A nested real-time PCR assay has an increased sensitivity suitable for detection of viruses in aerosol studies

Aims: Influenza is commonly spread by infectious aerosols; however, detection of viruses in aerosols is not sensitive enough to confirm the characteristics of virus aerosols. The aim of this study was to develop an assay for respiratory viruses sufficiently sensitive to be used in epidemiological studies. Method: A two-step, nested real-time PCR assay was developed for MS2 bacteriophage, and for influenza A and B, parainfluenza 1 and human respiratory syncytial virus. Outer primer pairs were designed to nest each existing real-time PCR assay. The sensitivities of the nested real-time PCR assays were compared to those of existing real-time PCR assays. Both assays were applied in an aerosol study to compare their detection limits in air samples. Conclusions: The nested real-time PCR assays were found to be several logs more sensitive than the real-time PCR assays, with lower levels of virus detected at lower Ct values. The nested real-time PCR assay successfully detected MS2 in air samples, whereas the real-time assay did not. Significance and Impact of the Study: The sensitive assays for respiratory viruses will permit further research using air samples from naturally generated virus aerosols. This will inform current knowledge regarding the risks associated with the spread of viruses through aerosol transmission.

Quantitative PCR assay of sewage-associated Bacteroides markers to assess sewage pollution in an urban lake in Dhaka, Bangladesh

This paper aimed to assess the magnitude of sewage pollution in an urban lake in Dhaka, Bangladesh by using Quantitative PCR (qPCR) of sewage-associated Bacteroides HF183 markers. PCR was also used for the quantitative detection of ruminant wastewater-associated CF128 markers along with the enumeration of traditional fecal indicator bacteria, namely, enterococci. The number of enterococci in lake water samples ranged from 1.1 x 104 to 1.9 x 105 CFU/100 ml of water. From the 20 water samples tested, 14 (70%) and 7 (35%) were PCR positive for the HF183 and CF128 markers, respectively. The numbers of the HF183 and CF128 markers in lake water samples were 3.9 x 104 to 6.3 × 107 and 9.3 x 103 to 6.3 x 105 genomic units (GU)/100 ml of water, respectively. The high numbers of enterococci and the HF183 markers indicate sewage pollution and potential health risks to those who use the lake water for non-potable purposes such as bathing and washing clothes. This is the first study that investigated the presence of microbial source tracking (MST) markers in Dhaka, Bangladesh where diarrhoeal diseases is one of the major causes of childhood mortality. The molecular assay as used in this study can provide valuable information on the extent of sewage pollution, thus facilitating the development of robust strategies to minimise potential health risks.

IS-RT-PCR assay detection of MT-MMP in a human breast cancer cell line

The in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) is a method that allows the direct localisation of gene expression. The method utilises the dual buffer mediated activity of the enzyme rTth DNA polymerase enabling both reverse transcription and DNA amplification. Labelled nucleoside triphosphates allow the site of expression to be labelled, rather than the PCR primers themselves, giving a more accurate localisation of transcript expression and decreased background than standard in situ hybridisation (ISH) assays. The MDA-MB-231 human breast carcinoma (HBC) cell line was assayed via the IS-RT-PCR technique, using primers encoding MT-MMP (membrane-type matrix metalloproteinase) and human β-actin. Our results clearly indicate baseline expression of MT-MMP in the relatively invasive MDA-MB-231 cell line at a signal intensity similar to the housekeeping gene β-actin, and results following induction with Concanavalin A (Con A) are consistent with our previous results obtained via Northern blotting.

Comparison of PCR, nested PCR, and random amplified polymorphic DNA PCR for detection and typing of Ureaplasma urealyticum in specimens from pregnant women

A PCR assay, using three primer pairs, was developed for the detection of Ureaplasma urealyticum, parvo biovar, mba types 1, 3, and 6, in cultured clinical specimens. The primer pairs were designed by using the polymorphic base positions within a 310- to 311-bp fragment of the 5* end and upstream control region of the mba gene. The specificity of the assay was confirmed with reference serovars 1, 3, 6, and 14 and by the amplified-fragment sizes (81 bp for mba 1, 262 bp for mba 3, and 193 bp for mba 6). A more sensitive nested PCR was also developed. This involved a first-step PCR, using the primers UMS-125 and UMA226, followed by the nested mba-type PCR described above. This nested PCR enabled the detection and typing of small numbers of U. urealyticum cells, including mixtures, directly in original clinical specimens. By using random amplified polymorphic DNA (RAPD) PCR with seven arbitrary primers, we were also able to differentiate the two biovars of U. urealyticum and to identify 13 RAPD-PCR subtypes. By applying these subtyping techniques to clinical samples collected from pregnant women, we established that (i) U. urealyticum is often a persistent colonizer of the lower genital tract from early midtrimester until the third trimester of pregnancy, (ii) mba type 6 was isolated significantly more often (P 5 0.048) from women who delivered preterm than from women who delivered at term, (iii) no particular ureaplasma subtype(s) was associated with placental infections and/or adverse pregnancy outcomes, and (iv) the ureaplasma subtypes most frequently isolated from women were the same subtypes most often isolated from infected placentas.

Protocol : A highly sensitive RT-PCR method for detection and quantification of microRNAs

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 l of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.

The severity of chorioamnionitis in pregnant sheep is associated with in vivo variation of the surface-exposed multiple-banded antigen/gene of ureaplasma parvum

Ureaplasma species are the bacteria most frequently isolated from human amniotic fluid in asymptomatic pregnancies and placental infections. Ureaplasma parvum serovars 3 and 6 are the most prevalent serovars isolated from men and women. We hypothesized that the effects on the fetus and chorioamnion of chronic ureaplasma infection in amniotic fluid are dependent on the serovar, dose, and variation of the ureaplasma multiple banded antigen (MBA) and mba gene. We injected high- or low dose U. parvum serovar 3, serovar 6, or vehicle intra-amniotically into pregnant ewes at 55 days of gestation (term = 150 days) and examined the chorioamnion, amniotic fluid, and fetal lung tissue of animals delivered by cesarean section at 125 days of gestation. Variation of the multiple banded antigen/mba generated by serovar 3 and serovar 6 ureaplasmas in vivo were compared by PCR assay and Western blot. Ureaplasma inoculums demonstrated only one (serovar 3) or two (serovar 6) MBA variants in vitro, but numerous antigenic variants were generated in vivo: serovar 6 passage 1 amniotic fluid cultures contained more MBA size variants than serovar 3 (P = 0.005),and ureaplasma titers were inversely related to the number of variants (P = 0.025). The severity of chorioamnionitis varied between animals. Low numbers of mba size variants (five or fewer) within amniotic fluid were associated with severe inflammation, whereas the chorioamnion from animals with nine or more mba variants showed little or no inflammation. These differences in chorioamnion inflammation may explain why not all women with in utero Ureaplasma spp. experience adverse pregnancy outcomes.

Detection of bacteriophage and respiratory viruses in droplets

Influenza is a widespread disease occurring in seasonal epidemics, and each year is responsible for up to 500,000 deaths worldwide. Influenza can develop into strains which cause severe symptoms and high mortality rates, and could potentially reach pandemic status if the virus’ properties allow easy transmission. Influenza is transmissible via contact with the virus, either directly (infected people) or indirectly (contaminated objects); via reception of large droplets over short distances (one metre or less); or through inhalation of aerosols containing the virus expelled by infected individuals during respiratory activities, that can remain suspended in the air and travel distances of more than one metre (the aerosol route). Aerosol transmission of viruses involves three stages: production of the droplets containing viruses; transport of the droplets and ability of a virus to remain intact and infectious; and reception of the droplets (via inhalation). Our understanding of the transmission of influenza viruses via the aerosol route is poor, and thus our ability to prevent a widespread outbreak is limited. This study explored the fate of viruses in droplets by investigating the effects of some physical factors on the recovery of both a bacteriophage model and influenza virus. Experiments simulating respiratory droplets were carried out using different types of droplets, generated from a commonly used water-like matrix, and also from an ‘artificial mucous’ matrix which was used to more closely resemble respiratory fluids. To detect viruses in droplets, we used the traditional plaque assay techniques, and also a sensitive, quantitative PCR assay specifically developed for this study. Our results showed that the artificial mucous suspension enhanced the recovery of infectious bacteriophage. We were able to report detection limits of infectious bacteriophage (no bacteriophage was detected by the plaque assay when aerosolised from a suspension of 103 PFU/mL, for three of the four droplet types tested), and that bacteriophage could remain infectious in suspended droplets for up to 20 minutes. We also showed that the nested real-time PCR assay was able to detect the presence of bacteriophage RNA where the plaque assay could not detect any intact particles. Finally, when applying knowledge from the bacteriophage experiments, we reported the quantitative recoveries of influenza viruses in droplets, which were more consistent and stable than we had anticipated. Influenza viruses can be detected up to 20 minutes (after aerosolisation) in suspended aerosols and possibly beyond. It also was detectable from nebulising suspensions with relatively low concentrations of viruses.

Detection of banana bunchy top virus in virus-infected plants using polymerase chain reaction

Polymerase chain reaction (PCR) was developed for the detection of Banana bunchy top virus (BBTV) at maximum after 210 min and at minimum after 90 min using Pc-1 and Pc-2, respectively. PCR detection of BBTV in crude sap indicated that the freezing of banana tissue in liquid nitrogen (LN2) before extraction was more effective than using sand as the extraction technique. BBTV was also detected using PCR assay in 69 healthy and diseased plants using Na-PO4 buffer containing 1 % SDS. PCR detection of BBTV in nucleic acid extracts using seven different extraction buffers to adapt the use of PCR in routine detection in the field was studied. Results proved that BBTV was detected with high sensitivity in nucleic acid extracts more than in infectious sap. The results also suggested the common aetiology for the BBTV by the PCR reactions of BBTV in nucleic acid extracts from Australia, Burundi, Egypt, France, Gabon, Philippines and Taiwan. Results also proved a positive relation between the Egyptian-BBTV isolate and abaca bunchy top isolate from the Philippines, but there no relation was found with the Cucumber mosaic cucumovirus (CMV) isolates from Egypt and Philippines and Banana bract mosaic virus (BBMV) were found.

Strategies for improved disease resistance in micro-propagated bananas

Bananas (Musa sp) are one of the most important food crops in the world and provide a staple food and source of income in many households especially in Africa. Diseases are a major constraint to production with bunchy top, caused by Banana bunchy top virus (BBTV) generally considered the most important virus disease of bananas worldwide. Of the fungal diseases, Fusarium wilt, caused by the Fusarium oxysporum f.sp cubense (Foc), and black Sigatoka, caused by Mycosphaerella fijiensis, are arguably two of the most important and cause significant yield losses. The low fertility of commercially important banana cultivars has hampered efforts to generate disease resistance using conventional breeding. Possible alternative strategies to generate or increase disease resistance are through genetic engineering or by manipulation of the innate plant defence mechanisms, namely systemic acquired resistance (SAR). The first research component of this thesis describes attempts to generate BBTV-resistant banana plants using a genetic modification approach. The second research component of the thesis focused on the identification of a potential marker gene associated with SAR in banana plants and a comparison of the expression levels of the marker gene in response to biotic and abiotic stresses, and chemical inducers. Previous research at QUT CTCB showed that replication of BBTV DNA components in banana embryogenic cell suspensions (ECS) was abolished following co-bombardment with 1.1mers of mutated BBTV DNA-R. BBTV DNA-R encodes the master replication protein (Rep) and is the only viral protein essential for BBTV replication. In this study, ECS of banana were stably transformed with the same constructs, each containing a different mutation in BBTV DNA-R, namely H41G, Y79F and K187M, to examine the effect on virus replication in stably transformed plants. Cells were also transformed with a construct containing a native BBTV Rep. A total of 16, 16, 11 and five lines of stably transformed banana plants containing the Y79F, H41G, K187M and native Rep constructs, respectively, were generated. Of these, up to nine replicates from Y79F lines, four H41G lines, seven K187M lines and three native Rep lines were inoculated with BBTV by exposure to viruliferous aphids in two separate experiments. At least one replicate from each of the nine Y79F lines developed typical bunchy top symptoms and all tested positive for BBTV using PCR. Of the four H41G lines tested, at least one replicate from three of the lines showed symptoms of bunchy top and tested positive using PCR. However, none of the five replicates of one H41G line (H41G-3) developed symptoms of bunchy top and none of the plants tested positive for BBTV using PCR. Of the seven K187M lines, at least one replicate of all lines except one (K187M-1) developed symptoms of bunchy top and tested positive for BBTV. Importantly, none of the four replicates of line K187M-1 showed symptoms or tested positive for BBTV. At least one replicate from each of the three native Rep lines developed symptoms and tested positive for BBTV. The H41G-3 and K187M-1 lines possibly represent the first transgenic banana plants generated using a mutated Rep strategy. The second research component of this thesis focused on the identification of SAR-associated genes in banana and their expression levels in response to biotic and abiotic stresses and chemical inducers. The impetus for this research was the observation that tissue-cultured (TC) banana plants were more susceptible to Fusarium wilt disease (and possibly bunchy top disease) than plants grown from field-derived suckers, possibly due to decreased levels of SAR gene expression in the former. In this study, the pathogenesis-related protein 1 (PR-1) gene was identified as a potential marker for SAR gene expression in banana. A quantitative real-time PCR assay was developed and optimised in order to determine the expression of PR-1, with polyubiquitin (Ubi-1) found to be the most suitable reference gene to enable relative quantification. The levels of PR-1 expression were subsequently compared in Lady Finger and Cavendish (cv. Williams) banana plants grown under three different environmental conditions, namely in the field, the glass house and in tissue-culture. PR-1 was shown to be expressed in both cultivars growing under different conditions. While PR-1 expression was highest in the field grown bananas and lowest in the TC bananas in Lady Finger cultivar, this was not the case in the Cavendish cultivar with glass house plants exhibiting the lowest PR-1 expression compared with tissue culture and field grown plants. The important outcomes of this work were the establishment of a qPCR-based assay to monitor PR-1 expression levels in banana and a preliminary assessment of the baseline PR-1 expression levels in two banana cultivars under three different growing conditions. After establishing the baseline PR-1 expression levels in Cavendish bananas, a study was done to determine whether PR-1 levels could be increased in these plants by exposure to known banana pathogens and non-pathogens, and a known chemical inducer of SAR. Cavendish banana plants were exposed to pathogenic Foc subtropical race 4 (FocSR4) and non-pathogenic Foc race 1 (Foc1), as well as two putative inducers of resistance, Fusarium lycopersici (Fol) and the chemical, acibenzolar-S-methyl (BION®). Tissue culture bananas were acclimatised under either glass house (TCS) or field (TCH) conditions and treatments were carried out in a randomised complete block design. PR-1 expression was determined using qPCR for both TCS and TCH samples for the period 12-72h post-exposure. Treatment of TCH plants using Foc1 and FocSR4 resulted in 120 and 80 times higher PR-1 expression than baseline levels, respectively. For TCS plants treated with Foc1, PR-1 expression was 30 times higher than baseline levels at 12h post-exposure, while TCS plants treated with FocSR4 showed the highest PR-1 expression (20 times higher than baseline levels) at 72h post-exposure. Interestingly, when TCS plants were treated with Fol there was a marked increase of PR-1 expression at 12 h and 48 h following treatment which was 4 and 8 times higher than the levels observed when TCS plants were treated with Foc1 and FocSR4, respectively. In contrast, when TCH plants were treated with Fol only a slight increase in PR-1 expression was observed at 12 h, which eventually returned to baseline levels. Exposure of both TCS and TCH plants to BION® resulted in no effect on PR-1 expression levels at any time-point. The major outcome of the SAR study was that the glass house acclimatised tissue culture bananas exhibited lower PR-1 gene expression compared to field acclimatised tissue culture plants and the identification of Fol as a good candidate for SAR induction in banana plants exhibiting low PR-1 levels. A number of outcomes that foster understanding of both pathogen-derived and plant innate resistance strategies in order to potentially improve banana resistance to diseases were explored in this study and include identification of potential inducers of systemic acquired resistance and a promising mutated Rep approach for BBTV resistance. The work presented in this thesis is the first report on the generation of potential BBTV resistant bananas using the mutated Rep approach. In addition, this is the first report on the status of SAR in banana grown under different conditions of exposure to the biotic and abiotic environment. Further, a robust qPCR assay for the study of gene expression using banana leaf samples was developed and a potential inducer of SAR in tissue culture bananas identified which could be harnessed to increase resistance in tissue culture bananas.

Molecular characterisation and expression analysis of interferon gamma in response to natural chlamydia infection in the koala, phascolarctos cinereus

Interferon gamma (IFNγ) is a key Th1 cytokine, with a principal role in the immune response against intracellular organisms such as Chlamydia. Along with being responsible for significant morbidity in human populations, Chlamydia is also responsible for wide spread infection and disease in many animal hosts, with reports that many Australian koala subpopulations are endemically infected. An understanding of the role played by IFNγ in koala chlamydial diseases is important for the establishment of better prophylactic and therapeutic approaches against chlamydial infection in this host. A limited number of IFNγ sequences have been published from marsupials and no immune reagents to measure expression have been developed. Through preliminary analysis of the koala transcriptome, we have identified the full coding sequence of the koala IFNγ gene. Transcripts were identified in spleen and lymph node tissue samples. Phylogenetic analysis demonstrated that koala IFNγ is closely related to other marsupial IFNγ sequences and more distantly related to eutherian mammals. To begin to characterise the role of this important cytokine in the koala's response to chlamydial infection, we developed a quantitative real time PCR assay and applied it to a small cohort of koalas with and without active chlamydial disease, revealing significant differences in expression patterns between the groups. Description of the IFNγ sequence from the koala will not only assist in understanding this species' response to its most important pathogen but will also provide further insight into the evolution of the marsupial immune system

A dynamic in vivo model of epithelial-to-mesenchymal transitions in circulating tumor cells and metastases of breast cancer

Epithelial-to-mesenchymal transition (EMT) processes endow epithelial cells with enhanced migratory/invasive properties and are therefore likely to contribute to tumor invasion and metastatic spread. Because of the difficulty in following EMT processes in human tumors, we have developed and characterized an animal model with transplantable human breast tumor cells (MDA-MB-468) uniquely showing spontaneous EMT events to occur. Using vimentin as a marker of EMT, heterogeneity was revealed in the primary MDA-MB-468 xenografts with vimentin-negative and vimentin-positive areas, as also observed on clinical human invasive breast tumor specimens. Reverse transcriptase-PCR after microdissection of these populations from the xenografts revealed EMT traits in the vimentin-positive zones characterized by enhanced 'mesenchymal gene' expression (Snail, Slug and fibroblast-specific protein-1) and diminished expression of epithelial molecules (E-cadherin, ZO-3 and JAM-A). Circulating tumor cells (CTCs) were detected in the blood as soon as 8 days after s.c. injection, and lung metastases developed in all animals injected as examined by in vivo imaging analyses and histology. High levels of vimentin RNA were detected in CTCs by reverse transcriptase-quantitative PCR as well as, to a lesser extent, Snail and Slug RNA. Von Willebrand Factor/vimentin double immunostainings further showed that tumor cells in vascular tumoral emboli all expressed vimentin. Tumoral emboli in the lungs also expressed vimentin whereas macrometastases displayed heterogenous vimentin expression, as seen in the primary xenografts. In conclusion, our data uniquely demonstrate in an in vivo context that EMT occurs in the primary tumors, and associates with an enhanced ability to intravasate and generate CTCs. They further suggest that mesenchymal-to-epithelial phenomena occur in secondary organs, facilitating the metastatic growth.

Inducible resistance to maize streak virus

Maize streak virus (MSV), which causes maize streak disease (MSD), is the major viral pathogenic constraint on maize production in Africa. Type member of the Mastrevirus genus in the family Geminiviridae, MSV has a 2.7 kb, single-stranded circular DNA genome encoding a coat protein, movement protein, and the two replication-associated proteins Rep and RepA. While we have previously developed MSV-resistant transgenic maize lines constitutively expressing ‘‘dominant negative mutant’’ versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or ‘‘leaky’’ expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture. Split gene cassette constructs (SGCs) were developed containing three different transgenes in combination with three different promoter sequences. In each SGC, the transgene was split such that it would be translatable only in the presence of an infecting MSV’s replication associated protein. We used a quantitative real-time PCR assay to show that one of these SGCs (pSPLITrepIII-Rb-Ubi) inducibly inhibits MSV replication as efficiently as does a constitutively expressed transgene that has previously proven effective in protecting transgenic maize from MSV. In addition, in our cell-culture based assay pSPLITrepIII-Rb-Ubi inhibited replication of diverse MSV strains, and even, albeit to a lesser extent, of a different mastrevirus species. The application of this new technology to MSV resistance in maize could allow a better, more acceptable product.

A mouse with an N-ethyl-N-nitrosourea (ENU) induced Trp589Arg Galnt3 mutation represents a model for hyperphosphataemic familial tumoural calcinosis

Mutations of UDP-N-acetyl-alpha-D-galactosamine polypeptide N-acetyl galactosaminyl transferase 3 (GALNT3) result in familial tumoural calcinosis (FTC) and the hyperostosis-hyperphosphataemia syndrome (HHS), which are autosomal recessive disorders characterised by soft-tissue calcification and hyperphosphataemia. To facilitate in vivo studies of these heritable disorders of phosphate homeostasis, we embarked on establishing a mouse model by assessing progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU), and identified a mutant mouse, TCAL, with autosomal recessive inheritance of ectopic calcification, which involved multiple tissues, and hyperphosphataemia; the phenotype was designated TCAL and the locus, Tcal. TCAL males were infertile with loss of Sertoli cells and spermatozoa, and increased testicular apoptosis. Genetic mapping localized Tcal to chromosome 2 (62.64-71.11 Mb) which contained the Galnt3. DNA sequence analysis identified a Galnt3 missense mutation (Trp589Arg) in TCAL mice. Transient transfection of wild-type and mutant Galnt3-enhanced green fluorescent protein (EGFP) constructs in COS-7 cells revealed endoplasmic reticulum retention of the Trp589Arg mutant and Western blot analysis of kidney homogenates demonstrated defective glycosylation of Galnt3 in Tcal/Tcal mice. Tcal/Tcal mice had normal plasma calcium and parathyroid hormone concentrations; decreased alkaline phosphatase activity and intact Fgf23 concentrations; and elevation of circulating 1,25-dihydroxyvitamin D. Quantitative reverse transcriptase-PCR (qRT-PCR) revealed that Tcal/Tcal mice had increased expression of Galnt3 and Fgf23 in bone, but that renal expression of Klotho, 25-hydroxyvitamin D-1α-hydroxylase (Cyp27b1), and the sodium-phosphate co-transporters type-IIa and -IIc was similar to that in wild-type mice. Thus, TCAL mice have the phenotypic features of FTC and HHS, and provide a model for these disorders of phosphate metabolism. © 2012 Esapa et al.

Protein complementation assay as a display system for screening protein libraries in the intracellular environment

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) *Escherichia coli* proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

Identification of circulating tumour cells in early stage breast cancer patients using multi marker immunobead RT-PCR

Introduction The ability to screen blood of early stage operable breast cancer patients for circulating tumour cells is of potential importance for identifying patients at risk of developing distant relapse. We present the results of a study of the efficacy of the immunobead RT-PCR method in identifying patients with circulating tumour cells. Results Immunomagnetic enrichment of circulating tumour cells followed by RT-PCR (immunobead RT-PCR) with a panel of five epithelial specific markers (ELF3, EPHB4, EGFR, MGB1 and TACSTD1) was used to screen for circulating tumour cells in the peripheral blood of 56 breast cancer patients. Twenty patients were positive for two or more RT-PCR markers, including seven patients who were node negative by conventional techniques. Significant increases in the frequency of marker positivity was seen in lymph node positive patients, in patients with high grade tumours and in patients with lymphovascular invasion. A strong trend towards improved disease free survival was seen for marker negative patients although it did not reach significance (p = 0.08). Conclusion Multi-marker immunobead RT-PCR analysis of peripheral blood is a robust assay that is capable of detecting circulating tumour cells in early stage breast cancer patients.