Publications by authors named "Mark Tangney"

72 Publications

Arming Yourself for The In Silico Protein Design Revolution.

Trends Biotechnol 2020 Oct 30. Epub 2020 Oct 30.

CancerResearch@UCC, University College Cork, Cork, Ireland; SynBioCentre, University College Cork, Cork, Ireland; APC Microbiome Ireland, University College Cork, Cork, Ireland. Electronic address:

Proteins mediate many essential processes of life to a degree of functional precision unmatched by any synthetic device. While engineered proteins are currently used in biotech, food, biomedicine, and material technology-based industries, the true potential of proteins is practically untapped. The emerging field of in silico protein design is predicted to provide the next quantum leap in the biotech industry. Having predictive control over protein function and the ability to redefine these functions have driven the field of protein engineering into an era of unprecedented development. This article provides a holistic analysis of protein design R&D (current state-of-the-art tools and knowhow) and commercial landscape, as well as a one-stop-shop profile of in silico protein design technology for biotechnology stakeholders.
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http://dx.doi.org/10.1016/j.tibtech.2020.10.003DOI Listing
October 2020

A novel cell permeability assay for macromolecules.

BMC Mol Cell Biol 2020 Oct 30;21(1):75. Epub 2020 Oct 30.

CancerResearch@UCC, University College Cork, Cork, Ireland.

Background: Many cell permeabilisation methods to mediate internalisation of various molecules to mammalian or bacterial cells have been developed. However, no size-specific permeability assay suitable for both cell types exists.

Results: We report the use of intrinsically biotinylated cell components as the target for reporter molecules for assessing permeabilisation. Due to its well-described biotin binding activity, we developed an assay using Streptavidin (SAv) as a molecular weight marker for assessing eukaryotic and prokaryotic cell internalisation, using flow cytometry as a readout. This concept was tested here as part of the development of host DNA depletion strategies for microbiome analysis of formalin-fixed (FF) samples. Host depletion (HD) strategies require differential cell permeabilisation, where mammalian cells but not bacterial cells are permeabilised, and are subsequently treated with a nuclease. Here, the internalisation of a SAv-conjugate was used as a reference for nucleases of similar dimensions. With this assay, it was possible to demonstrate that formalin fixation does not generate pores which allow the introduction of 60 KDa molecules in mammalian or bacterial membranes/envelopes. Among surfactants tested, Saponin derived from Quillaja bark showed the best selectivity for mammalian cell permeabilisation, which, when coupled with Benzonase nuclease, provided the best results for host DNA depletion, representing a new HD strategy for formalin fixed samples.

Conclusion: The assay presented provides researchers with a sensitive and accessible tool for discerning membrane/cell envelop permeability for different size macromolecules.
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http://dx.doi.org/10.1186/s12860-020-00321-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7602297PMC
October 2020

Characterization of FFPE-induced bacterial DNA damage and development of a repair method.

Biol Methods Protoc 2020 27;5(1):bpaa015. Epub 2020 Jul 27.

CancerResearch@UCC, University College Cork, Cork, T12 XF62, Ireland.

Formalin-fixed, paraffin-embedded (FFPE) specimens have huge potential as source material in the field of human microbiome research. However, the effects of FFPE processing on bacterial DNA remain uncharacterized. Any effects are relevant for microbiome studies, where DNA template is often minimal and sequences studied are not limited to one genome. As such, we aimed to both characterize this FFPE-induced bacterial DNA damage and develop strategies to reduce and repair this damage. Our analyses indicate that bacterial FFPE DNA is highly fragmented, a poor template for PCR, crosslinked and bears sequence artefacts derived predominantly from oxidative DNA damage. Two strategies to reduce this damage were devised - an optimized decrosslinking procedure reducing sequence artefacts generated by high-temperature incubation, and secondly, an reconstitution of the base excision repair pathway. As evidenced by whole genome sequencing, treatment with these strategies significantly increased fragment length, reduced the appearance of sequence artefacts and improved the sequencing readability of bacterial and mammalian FFPE DNA. This study provides a new understanding of the condition of bacterial DNA in FFPE specimens and how this impacts downstream analyses, in addition to a strategy to improve the sequencing quality of bacterial and possibly mammalian FFPE DNA.
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http://dx.doi.org/10.1093/biomethods/bpaa015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7548031PMC
July 2020

Non-specific amplification of human DNA is a major challenge for 16S rRNA gene sequence analysis.

Sci Rep 2020 10 1;10(1):16356. Epub 2020 Oct 1.

CancerResearch@UCC, University College Cork, Cork, Ireland.

The targeted sequencing of the 16S rRNA gene is one of the most frequently employed techniques in the field of microbial ecology, with the bacterial communities of a wide variety of niches in the human body have been characterised in this way. This is performed by targeting one or more hypervariable (V) regions within the 16S rRNA gene in order to produce an amplicon suitable in size for next generation sequencing. To date, all technical research has focused on the ability of different V regions to accurately resolve the composition of bacterial communities. We present here an underreported artefact associated with 16S rRNA gene sequencing, namely the off-target amplification of human DNA. By analysing 16S rRNA gene sequencing data from a selection of human sites we highlighted samples susceptible to this off-target amplification when using the popular primer pair targeting the V3-V4 region of the gene. The most severely affected sample type identified (breast tumour samples) were then re-analysed using the V1-V2 primer set, showing considerable reduction in off target amplification. Our data indicate that human biopsy samples should preferably be amplified using primers targeting the V1-V2 region. It is shown here that these primers result in on average 80% less human genome aligning reads, allowing for more statistically significant analysis of the bacterial communities residing in these samples.
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http://dx.doi.org/10.1038/s41598-020-73403-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529756PMC
October 2020

Seeding sustainable education in developing countries: Teaching biotech in low-income areas.

EMBO Rep 2020 09 1;21(9):e50587. Epub 2020 Sep 1.

SynBioCentre, University College Cork, Cork, Ireland.

Synthetic biology has the potential to seed research in impoverished countries. Teaching students and academics has to take into account reality - lessons from Honduras.
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http://dx.doi.org/10.15252/embr.202050587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507435PMC
September 2020

Protoblock - A biological standard for formalin fixed samples.

Microbiome 2020 08 22;8(1):122. Epub 2020 Aug 22.

CancerResearch@UCC, University College Cork, Cork, Ireland.

Background: Formalin-fixed, paraffin-embedded (FFPE) tissue is the gold standard in pathology tissue storage, representing the largest collections of patient material. Their reliable use for DNA analyses could open a trove of potential samples for research and are currently being recognised as a viable source material for bacterial analysis. There are several key features which limit bacterial-related data generation from this material: (i) DNA damage inherent to the fixing process, (ii) low bacterial biomass that increases the vulnerability to contamination and exacerbates the host DNA effects and (iii) lack of suitable DNA extraction methods, leading to data bias. The development and systematic use of reliable standards is a key priority for microbiome research. More than perhaps any other sample type, FFPE material urgently requires the development of standards to ensure the validity of results and to promote reproducibility.

Results: To address these limitations and concerns, we have developed the Protoblock as a biological standard for FFPE tissue-based research and method optimisation. This is a novel system designed to generate bespoke mock FFPE 'blocks' with a cell content that is user-defined and which undergoes the same treatment conditions as clinical FFPE tissues. The 'Protoblock' features a mix of formalin-fixed cells, of known number, embedded in an agar matrix which is solidified to form a defined shape that is paraffin embedded. The contents of various Protoblocks populated with mammalian and bacterial cells were verified by microscopy. The quantity and condition of DNA purified from blocks was evaluated by qPCR, 16S rRNA gene amplicon sequencing and whole genome sequencing. These analyses validated the capability of the Protoblock system to determine the extent to which each of the three stated confounding features impacts on eventual analysis of cellular DNA present in FFPE samples.

Conclusion: The Protoblock provides a representation of biological material after FFPE treatment. Use of this standard will greatly assist the stratification of biological variations detected into those legitimately resulting from experimental conditions, and those that are artefacts of the processed nature of the samples, thus enabling users to relate the outputs of laboratory analyses to reality. Video Abstract.
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http://dx.doi.org/10.1186/s40168-020-00901-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443293PMC
August 2020

Sequence-Based Characterization of Intratumoral Bacteria-A Guide to Best Practice.

Front Oncol 2020 21;10:179. Epub 2020 Feb 21.

APC Microbiome Ireland, University College Cork, Cork, Ireland.

Tumors are hospitable environments to bacteria and several recent studies on cancer patient samples have introduced the concept of an endogenous tumor microbiome. For a variety of reasons, this putative tumor microbiome is particularly challenging to investigate, and a failure to account for the various potential pitfalls will result in erroneous results and claims. Before this potentially extremely medically-significant habitat can be accurately characterized, a clear understanding of all potential confounding factors is required, and a best-practice approach should be developed and adopted. This review summarizes all of the potential issues confounding accurate bacterial DNA sequence analysis of the putative tumor microbiome, and offers solutions based on related research with the hope of assisting in the progression of research in this field.
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http://dx.doi.org/10.3389/fonc.2020.00179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046755PMC
February 2020

Function2Form Bridge-Toward synthetic protein holistic performance prediction.

Proteins 2020 03 29;88(3):462-475. Epub 2019 Oct 29.

Cancer Research at UCC, University College Cork, Cork, Ireland.

Protein engineering and synthetic biology stand to benefit immensely from recent advances in silico tools for structural and functional analyses of proteins. In the context of designing novel proteins, current in silico tools inform the user on individual parameters of a query protein, with output scores/metrics unique to each parameter. In reality, proteins feature multiple "parts"/functions and modification of a protein aimed at altering a given part, typically has collateral impact on other protein parts. A system for prediction of the combined effect of design parameters on the overall performance of the final protein does not exist. Function2Form Bridge (F2F-Bridge) attempts to address this by combining the scores of different design parameters pertaining to the protein being analyzed into a single easily interpreted output describing overall performance. The strategy comprises of (a) a mathematical strategy combining data from a myriad of in silico tools into an OP-score (a singular score informing on a user-defined overall performance) and (b) the F2F Plot, a graphical means of informing the wetlab biologist holistically on designed construct suitability in the context of multiple parameters, highlighting scope for improvement. F2F predictive output was compared with wetlab data from a range of synthetic proteins designed, built, and tested for this study. Statistical/machine learning approaches for predicting overall performance, for use alongside the F2F plot, were also examined. Comparisons between wetlab performance and F2F predictions demonstrated close and reliable correlations. This user-friendly strategy represents a pivotal enabler in increasing the accessibility of synthetic protein building and de novo protein design.
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http://dx.doi.org/10.1002/prot.25825DOI Listing
March 2020

ODX: A Fitness Tracker-Based Device for Continuous Bacterial Growth Monitoring.

Anal Chem 2019 10 12;91(19):12329-12335. Epub 2019 Sep 12.

Centre for Advanced Photonics and Process Analysis , Cork Institute of Technology , Cork T12 P928 , Ireland.

Continuous monitoring of bacterial growth in aqueous media is a crucial process in academic research as well as in the biotechnology industry. Bacterial growth is usually monitored by measuring the optical density of bacteria in liquid media, using benchtop spectrophotometers. Due to the large form factor of the existing spectrophotometers, they cannot be used for live monitoring of the bacteria inside bacterial incubation chambers. Additionally, the use of benchtop spectrometers for continuous monitoring requires multiple samplings and is labor intensive. To overcome these challenges, we have developed an optical density measuring device (ODX) by modifying a generic fitness tracker. The resulting ODX device is an ultraportable and low-cost device that can be used inside bacterial incubators for real-time monitoring even while shaking is in progress. We evaluated the performance of ODX with different bacterial types and growth conditions and compared it with a commercial benchtop spectrophotometer. In all cases, ODX showed comparable performance to that of the standard benchtop spectrophotometer. Finally, we demonstrate a simple and useful smartphone application whereby the user is notified when the bacterial concentration reaches the targeted value. Due to its potential for automation and mass production, we believe that the ODX has a wide range of applications in biotechnology research and industry.
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http://dx.doi.org/10.1021/acs.analchem.9b02628DOI Listing
October 2019

Microbiome analysis as a platform R&D tool for parasitic nematode disease management.

ISME J 2019 11 25;13(11):2664-2680. Epub 2019 Jun 25.

Moredun Research Institute, Pentlands Science Park, Penicuik, EH26 0PZ, UK.

The relationship between bacterial communities and their host is being extensively investigated for the potential to improve the host's health. Little is known about the interplay between the microbiota of parasites and the health of the infected host. Using nematode co-infection of lambs as a proof-of-concept model, the aim of this study was to characterise the microbiomes of nematodes and that of their host, enabling identification of candidate nematode-specific microbiota member(s) that could be exploited as drug development tools or for targeted therapy. Deep sequencing techniques were used to elucidate the microbiomes of different life stages of two parasitic nematodes of ruminants, Haemonchus contortus and Teladorsagia circumcincta, as well as that of the co-infected ovine hosts, pre- and post infection. Bioinformatic analyses demonstrated significant differences between the composition of the nematode and ovine microbiomes. The two nematode species also differed significantly. The data indicated a shift in the constitution of the larval nematode microbiome after exposure to the ovine microbiome, and in the ovine intestinal microbial community over time as a result of helminth co-infection. Several bacterial species were identified in nematodes that were absent from their surrounding abomasal environment, the most significant of which included Escherichia coli/Shigella. The ability to purposefully infect nematode species with engineered E. coli was demonstrated in vitro, validating the concept of using this bacterium as a nematode-specific drug development tool and/or drug delivery vehicle. To our knowledge, this is the first description of the concept of exploiting a parasite's microbiome for drug development and treatment purposes.
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http://dx.doi.org/10.1038/s41396-019-0462-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794328PMC
November 2019

Resident bacteria in breast cancer tissue: pathogenic agents or harmless commensals?

Discov Med 2018 09;26(142):93-102

UCD School of Medicine, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, UCD, Belfield, Dublin, Ireland.

Breast cancer is the second most common cancer in women. Recent evidence identifies a unique microbiome in breast tissue; a site previously thought to be sterile. The identification that this microbiome varies considerably from healthy subjects to cancer patients has prompted investigations into the role of specific bacterial species in oncogenesis. Indeed, certain bacteria have been shown to aid cancer development in vitro by promoting genomic instability, invasion, and chemotherapy resistance. However, the in vivo role of the breast microbiome in cancer appears to be more complex, involving numerous interactions between its constituent species and host cells. As such, reduced abundances of species which exert a protective effect against oncogenesis have come into focus and there is an emerging consensus that states of microbial dysbiosis, in which the normal balance of bacterial species is altered, can contribute to the development of cancer. This review summarizes the findings to date from the available literature pertaining to the microbiome in breast cancer and outlines areas worthy of further investigation.
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September 2018

The Who, What, and Why of Drug Discovery and Development.

Trends Pharmacol Sci 2018 10 6;39(10):848-852. Epub 2018 Sep 6.

SynBioCentre, University College Cork, Cork, Ireland; Cancer Research@UCC, University College Cork, Cork, Ireland; APC Microbiome Ireland, University College Cork, Cork, Ireland. Electronic address:

Inquiry into declining pharmaceutical R&D efficiency has focussed on 'what' can be improved, with only brief thought given to 'who' can be improved. Here, we argue that enabling people in the idea-to-product chain to have a more holistic knowledge of the behaviours and incentives of each other can optimise R&D.
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http://dx.doi.org/10.1016/j.tips.2018.08.002DOI Listing
October 2018

Ascending Vaginal Infection Using Bioluminescent Bacteria Evokes Intrauterine Inflammation, Preterm Birth, and Neonatal Brain Injury in Pregnant Mice.

Am J Pathol 2018 10 21;188(10):2164-2176. Epub 2018 Jul 21.

Gene Transfer Technology Group, University College London, London, United Kingdom; MRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.

Preterm birth is a serious global health problem and the leading cause of infant death before 5 years of age. At least 40% of cases are associated with infection. The most common way for pathogens to access the uterine cavity is by ascending from the vagina. Bioluminescent pathogens have revolutionized the understanding of infectious diseases. We hypothesized that bioluminescent Escherichia coli can be used to track and monitor ascending vaginal infections. Two bioluminescent strains were studied: E. coli K12 MG1655-lux, a nonpathogenic laboratory strain, and E. coli K1 A192PP-lux2, a pathogenic strain capable of causing neonatal meningitis and sepsis in neonatal rats. On embryonic day 16, mice received intravaginal E. coli K12, E. coli K1, or phosphate-buffered saline followed by whole-body bioluminescent imaging. In both cases, intravaginal delivery of E. coli K12 or E. coli K1 led to bacterial ascension into the uterine cavity, but only E. coli K1 induced preterm parturition. Intravaginal administration of E. coli K1 significantly reduced the proportion of pups born alive compared with E. coli K12 and phosphate-buffered saline controls. However, in both groups of viable pups born after bacterial inoculation, there was evidence of comparable brain inflammation by postnatal day 6. This study ascribes specific mechanisms by which exposure to intrauterine bacteria leads to premature delivery and neurologic inflammation in neonates.
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http://dx.doi.org/10.1016/j.ajpath.2018.06.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168615PMC
October 2018

In situ biomolecule production by bacteria; a synthetic biology approach to medicine.

J Control Release 2018 04 22;275:217-228. Epub 2018 Feb 22.

SynBioCentre, University College Cork, Cork, Ireland; Cork Cancer Research Centre, University College Cork, Cork, Ireland; APC Microbiome Institute, University College Cork, Cork, Ireland. Electronic address:

The ability to modify existing microbiota at different sites presents enormous potential for local or indirect management of various diseases. Because bacteria can be maintained for lengthy periods in various regions of the body, they represent a platform with enormous potential for targeted production of biomolecules, which offer tremendous promise for therapeutic and diagnostic approaches for various diseases. While biological medicines are currently limited in the clinic to patient administration of exogenously produced biomolecules from engineered cells, in situ production of biomolecules presents enormous scope in medicine and beyond. The slow pace and high expense of traditional research approaches has particularly hampered the development of biological medicines. It may be argued that bacterial-based medicine has been "waiting" for the advent of enabling technology. We propose that this technology is Synthetic Biology, and that the wait is over. Synthetic Biology facilitates a systematic approach to programming living entities and/or their products, using an approach to Research and Development (R&D) that facilitates rapid, cheap, accessible, yet sophisticated product development. Full engagement with the Synthetic Biology approach to R&D can unlock the potential for bacteria as medicines for cancer and other indications. In this review, we describe how by employing Synthetic Biology, designer bugs can be used as drugs, drug-production factories or diagnostic devices, using oncology as an exemplar for the concept of in situ biomolecule production in medicine.
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http://dx.doi.org/10.1016/j.jconrel.2018.02.023DOI Listing
April 2018

Development of a Click Beetle Luciferase Reporter System for Enhanced Bioluminescence Imaging of : Analysis in Cell Culture and Murine Infection Models.

Front Microbiol 2017 26;8:1797. Epub 2017 Sep 26.

APC Microbiome Institute, University College Cork, Cork, Ireland.

is a Gram-positive facultative intracellular pathogen that is widely used as a model organism for the analysis of infection biology. In this context, there is a current need to develop improved reporters for enhanced bioluminescence imaging (BLI) of the pathogen in infection models. We have developed a click beetle red luciferase (CBR-) based vector (pPL2CBR) expressing codon optimized CBR- under the control of a highly expressed Listerial promoter (P) for and have compared this to a -based system expressing bacterial luciferase for BLI of the pathogen using growth experiments and models. The CBR- plasmid stably integrates into the chromosome and can be used to label field isolates and laboratory strains of the pathogen. Growth experiments revealed that CBR- labeled emits a bright signal in exponential phase that is maintained during stationary phase. In contrast, -labeled bacteria produced a light signal that peaked during exponential phase and was significantly reduced during stationary phase. Light from CBR- labeled bacteria was more efficient than the signal from -labeled bacteria in penetrating an artificial tissue depth assay system. A cell invasion assay using C2Bbe1 cells and a systemic murine infection model revealed that CBR- is suited to BLI approaches and demonstrated enhanced sensitivity relative to in the context of infection models. Overall, we demonstrate that this novel CBR reporter system provides efficient, red-shifted light production relative to and may have significant applications in the analysis of pathogenesis.
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http://dx.doi.org/10.3389/fmicb.2017.01797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622934PMC
September 2017

Designer bacteria as intratumoural enzyme biofactories.

Adv Drug Deliv Rev 2017 09 12;118:8-23. Epub 2017 Sep 12.

Cork Cancer Research Centre, University College Cork, Cork, Ireland; SynBioCentre, University College Cork, Cork, Ireland; APC Microbiome Institute, University College Cork, Cork, Ireland. Electronic address:

Bacterial-directed enzyme prodrug therapy (BDEPT) is an emerging form of treatment for cancer. It is a biphasic variant of gene therapy in which a bacterium, armed with an enzyme that can convert an inert prodrug into a cytotoxic compound, induces tumour cell death following tumour-specific prodrug activation. BDEPT combines the innate ability of bacteria to selectively proliferate in tumours, with the capacity of prodrugs to undergo contained, compartmentalised conversion into active metabolites in vivo. Although BDEPT has undergone clinical testing, it has received limited clinical exposure, and has yet to achieve regulatory approval. In this article, we review BDEPT from the system designer's perspective, and provide detailed commentary on how the designer should strategize its development de novo. We report on contemporary advancements in this field which aim to enhance BDEPT in terms of safety and efficacy. Finally, we discuss clinical and regulatory barriers facing BDEPT, and propose promising approaches through which these hurdles may best be tackled.
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http://dx.doi.org/10.1016/j.addr.2017.09.012DOI Listing
September 2017

Intratumoural production of TNFα by bacteria mediates cancer therapy.

PLoS One 2017 29;12(6):e0180034. Epub 2017 Jun 29.

Cork Cancer Research Centre, University College Cork, Cork, Ireland.

Systemic administration of the highly potent anticancer therapeutic, tumour necrosis factor alpha (TNFα) induces high levels of toxicity and is responsible for serious side effects. Consequently, tumour targeting is required in order to confine this toxicity within the locality of the tumour. Bacteria have a natural capacity to grow within tumours and deliver therapeutic molecules in a controlled fashion. The non-pathogenic E. coli strain MG1655 was investigated as a tumour targeting system in order to produce TNFα specifically within murine tumours. In vivo bioluminescence imaging studies and ex vivo immunofluorescence analysis demonstrated rapid targeting dynamics and prolonged survival, replication and spread of this bacterial platform within tumours. An engineered TNFα producing construct deployed in mouse models via either intra-tumoural (i.t.) or intravenous (i.v.) administration facilitated robust TNFα production, as evidenced by ELISA of tumour extracts. Tumour growth was impeded in three subcutaneous murine tumour models (CT26 colon, RENCA renal, and TRAMP prostate) as evidenced by tumour volume and survival analyses. A pattern of pro-inflammatory cytokine induction was observed in tumours of treated mice vs.

Controls: Mice remained healthy throughout experiments. This study indicates the therapeutic efficacy and safety of TNFα expressing bacteria in vivo, highlighting the potential of non-pathogenic bacteria as a platform for restricting the activity of highly potent cancer agents to tumours.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180034PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491124PMC
October 2017

Synthetic Biology in the Driving Seat of the Bioeconomy.

Trends Biotechnol 2017 05 27;35(5):373-378. Epub 2017 Feb 27.

Cork Cancer Research Centre, University College Cork, Cork, Ireland; SynBioCentre, University College Cork, Cork, Ireland; APC Microbiome Institute, University College Cork, Cork, Ireland. Electronic address:

Synthetic biology is revolutionising the biotech industry and is increasingly applied in previously unthought-of markets. Here, we discuss the importance of this industry to the bioeconomy and two of its key factors: the synthetic biology approach to research and development (R&D), and the unique nature of the carefully designed, stakeholder-inclusive, community-directed evolution of the field.
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http://dx.doi.org/10.1016/j.tibtech.2017.02.002DOI Listing
May 2017

The Microbiota of Breast Tissue and Its Association with Breast Cancer.

Appl Environ Microbiol 2016 08 29;82(16):5039-48. Epub 2016 Jul 29.

Lawson Health Research Institute, London, Ontario, Canada Department of Microbiology and Immunology, Western University, London, Ontario, Canada

Unlabelled: In the United States, 1 in 8 women will be diagnosed with breast cancer in her lifetime. Along with genetics, the environment contributes to disease development, but what these exact environmental factors are remains unknown. We have previously shown that breast tissue is not sterile but contains a diverse population of bacteria. We thus believe that the host's local microbiome could be modulating the risk of breast cancer development. Using 16S rRNA amplicon sequencing, we show that bacterial profiles differ between normal adjacent tissue from women with breast cancer and tissue from healthy controls. Women with breast cancer had higher relative abundances of Bacillus, Enterobacteriaceae and Staphylococcus Escherichia coli (a member of the Enterobacteriaceae family) and Staphylococcus epidermidis, isolated from breast cancer patients, were shown to induce DNA double-stranded breaks in HeLa cells using the histone-2AX (H2AX) phosphorylation (γ-H2AX) assay. We also found that microbial profiles are similar between normal adjacent tissue and tissue sampled directly from the tumor. This study raises important questions as to what role the breast microbiome plays in disease development or progression and how we can manipulate this for possible therapeutics or prevention.

Importance: This study shows that different bacterial profiles in breast tissue exist between healthy women and those with breast cancer. Higher relative abundances of bacteria that had the ability to cause DNA damage in vitro were detected in breast cancer patients, as was a decrease in some lactic acid bacteria, known for their beneficial health effects, including anticarcinogenic properties. This study raises important questions as to the role of the mammary microbiome in modulating the risk of breast cancer development.
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http://dx.doi.org/10.1128/AEM.01235-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968547PMC
August 2016

In Vivo Bioluminescence Imaging of Intratumoral Bacteria.

Methods Mol Biol 2016 ;1409:69-77

Cork Cancer Research Centre, University College Cork, Cork, Ireland.

This chapter describes the use of whole-body bioluminescent imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in therapy of cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and tumor sites concurrently. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor-bearing mice. This powerful, and inexpensive, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research. This protocol outlines the procedure for studying lux-tagged Escherichia coli and Bifidobacterium breve in mice, demonstrating the spatial and temporal readout from 2D and 3D BLI achievable with whole-body in vivo luminescence imaging.
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http://dx.doi.org/10.1007/978-1-4939-3515-4_7DOI Listing
November 2016

Activation of multiple chemotherapeutic prodrugs by the natural enzymolome of tumour-localised probiotic bacteria.

J Control Release 2016 Jan 2;222:9-17. Epub 2015 Dec 2.

Cork Cancer Research Centre, University College Cork, Cork, Ireland. Electronic address:

Some chemotherapeutic drugs (prodrugs) require activation by an enzyme for efficacy. We and others have demonstrated the ability of probiotic bacteria to grow specifically within solid tumours following systemic administration, and we hypothesised that the natural enzymatic activity of these tumour-localised bacteria may be suitable for activation of certain such chemotherapeutic drugs. Several wild-type probiotic bacteria; Escherichia coli Nissle, Bifidobacterium breve, Lactococcus lactis and Lactobacillus species, were screened against a panel of popular prodrugs. All strains were capable of activating at least one prodrug. E. coli Nissle 1917 was selected for further studies because of its ability to activate numerous prodrugs and its resistance to prodrug toxicity. HPLC data confirmed biochemical transformation of prodrugs to their toxic counterparts. Further analysis demonstrated that different enzymes can complement prodrug activation, while simultaneous activation of multiple prodrugs (CB1954, 5-FC, AQ4N and Fludarabine phosphate) by E. coli was confirmed, resulting in significant efficacy improvement. Experiments in mice harbouring murine tumours validated in vitro findings, with significant reduction in tumour growth and increase in survival of mice treated with probiotic bacteria and a combination of prodrugs. These findings demonstrate the ability of probiotic bacteria, without the requirement for genetic modification, to enable high-level activation of multiple prodrugs specifically at the site of action.
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http://dx.doi.org/10.1016/j.jconrel.2015.11.030DOI Listing
January 2016

Local bacteria affect the efficacy of chemotherapeutic drugs.

Sci Rep 2015 Sep 29;5:14554. Epub 2015 Sep 29.

Cork Cancer Research Centre, University College Cork, Cork, Ireland.

In this study, the potential effects of bacteria on the efficacy of frequently used chemotherapies was examined. Bacteria and cancer cell lines were examined in vitro and in vivo for changes in the efficacy of cancer cell killing mediated by chemotherapeutic agents. Of 30 drugs examined in vitro, the efficacy of 10 was found to be significantly inhibited by certain bacteria, while the same bacteria improved the efficacy of six others. HPLC and mass spectrometry analyses of sample drugs (gemcitabine, fludarabine, cladribine, CB1954) demonstrated modification of drug chemical structure. The chemoresistance or increased cytotoxicity observed in vitro with sample drugs (gemcitabine and CB1954) was replicated in in vivo murine subcutaneous tumour models. These findings suggest that bacterial presence in the body due to systemic or local infection may influence tumour responses or off-target toxicity during chemotherapy.
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http://dx.doi.org/10.1038/srep14554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4586607PMC
September 2015

Development of a Bioluminescent Nitroreductase Probe for Preclinical Imaging.

PLoS One 2015 25;10(6):e0131037. Epub 2015 Jun 25.

School of Basic Sciences, Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology of Lausanne, Lausanne, Switzerland.

Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131037PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4482324PMC
April 2016

In vivo bacterial imaging without engineering; A novel probe-based strategy facilitated by endogenous nitroreductase enzymes.

Curr Gene Ther 2015 ;15(3):277-88

Cork Cancer Research Centre, BioSciences Institute, University College Cork, Ireland.

The feasibility of utilising bacteria as vectors for gene therapy is becoming increasingly recognised. This is primarily due to a number of intrinsic properties of bacteria such as their tumour targeting capabilities, their ability to carry large genetic or protein loads and the availability of well-established genetic engineering tools for a range of common lab strains. However, a number of issues relating to the use of bacteria as vectors for gene therapy need to be addressed in order for the field to progress. Amongst these is the need for the development of non-invasive detection/imaging systems for bacteria within a living host. In vivo optical imaging has advanced preclinical research greatly, and typically involves engineering of bacteria with genetic expression constructs for luminescence (e.g. the lux operon) or fluorescent proteins (GFP etc.). This requirement for genetic modification can be restrictive, where engineering is not experimentally appropriate or technologically feasible (e.g. due to lack of suitable engineering tools). We describe a novel strategy exploiting endogenous bacterial enzymatic activity to specifically activate an exogenously administered fluorescent imaging probe. The red shifted, quenched fluorophore CytoCy5S is reduced to a fluorescent form by bacterial-specific nitroreductase (NTR) enzymes. NTR enzymes are present in a wide range of bacterial genera and absent in mammalian systems, permitting highly specific detection of Gram-negative and Gram-positive bacteria in vivo. In this study, dose-responsive bacterial-specific signals were observed in vitro from all genera examined - E. coli, Salmonella, Listeria, Bifidobacterium and Clostridium difficile. Examination of an NTR-knockout strain validated the enzyme specificity of the probe. In vivo whole-body imaging permitted specific, dose-responsive monitoring of bacteria over time in various infection models, and no toxicity to bacteria or host was observed. This study demonstrates the concept of exploiting innate NTR activity as a reporting strategy for wild-type bacteria using optical imaging, while the concept may also be extended to NTR-specific probes for use with other imaging modalities.
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http://dx.doi.org/10.2174/1566523215666150126122712DOI Listing
January 2016

Oral tolerance to cancer can be abrogated by T regulatory cell inhibition.

PLoS One 2014 15;9(5):e97602. Epub 2014 May 15.

Cork Cancer Research Centre, BioSciences Institute, University College Cork, Cork, Ireland.

Oral administration of tumour cells induces an immune hypo-responsiveness known as oral tolerance. We have previously shown that oral tolerance to a cancer is tumour antigen specific, non-cross-reactive and confers a tumour growth advantage. We investigated the utilisation of regulatory T cell (Treg) depletion on oral tolerance to a cancer and its ability to control tumour growth. Balb/C mice were gavage fed homogenised tumour tissue--JBS fibrosarcoma (to induce oral tolerance to a cancer), or PBS as control. Growth of subcutaneous JBS tumours were measured; splenic tissue excised and flow cytometry used to quantify and compare systemic Tregs and T effector (Teff) cell populations. Prior to and/or following tumour feeding, mice were intraperitoneally administered anti-CD25, to inactivate systemic Tregs, or given isotype antibody as a control. Mice which were orally tolerised prior to subcutaneous tumour induction, displayed significantly higher systemic Treg levels (14% vs 6%) and faster tumour growth rates than controls (p<0.05). Complete regression of tumours were only seen after Treg inactivation and occurred in all groups--this was not inhibited by tumour feeding. The cure rates for Treg inactivation were 60% during tolerisation, 75% during tumour growth and 100% during inactivation for both tolerisation and tumour growth. Depletion of Tregs gave rise to an increased number of Teff cells. Treg depletion post-tolerisation and post-tumour induction led to the complete regression of all tumours on tumour bearing mice. Oral administration of tumour tissue, confers a tumour growth advantage and is accompanied by an increase in systemic Treg levels. The administration of anti-CD25 Ab decreased Treg numbers and caused an increase in Teffs. Most notably Treg cell inhibition overcame established oral tolerance with consequent tumor regression, especially relevant to foregut cancers where oral tolerance is likely to be induced by the shedding of tumour tissue into the gut.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0097602PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4022586PMC
January 2015

Microbiota of human breast tissue.

Appl Environ Microbiol 2014 May 7;80(10):3007-14. Epub 2014 Mar 7.

Lawson Health Research Institute, London, Ontario, Canada.

In recent years, a greater appreciation for the microbes inhabiting human body sites has emerged. In the female mammary gland, milk has been shown to contain bacterial species, ostensibly reaching the ducts from the skin. We decided to investigate whether there is a microbiome within the mammary tissue. Using 16S rRNA sequencing and culture, we analyzed breast tissue from 81 women with and without cancer in Canada and Ireland. A diverse population of bacteria was detected within tissue collected from sites all around the breast in women aged 18 to 90, not all of whom had a history of lactation. The principal phylum was Proteobacteria. The most abundant taxa in the Canadian samples were Bacillus (11.4%), Acinetobacter (10.0%), Enterobacteriaceae (8.3%), Pseudomonas (6.5%), Staphylococcus (6.5%), Propionibacterium (5.8%), Comamonadaceae (5.7%), Gammaproteobacteria (5.0%), and Prevotella (5.0%). In the Irish samples the most abundant taxa were Enterobacteriaceae (30.8%), Staphylococcus (12.7%), Listeria welshimeri (12.1%), Propionibacterium (10.1%), and Pseudomonas (5.3%). None of the subjects had signs or symptoms of infection, but the presence of viable bacteria was confirmed in some samples by culture. The extent to which these organisms play a role in health or disease remains to be determined.
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http://dx.doi.org/10.1128/AEM.00242-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4018903PMC
May 2014

Bacterial-mediated knockdown of tumor resistance to an oncolytic virus enhances therapy.

Mol Ther 2014 Jun 26;22(6):1188-1197. Epub 2014 Feb 26.

Cork Cancer Research Centre, University College Cork, Cork, Ireland. Electronic address:

Oncolytic viruses (OVs) and bacteria share the property of tumor-selective replication following systemic administration. In the case of nonpathogenic bacteria, tumor selectivity relates to their ability to grow extracellularly within tumor stroma and is therefore ideally suited to restricting the production of bacterially produced therapeutic agents to tumors. We have previously shown the ability of the type 1 interferon antagonist B18R to enhance the replication and spread of vesicular stomatitis virus (VSV) by overcoming related cellular innate immunity. In this study, we utilized nonpathogenic bacteria (E. coli) expressing B18R to facilitate tumor-specific production of B18R, resulting in a microenvironment depleted of bioactive antiviral cytokine, thus "preconditioning" the tumor to enhance subsequent tumor destruction by the OV. Both in vitro and in vivo infection by VSVΔ51 was greatly enhanced by B18R produced from E. coli. Moreover, a significant increase in therapeutic efficacy resulted from intravenous (i.v.) injection of bacteria to tumor-bearing mice 5 days prior to i.v. VSVΔ51 administration, as evidenced by a significant reduction in tumor growth and increased survival in mice. Our strategy is the first example where two such diverse microorganisms are rationally combined and demonstrates the feasibility of combining complementary microorganisms to improve therapeutic outcome.
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http://dx.doi.org/10.1038/mt.2014.23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4048890PMC
June 2014

Bacterial systems for gene delivery to systemic tumors.

Methods Mol Biol 2014 ;1141:201-9

Cork Cancer Research Centre, BioSciences Institute, University College Cork, Cork, Ireland.

Certain bacteria have emerged as biological gene vectors with natural tumor specificity, capable of specifically delivering genes or gene products to the tumor environment when intravenously (i.v.) administered to rodent models. Here, we describe procedures for studying this phenomenon in vitro and in vivo for both invasive and noninvasive bacteria suitable for exploitation as tumor-specific therapeutic delivery vehicles, due to their ability to replicate specifically within tumors and/or mediate bacterial-mediated transfer of plasmid DNA to mammalian cells (bactofection).
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http://dx.doi.org/10.1007/978-1-4939-0363-4_13DOI Listing
May 2015

Bacterial-directed enzyme prodrug therapy.

J Control Release 2013 Aug 17;170(1):120-31. Epub 2013 May 17.

Cork Cancer Research Centre, Biosciences Institute, University College Cork, Cork, Ireland.

Current conventional treatments for cancer lack tumour selectivity resulting in the destruction of healthy tissue and severe adverse effects to the patient in addition to limiting the administration dose and efficacy. Hence, it is imperative that we seek alternative approaches to treat cancer that localise therapeutic agents to the site of the tumour and spare normal tissue. The use of bacteria in cancer therapy represents one such approach. Bacteria were first used as anti-cancer agents over a century ago. Today, this field has re-emerged from the past and is progressing at a rapid rate. Bacteria are used as anticancer agents either alone or in combination with conventional treatments and have been armed with an arsenal of therapeutic genes, which enhance their efficacy. Bacterial directed enzyme prodrug therapy (BDEPT) is one of the most promising approaches, which harnesses the tumour-specific location of bacteria to locally activate systemically administered 'prodrugs' within the tumour in order to induce selective tumour destruction. BDEPT is a relatively new concept. It was originally conceived more than 10years ago but it is only until recently that we witness a surge in activity in this field. In this review, we provide a full account of developments in the field of BDEPT since its inception. We share technical knowhow and discuss optimization strategies for vector and enzyme combinations, provide a clear view of the research landscape and suggest possible directions for the field.
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http://dx.doi.org/10.1016/j.jconrel.2013.05.005DOI Listing
August 2013

Use of optical imaging to progress novel therapeutics to the clinic.

J Control Release 2013 Dec 13;172(2):523-34. Epub 2013 May 13.

Cork Cancer Research Centre, BioScience Institute, University College Cork, Cork, Ireland.

There is an undisputed need for employment and improvement of robust technology for real-time analyses of therapeutic delivery and responses in clinical translation of gene and cell therapies. Over the past decade, optical imaging has become the in vivo imaging modality of choice for many preclinical laboratories due to its efficiency, practicality and affordability, while more recently, the clinical potential for this technology is becoming apparent. This review provides an update on the current state of the art in in vivo optical imaging and discusses this rapidly improving technology in the context of it representing a translation enabler or indeed a future clinical imaging modality in its own right.
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http://dx.doi.org/10.1016/j.jconrel.2013.05.004DOI Listing
December 2013