1,721,001 research outputs found
Molecular imaging in TB: from the bench to the clinic
Get PDFDespite all efforts, tuberculosis (TB) still constitutes a serious global health threat with 9.4 million new cases and 1.7 million deaths worldwide in 2009 (World Health Organisation,2010). Furthermore, an estimated one third of the worlds’ population is infected with the bacterium responsible, Mycobacterium tuberculosis. The main handicaps in fighting TB include a vaccine which works poorly in the most affected populations, and an arduous treatment regimen, involving a combination of several drugs taken over many months. This is further complicated by the emergence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) M. tuberculosis strains, which require even longer treatment times with less well-tolerated drugs. Eradication of TB will require the development of new drugs and vaccines, alongside improved methods for diagnosis and monitoring treatment efficacy. With the vast burden of disease falling in resource poor settings, the challenge will also be to develop methodologies that can be deployed with minimal investment in infrastructure, maintenance and staff expertise. Recent decades have seen the emergence of the new discipline of molecular imaging. In essence, molecular imaging enables the non-invasive visualisation, characterisation, and quantification of biological processes taking place within intact living subjects, be it a mouse or man (Filippi & Rocca, 2011; Horky & Treves, 2011; Pysz et al., 2010; Sandhu et al., 2010). Imaging has long been applied to managing TB; simple chest x-rays have allowed clinicians to visualise TB in people for over a century (Singh & Nath, 1994). However, the new molecular imaging techniques are revolutionising medical research, with the potential to translate into significant changes in clinical practice. In this chapter we describe the new generation of imaging modalities and how these are being applied to eradicating TB, from the laboratory bench and in to the clinic
Sparking Fungal Activity with Bacterial Components: Can co-culture with bacterial components induce antibacterial activity in New Zealand fungi?
No full textFull Text is available to authenticated members of The University of Auckland only.Methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum β-lactamase (ESBL) and carbapenemase-producing bacteria cause deadly multi-drug resistant infections, against which most of the current antibiotics we use are ineffective. To replenish our banks of antibiotics, the biodiscovery of natural products in the form of fungi secondary metabolites is attractive and has resulted in several interesting antibacterial compounds. The OSMAC (One Strain, Many Compounds) approach has been undertaken to induce secondary metabolite production by fungi. More recently, bacterial components have also been used to activate fungal secondary metabolite function.
For this project, I investigated whether co-culturing New Zealand fungi with bacterial components such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), or autoclaved bacteria would impact fungal secondary metabolite production. I made use of bioluminescent derivatives of Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and S. aureus to detect any antibacterial activity by the co-culture supernatant. As these tagged bacteria only glow when they are alive, bioluminescence is a sensitive, rapid, and non-destructive way to measure bacteria metabolism and viability. Finally, I used liquid chromatography coupled with mass spectrometry (LC-MS) to help elucidate changes in the secondary metabolite profile of the fungi.
I screened 30 New Zealand fungi, a mixture of previously tested and untested isolates, and identified eight that were active under at least one of the conditions I grew them under. Of these fungi, the addition of autoclaved bacteria induced antibacterial activity in Neofusicoccum cryptoaustrale and Heterobasidion araucariae, and the presence of LTA potentially enhanced antibacterial activity of Stropharia rugosoannulata. All other active isolates displayed activity both in the presence and absence of the additives, with some additive conditions prolonging or enhancing antibacterial activity. Four isolates retained active fractions after LC-MS allowing me to identify two known antibacterial compounds, patulin from Penicillium expansum and piptamine from Stropharia rugosoannulata.
While I did not discover any novel antibacterial compounds during my studies, my findings confirm the potential for bacterial components to induce changes in fungal secondary metabolite production, and highlight the utility of LC-MS as a complementary dereplication tool
Modelling nasopharyngeal colonisation by Streptococcus pyogenes : Bioluminescence and other longitudinal techniques
Get PDFStreptococcus pyogenes causes an estimated 616 million pharyngitis cases per year
and a variety of invasive diseases such as necrotising fasciitis and toxic shock
syndrome. The human nasopharynx is the major reservoir for all S. pyogenes
infection, including severe invasive disease. A combination of biophotonic imaging
(BPI) and direct nasal sampling techniques were used to longitudinally measure the
in vivo carriage of S. pyogenes, looking at the effects of virulence factor expression
on carriage and transmission and to enable vaccine evaluation.
Direct nasal sampling demonstrated that the two component regulatory system,
CovR/S, is required for infection and transmission from the nasopharynx. The
fitness cost conferred by covR/S mutation in the nasopharynx may explain why S.
pyogenes with altered covR/S have not become prevalent in community infections
despite conferring a selective advantage in invasive infection.
Bioluminescent S. pyogenes strains expressing the luxABCDE operon demonstrated
a growth deficit independent of the target site for integration in vitro that manifested
as a fitness cost during infection in vivo. Notwithstanding this, bioluminescence
expression permitted longitudinal quantitation of S. pyogenes within the nasopharynx
using BPI. Intramuscular vaccination with heat killed streptococci or the
streptococcal chemokine protease SpyCEP conferred protection against pharyngeal
infection in this model.
These longitudinal techniques allow for S. pyogenes to be tracked in the
nasopharynx non-invasively, and allow for new insights into the pathogenesis of this
disease
In vitro and in vivo characterisation of a panel of clinical and environmental isolates of Mycobacterium avium complex (MAC)
No full textNon-tuberculous Mycobacteria (NTM) are considered opportunistic pathogens of humans and animals, causing a variety of infectious diseases which are difficult to diagnose and treat. Among the most prevalent and important NTM species are those belonging to the M. avium complex (MAC), which includes M. avium, M. intracellulare, and M. chimaera.
This thesis aims to investigate the genomic and phenotypic differences of a panel of environmental and clinical isolates previously identified as M. chimaera. Environmental isolates were recovered from soil and households in the Hawaiian Islands, while clinical isolates were recovered from the sputum of patients in the Hawaiian Islands being investigated for suspected NTM lung disease. As NTM infections are thought to originate from environmental exposure, I hypothesised that there would be no difference in the virulence of the isolates by their source.
Using whole genome sequencing and molecular diagnostics tests, I identified 9/13 clinical and 12/14 environmental isolates as M. chimaera and the remaining isolates as either M. intracellulare or M. avium. I determined that 7/20 isolates produced colony variants, giving me a total of 27 isolates to investigate further. I characterised the isolates based on their cellular and colony morphology, growth in Middlebrook 7H9 broth, and antibiotic susceptibility profiles. I determined that 16 isolates produced smooth opaque (SmO) colonies, nine produced smooth transparent (SmT) colonies, and two produced rough colonies. I further determined that environmental isolates had significantly shorter lag times when grown in 7H9 broth and that the MICs for some antibiotics differed between colony types and MAC species.
I used larvae of the Greater wax moth Galleria mellonella as a surrogate host to assess the in vivo virulence of the panel of MAC isolates. I was able to categorise the panel of isolates as either high-, intermediate-, or low-virulence based on larval survival. In addition, I observed that virulence differed significantly between colony types and colony variants, with higher virulence being associated with the SmO colony phenotype. High- and intermediate-virulence isolates and those isolates producing SmO colonies also caused a significant decrease in haemocyte cell numbers in the first 24-48 h of infection of larvae. The work I present in this thesis confirms the suitability of using G. mellonella to investigate M. avium complex species and sheds further light on the virulence capacity of environmental M. chimaera isolates
Characterization of Mycobacterial Membrane Vesicles
No full textTuberculous and non-tuberculous mycobacteria release membrane vesicles (MMVs) that contain protein, lipoproteins, and polar lipids. It is hypothesized that membrane vesicles facilitate both the delivery of virulence factors and function as “immune decoys”. To better understand the biology of MMVs, I undertook the analysis of a panel of mycobacterial species: Mycobacterium smegmatis (non-pathogenic, fast-grower), Mycobacterium abscessus (human pathogen, fast-grower), Mycobacterium marinum (fish and opportunistic human pathogen, slow-grower) and Mycobacterium tuberculosis (human pathogenic, slow grower).
The main aims of this study are to establish a methodology for isolation and reproducible production of purified MMVs, to determine MMV cargo, to compare MMV production in the presence of stress factors and to analyse the response of host immune cells to MMV challenge.
MMVs were observed to be produced throughout mycobacterial growth in detergent and serum-free Sauton’s media, with most produced at exponential and stationary phases. Live/dead staining by SYTO9 and PI showed that MMVs were produced by cultures comprised of predominantly live cells. Crude MMVs, obtained from 2 L cell-free culture supernatants were purified by Density Gradient Centrifugation, with the successful identification of MMV-rich fractions for M. marinum and M. smegmatis but not for M. abscessus and M. tuberculosis due to the wide variability in density of the crude MMV preparation. MMVs were isolated with modal diameters between 90-150 nm (measured by Nanoparticle Tracking Analysis). Protein content was analysed using SDS-PAGE and proteomics of purified MMVs from M. marinum, by LC-MS, revealed 366 proteins, while 184 proteins in crude MMVs were found to be extra-vesicular. DNA, RNA, and protein content were quantified using commercial kits, and lipids by DI-MS. Different amounts of protein, lipids, DNA and RNA were isolated from each species suggesting the existence of a selective packaging mechanism. Application of a temperature and sub-lethal isoniazid stress revealed production of larger MMVs. However, sub-lethal rifampicin stress revealed MMVs with significantly higher amounts of DNA and MMV numbers produced per mycobacterial cell, suggesting stress factors may affect MMV size, numbers and associated molecular cargo. Confocal microscopy demonstrated that M. marinum MVs were taken up by THP-1 cells and localized within their cytoplasm. In vitro experiments challenging THP-1 cells with M. marinum MVs showed a positive dose dependent effect on the metabolic activity of THP-1 cells. Transcriptomic analysis of the effect of M. marinum MVs on THP-1 cells revealed the MMV dose-dependent upregulation of genes known to be involved in an innate immune response to invading mycobacteria and those facilitating conversion of monocytes to activated macrophages and stimulating granuloma formation. Treatment of THP1-Dual macrophages with MMVs demonstrated induction of NF-κB signalling pathway but not IRF pathway or increased secretion of the chemokine IP10.
Taken together, these results demonstrate: that MMVs are released by all tested species of mycobacteria; that MMVs contain a selected cargo that could be used to identify and develop new vaccines, adjuvants, and biomarkers; a new understanding of how MMVs may have a role in pro-inflammatory signalling via NF-κB, further elucidating their functional effects, and potentially contributing to a better understanding of disease pathogenesis
A Helping Hand during -Testing Times.
No full textSiouxsie Wiles reviews by cartoonist Grady Klein and statistician Alan Dabney
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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