrine disruption hypothesis to date. Yet, one learn from vast amounts of literature that in cases where sex is not exclusively determined by the genetic fraction of an individual, a number other characteristics may very well be used to determine estrogenic disruption in ecosystems. Quantitative production of the egg yolk precursor protein (vitellogenin) resides under these characteristics, and in the proposed sentinel, South African tilapiine, Oreochromis mossambicus phenotypic sex can be altered by environmental sex determination. The present study therefore targeted firstly the product most often used in tier I screening processes, vitellogenin (VTG). Specimens of O. mossambicus were cultured for this purpose from wild breeding stock, sampled at 5 day intervals and the transcription levels of vitellogenin gene (vtg) studied in those. Hereby, Chapter 2 describes the cloning of partial vtg gene and subsequent temporal expression of vtg quantitatively in O. mossambicus. To shed light on the state of gonadal differentiation sub‐samples were subjected to histology, illustrated in Chapter 3. In addition the quantitative vtg responses has been described in this study at a transcriptional level, both of adult males and juveniles subjected to low and very high levels of natural estrogens. In addition, a 3 kb 5’ flanking region of vtg was cloned and sequenced, and several putative binding sites identified for transcription factors of vtg, including several estrogen responsive elements (EREs). These indicate the expected regulational process of vtg by estrogens. Subsequently I measured the transcription levels of the only enzyme capable of aromatizing androgens into estrogens, Cytochrome P450 19 (cyp19) as has been characterized in Chapter 3. For stable binding of an estrogen to an ERE, binding of the ligand to its specific nuclear receptor (Estrogen receptor, ESR) is required. Since E2 is known to have different mechanisms of action in vertebrates, the expression levels of the ESRs were evaluated in our sample set after cloning 3 different homologues of ESR in O. mossambicus. The results on this matter is discussed in Chapter 4 and provides in addition to data on vtg and cyp19 a platform of “normal” transcription levels of these candidate genes involved in estrogenic endocrine disruption of O. mossambicus. Ultimately, characterization of those candidate genes involved extensively in phenotypic sex, contribute to our understanding of sex determination and differentiation in this species in a small way

Die ontwikkeling en evaluering van 'n opleidingskurrikulum vir geskrewe kommunikasie

Tesis (M. Ed.) -- Universiteit van Stellenbosch, 1998.Een kopie mikrofiche.Full text to be digitised and attached to bibliographic record

n Geïntegreerde bevoegdheidsgebaseerde model vir die ontwikkeling van menslike hulpbronne in 'n gesondheidsorganisasie : 'n onderwys- en opleidingsperspektief

Thesis (PhD)--University of Stellenbosch, 2002.ENGLISH ABSTRACT: The problem that lead to this study is whether the view of the personnel in the utilisation review centre in the organisation with reference to the management of human resources as well as their work performance increased after implementation of the integrated competencybased human resource model. The general purpose of this study is to develop through extensive literature survey an integrated competency-based human resource model, to implement it in the organisation and to measure the impact the model had on the personnel in the utilisation review centre in the organisation. The dimensions according to which the impact was measured, are the views of the personnel in the organisation and their work performance. The following methods were used in developing and implementing the integrated competency-based human resource model: an analysis of the work processes and tasks was done to determine the flow of work through the utilisation review centre. Thereafter the different roles were identified. Simultaneously an analysis of the strategy of the organisation was conducted to determine what impact it would have on the work processes and tasks in future. Once the analyses were completed, role descriptions were compiled for the identified roles. The role descriptions are used in the development of the various human resource management activities, viz. recruitment and selection, education and development, performance management and remuneration. Throughout the implementation process the human resource management activities are evaluated to determine that it complies with the regulations of the labour laws. An opinion survey questionnaire was used to determine the view of the personnel in the utilisation review centre with reference to the methods used to implement the integrated competency-based human resource model and whether the methods were successfully executed. Thirty of the 50 respondents partook in this study. A one-group first measurementsecond measurement design was used to determine whether the model contributed to the expected results. The design allowed a single group to complete a first measurement twelve months and a second measurement 24 months after the model was implemented (January 2002 to December 2001). The outcome of this study is based on the degree of variance between the first and second measurements. The quality requirements were used to evaluate the work performance of the participants (40 respondents) on a six-monthly basis. Forty of the 50 respondents partook in this study. This was done in order to determine whether there was an improvement in the work performance of the personnel. The most significant conclusions of this study can be summarised as follows: o There was a significant difference between the first and second measurements, which indicated that the participants were of the opinion that the model had a positive impact in the organisation. o There was a significant improvement in the work performance of the participants, which indicates that the modelled to an improvement in the work performance in the organisation where the study was carried out. Based on the findings of the study, recommendations are made for further research in this field. The study succeeded in developing and implementing an integrated competency-based human resource model, which can be used with certainty by organisations to develop their personnel and which can therefore enable organisations to reach and retain a competitive advantage.AFRIKAANSE OPSOMMING: Die probleem wat aanleiding gegee het tot die studie is of personeellede verbonde aan die benuttingsoorsigsentrum in die organisasie se mening rakende die bestuur van menslike hulpbronne, asook hulle werksprestasie sal verbeter ná implementering van die geïntegreerde bevoegdheidsgebaseerde menslikehulpbronmodel. Die algemene doelstelling van hierdie studie is om aan die hand van 'n omvattende literatuurstudie 'n geïntegreerde bevoegdheidsgebaseerde menslikehulpbronmodel te ontwikkel, dit in die organisasie te implementeer en die impak daarvan op die personeel in die benuttingsoorsigsentrum in die organisasie te takseer. Die dimensies waarteen die impak getakseer word, is die mening van die personeel in die organisasie en hulle werksprestasie. Die volgende metodes is aangewend m die ontwikkeling en implementering van die geïntegreerde bevoegdheidsgebaseerde menslikehulpbronmodel: 'n analise van die werksprosesse en take is uitgevoer om te bepaal wat die verloop van werk deur die benuttingsoorsigsentrum is. Daarna is die verskillende rolle geïdentifiseer. Terselfdertyd is daar ook 'n analise van die organisasiestrategie gedoen om te bepaal watter impak dit in die toekoms op die werksprosesse en take gaan hê. Nadat die analises afgehandel is, is rolbeskrywings opgestel vir die geïdentifiseerde rolle. Die rolbeskrywings word gebruik vir die ontwikkeling van die verskillende menslikehulpbronbestuursaktiwiteite, naamlik werwing, keuring en seleksie, opvoeding en ontwikkeling, prestasiebestuur en vergoeding. Deurlopend deur die implementeringsproses word daar seker gemaak dat die menslikehulpbronbestuursaktiwiteite voldoen aan die bepalings van die verskillende arbeidswette. 'n Meningsopnamevraelys is gebruik om te bepaal wat die mening van personeel in die benuttingsoorsigsentrum is oor die metodes wat gebruik is om die geïntegreerde bevoegdheidsgebaseerde menslikehulpbronmodel te implementeer en of die metodes suksesvol deurgevoer is. Dertig van die 50 deelnemers het aan die ondersoek deelgeneem. Om te bepaal of die beoogde resultate wel bereik is, is 'n enkelgroep-eerste-tweede-metingontwerp vir die evaluering daarvan gebruik. Inhierdie tipe ontwerp lê 'n enkelgroep 'n eerste meting twaalf maande en 'n tweede meting 24 maande ná die implementering van die model af (Januarie 2000 tot Desember 2001). Die resultaat van die ondersoek is die mate van verandering tussen die eerste en tweede meting. Evaluering van die ondersoekgroep se werksprestasie is op 'n sesmaandelikse basis aan die hand van die kwaliteitsvereistes gedoen. Veertig van die 50 deelnemers het aan die ondersoek deelgeneem. Die doel hiermee is om ondersoek in te stelof daar 'n verbetering in die werksprestasie van die personeel voorgekom het. Die vernaamste bevindinge van die studie kan soos volg opgesom word: D Daar is beduidende verskille tussen die eerste en tweede metings, wat daarop dui dat die ondersoekgroep van mening is dat die model wel 'n positiewe impak in die organisasie het. D Daar is 'n beduidende verbetering in die werksprestasie van die ondersoekgroep, wat daarop dui dat die model tot 'n verbetering in die werksprestasie binne die organisasie waar die studie uitgevoer is, gelei het. Gebaseer op die bevindinge van die studie word sekere aanbevelings aan die hand gedoen met die oog op toekomstige navorsing. Die studie het daarin geslaag om 'n geïntegreerde bevoegdheidsgebaseerde menslikehulpbronmodel te ontwikkel en te implementeer wat met sekerheid deur organisasies gebruik kan word om hulle personeel te ontwikkel. Sodoende word die organisasies in staat gestel om 'n mededingende voordeel te bekom en te behou

Epigenetics and Proteomics Join Transcriptomics in the Quest for Tuberculosis Biomarkers

An estimated one-third of the world's population is currently latently infected with Mycobacterium tuberculosis. Latent M. tuberculosis infection (LTBI) progresses into active tuberculosis (TB) disease in ~5 to 10% of infected individuals. Diagnostic and prognostic biomarkers to monitor disease progression are urgently needed to ensure better care for TB patients and to decrease the spread of TB. Biomarker development is primarily based on transcriptomics. Our understanding of biology combined with evolving technical advances in high-throughput techniques led us to investigate the possibility of additional platforms (epigenetics and proteomics) in the quest to (i) understand the biology of the TB host response and (ii) search for multiplatform biosignatures in TB. We engaged in a pilot study to interrogate the DNA methylome, transcriptome, and proteome in selected monocytes and granulocytes from TB patients and healthy LTBI participants. Our study provides first insights into the levels and sources of diversity in the epigenome and proteome among TB patients and LTBI controls, despite limitations due to small sample size. Functionally the differences between the infection phenotypes (LTBI versus active TB) observed in the different platforms were congruent, thereby suggesting regulation of function not only at the transcriptional level but also by DNA methylation and microRNA. Thus, our data argue for the development of a large-scale study of the DNA methylome, with particular attention to study design in accounting for variation based on gender, age, and cell type

Africa-wide evaluation of host biomarkers in QuantiFERON supernatants for the diagnosis of pulmonary tuberculosis

Abstract We investigated host-derived biomarkers that were previously identified in QuantiFERON supernatants, in a large pan-African study. We recruited individuals presenting with symptoms of pulmonary TB at seven peripheral healthcare facilities in six African countries, prior to assessment for TB disease. We then evaluated the concentrations of 12 biomarkers in stored QuantiFERON supernatants using the Luminex platform. Based on laboratory, clinical and radiological findings and a pre-established algorithm, participants were classified as TB disease or other respiratory diseases(ORD). Of the 514 individuals included in the study, 179(34.8%) had TB disease, 274(51.5%) had ORD and 61(11.5%) had an uncertain diagnosis. A biosignature comprising unstimulated IFN-γ, MIP-1β, TGF-α and antigen-specific levels of TGF-α and VEGF, identified on a training sample set (n = 311), validated by diagnosing TB disease in the test set (n = 134) with an AUC of 0.81(95% CI, 0.76–0.86), corresponding to a sensitivity of 64.2%(95% CI, 49.7–76.5%) and specificity of 82.7%(95% CI, 72.4–89.9%). Host biomarkers detected in QuantiFERON supernatants can contribute to the diagnosis of active TB disease amongst people presenting with symptoms requiring investigation for TB disease, regardless of HIV status or ethnicity in Africa

Evaluation of cytokine responses against novel Mtb antigens as diagnostic markers for TB disease

Objective: We investigated the accuracy of host markers detected in Mtb antigen-stimulated whole blood culture supernatant in the diagnosis of TB. Methods: Prospectively, blood from 322 individuals with presumed TB disease from six African sites was stimulated with four different Mtb antigens (Rv0081, Rv1284, ESAT-6/CFP-10, and Rv2034) in a 24 h whole blood stimulation assay (WBA). The concentrations of 42 host markers in the supernatants were measured using the Luminex multiplex platform. Diagnostic biosignatures were investigated through the use of multivariate analysis techniques. Results: 17% of the participants were HIV infected, 106 had active TB disease and in 216 TB was excluded. Unstimulated concentrations of CRP, SAA, ferritin and IP-10 had better discriminating ability than markers from stimulated samples. Accuracy of marker combinations by general discriminant analysis (GDA) identified a six analyte model with 77% accuracy for TB cases and 84% for non TB cases, with a better performance in HIV uninfected patients. Conclusions: A biosignature of 6 cytokines obtained after stimulation with four Mtb antigens has moderate potential as a diagnostic tool for pulmonary TB disease individuals and stimulated marker expression had no added value to unstimulated marker performance