Combined effects of short-term exposure to elevated CO2 and decreased O2 on the physiology and energy budget of the thick shell mussel Mytilus coruscus

Hypoxia and ocean acidification are two consequences of anthropogenic activities. These global trends occur on top of natural variability. In environments such as estuarine areas, short-term acute pH and O2 fluctuations are occurring simultaneously. The present study tested the combined effects of short-term seawater acidification and hypoxia on the physiology and energy budget of the thick shell mussel Mytilus coruscus. Mussels were exposed for 72 h to six combined treatments with three pH levels (8.1, 7.7 and 7.3) and two dissolved oxygen (DO) levels (2 mg/L, 6 mg/L). Clearance rate (CR), food absorption efficiency (AE), respiration rate (RR), ammonium excretion rate (ER), O:N ratio and scope for growth (SFG) were significantly reduced, and faecal organic dry weight ratio (E) was significantly increased at low DO. Low pH did not lead to a reduced SFG. Interactive effects of pH and DO were observed for CR, E and RR. Principal component analysis (PCA) revealed positive relationships among most physiological indicators, especially between SFG and CR under normal DO conditions. These results demonstrate that Mytilus coruscus was sensitive to short-term (72 h) exposure to decreased O2 especially if combined with decreased pH levels. In conclusion, the short-term oxygen and pH variation significantly induced physiological changes of mussels with some interactive effects.

Data compilation on the biological response to ocean acidification: environmental and experimental context of data sets and related literature

The exponential growth of studies on the biological response to ocean acidification over the last few decades has generated a large amount of data. To facilitate data comparison, a data compilation hosted at the data publisher PANGAEA was initiated in 2008 and is updated on a regular basis (doi:10.1594/PANGAEA.149999). By January 2015, a total of 581 data sets (over 4 000 000 data points) from 539 papers had been archived. Here we present the developments of this data compilation five years since its first description by Nisumaa et al. (2010). Most of study sites from which data archived are still in the Northern Hemisphere and the number of archived data from studies from the Southern Hemisphere and polar oceans are still relatively low. Data from 60 studies that investigated the response of a mix of organisms or natural communities were all added after 2010, indicating a welcomed shift from the study of individual organisms to communities and ecosystems. The initial imbalance of considerably more data archived on calcification and primary production than on other processes has improved. There is also a clear tendency towards more data archived from multifactorial studies after 2010. For easier and more effective access to ocean acidification data, the ocean acidification community is strongly encouraged to contribute to the data archiving effort, and help develop standard vocabularies describing the variables and define best practices for archiving ocean acidification data.

Virtual reality rehabilitation as a treatment approach for older women with mixed urinary incontinence : a feasibility study

Background Motivated patients are more likely to adhere to treatment resulting in better outcomes. Virtual reality rehabilitation (VRR) is a treatment approach that includes video gaming to enhance motivation and functional training. Aims The study objectives were (1) to evaluate the feasibility of using a combination of pelvic floor muscles (PFM) exercises and VRR (PFM/VRR) to treat mixed urinary incontinence (MUI) in older women, (2) to evaluate the effectiveness of the PFM/VRR program on MUI symptoms, quality of life (QoL), and (3) gather quantitative information regarding patient satisfaction with this new combined training program. Methods Women 65 years and older with at least 2 weekly episodes of MUI were recruited. Participants were evaluated two times before and one time after a 12-week PFM/VRR training program. Feasibility was defined as the participants' rate of participation in and completion of both the PFM/VRR training program and the home exercise. Effectiveness was evaluated through a bladder diary, pad test, symptom and QoL questionnaire, and participant's satisfaction through a questionnaire. Results Twenty-four women (70.5 ± 3.6 years) participated. The participants complied with the study demands in terms of attendance at the weekly treatment sessions (91%), adherence to home exercise (92%) and completion of the three evaluations (96%). Post-intervention, the frequency and quantity of urine leakage decreased and patientreported symptoms and QoL improved significantly. Most participants were very satisfied with treatment (91%). Conclusion A combined PFM/VRR program is an acceptable, efficient, and satisfying functional treatment for older women with MUI and should be explore through further RCTs.

An investigation of the molecular interactions between statins and bacterial pathogens, and their combined impact on the human immune system

Statins are a class of drug that inhibits cholesterol biosynthesis, and are used to treat patients with high serum cholesterol levels. They exert this function by competitively binding to the enzyme 3-hydroxy-3-methylglutaryl-CoenzymeA reductase (HMGR), which catalyses the formation of mevalonate, a rate-limiting step in cholesterol biosynthesis. In addition, statins have what are called “pleiotropic effects”, which include the reduction of inflammation, immunomodulation, and antimicrobial effects. Statins can also improve survival of patients with sepsis and pneumonia. Cystic fibrosis (CF) is the most common recessive inherited disease in the Caucasian population, which is characterised by factors including, but not limited to, excessive lung inflammation and increased susceptibility to infection. Therefore, the overall objective of this study was to examine the effects of statins on CFassociated bacterial pathogens and the host response. In this work, the prevalence of HMGR was examined in respiratory pathogens, and several CF-associated pathogens were found to possess homologues of this enzyme. HMGR homology was analysed in Staphylococcus aureus, Burkholderia cenocepacia and Streptococcus pneumoniae, and the HMGR of B. cenocepacia was found to have significant conservation to that of Pseudomonas mevalonii, which is the most widely-characterised bacterial HMGR. However, in silico analysis revealed that, unlike S. aureus and S. pneumoniae, B. cenocepacia did not possess homologues of other mevalonate pathway proteins, and that the HMGR of B. cenocepacia appeared to be involved in an alternative metabolic pathway. The effect of simvastatin was subsequently tested on the growth and virulence of S. aureus, B. cenocepacia and S. pneumoniae. Simvastatin inhibited the growth of all 3 species in a dose-dependent manner. In addition, statin treatment also attenuated biofilm formation of all 3 species, and reduced in vitro motility of S. aureus. Interestingly, simvastatin also increased the potency of the aminoglycoside antibiotic gentamicin against B. cenocepacia. The impact of statins was subsequently tested on the predominant CF-associated pathogen Pseudomonas aeruginosa, which does not possess a HMGR homologue. Mevastatin, lovastatin and simvastatin did not influence the growth of this species. However, sub-inhibitory statin concentrations reduced the swarming motility and biofilm formation of P. aeruginosa. The influence of statins was also examined on Type 3 toxin secretion, quorum sensing and chemotaxis, and no statin effect was observed on any of these phenotypes. Statins did not appear to have a characteristic effect on the P. aeruginosa transcriptome. However, a mutant library screen revealed that the effect of statins on P. aeruginosa biofilm was mediated through the PvrR regulator and the Cup fimbrial biosynthesis genes. Furthermore, proteomic analysis demonstrated that 6 proteins were reproducibly induced by simvastatin in the P. aeruginosa swarming cells. The effect of statins on the regulation of the host-P. aeruginosa immune response was also investigated. Statin treatment increased expression of the pro-inflammatory cytokines IL-8 and CCL20 in lung epithelial cells, but did not attenuate P. aeruginosa-mediated inflammatory gene induction. In fact, simvastatin and P. aeruginosa caused a synergistic effect on CCL20 expression. The expression of the transcriptional regulators KLF2 and KLF6 was also increased by statins and P. aeruginosa, with the induction of KLF6 by simvastatin proving to be a novel effect. Interestingly, both statins and P. aeruginosa were capable of inducing alternative splicing of KLF6. P. aeruginosa was found to induce KLF6 alternative splicing by way of the type 3 secreted toxin ExoS. In addition, a mechanistic role was elucidated for KLF6 in the lung, as it was determined that statin-mediated induction of this protein was responsible for the induction of the host response genes CCL20 and iNOS. Moreover, statin treatment caused a slight increase in infection-related cytotoxicity, and increased bacterial adhesion to cells. Taken together, these data demonstrate that statins can reduce the virulence of CFassociated bacterial pathogens and alter host response effectors. Furthermore, novel statin effectors were identified in both bacterial and host cells.

Virtual reality rehabilitation as a treatment approach for older women with mixed urinary incontinence : a feasibility study

Background Motivated patients are more likely to adhere to treatment resulting in better outcomes. Virtual reality rehabilitation (VRR) is a treatment approach that includes video gaming to enhance motivation and functional training. Aims The study objectives were (1) to evaluate the feasibility of using a combination of pelvic floor muscles (PFM) exercises and VRR (PFM/VRR) to treat mixed urinary incontinence (MUI) in older women, (2) to evaluate the effectiveness of the PFM/VRR program on MUI symptoms, quality of life (QoL), and (3) gather quantitative information regarding patient satisfaction with this new combined training program. Methods Women 65 years and older with at least 2 weekly episodes of MUI were recruited. Participants were evaluated two times before and one time after a 12-week PFM/VRR training program. Feasibility was defined as the participants' rate of participation in and completion of both the PFM/VRR training program and the home exercise. Effectiveness was evaluated through a bladder diary, pad test, symptom and QoL questionnaire, and participant's satisfaction through a questionnaire. Results Twenty-four women (70.5 ± 3.6 years) participated. The participants complied with the study demands in terms of attendance at the weekly treatment sessions (91%), adherence to home exercise (92%) and completion of the three evaluations (96%). Post-intervention, the frequency and quantity of urine leakage decreased and patientreported symptoms and QoL improved significantly. Most participants were very satisfied with treatment (91%). Conclusion A combined PFM/VRR program is an acceptable, efficient, and satisfying functional treatment for older women with MUI and should be explore through further RCTs.

Combined effects of solar UV radiation and CO2-induced seawater acidification on photosynthetic carbon fixation of phytoplankton assemblages in the South China Sea, 2010

We carried out short term pCO2/pH perturbation experiments in the coastal waters of the South China Sea to evaluate the combined effects of seawater acidification (low pH/high pCO2) and solar UV radiation (UVR, 280-400 nm) on photosynthetic carbon fixation of phytoplankton assemblages. Under photosynthetically active radiation (PAR) alone treatments, reduced pCO2 (190 ppmv) with increased pH resulted in a significant decrease in the photosynthetic carbon fixation rate (about 23%), while enriched pCO2 (700 ppmv) with lowered pH had no significant effect on the photosynthetic performance compared to the ambient level. The apparent photosynthetic efficiency decreased under the reduced pCO2 level, probably due to C-limitation as well as energy being diverged for up-regulation of carbon concentrating mechanisms (CCMs). In the presence of UVR, both UV-A and UV-B caused photosynthetic inhibition, though UV-A appeared to enhance the photosynthetic efficiency under lower PAR levels. UV-B caused less inhibition of photosynthesis under the reduced pCO2 level, probably because of its contribution to the inorganic carbon (Ci)-acquisition processes. Under the seawater acidification conditions (enriched pCO2), both UV-A and UV-B reduced the photosynthetic carbon fixation to higher extents compared to the ambient pCO2 conditions. We conclude that solar UV and seawater acidification could synergistically inhibit photosynthesis.

Berry size and seed number of V. myrtillus and E. hermaphroditum according to treatment, and environmental parameters 1991-2009 at Abisko Research Station

This study investigated the effects of long-term-enhanced UV-B, and combined UV-B with elevated CO2 on dwarf shrub berry characteristics in a sub-arctic heath community. Germination of Vaccinium myrtillus was enhanced in seeds produced at elevated UV-B, but seed numbers and berry size were unaffected. Elevated UV-B and CO2 stimulated the abundance of V. myrtillus berries, whilst UV-B alone stimulated the berry abundance of V. vitis-idaea and Empetrum hermaphroditum. Enhanced UV-B reduced concentrations of several polyphenolics in V. myrtillus berries, whilst elevated CO2 increased quercetin glycosides in V. myrtillus, and syringetin glycosides and anthocyanins in E. hermaphroditum berries. UV-B x CO2 interactions were found for total anthocyanins, delphinidin-3-hexoside and peonidin-3-pentosidein in V. myrtillus berries but not E. hermaphroditum. Results suggest positive impacts of UV-B on the germination of V. myrtillus and species-specific impacts of UV-B x elevated CO2 on berry abundance and quality. The findings have relevance and implications for human and animal consumers plus seed dispersal and seedling establishment.

Seawater carbonate chemistry and combined physiological effects of CO2 and light on the N2-fixing cyanobacterium Trichodesmium IMS101 during experiments, 2010

Recent studies on the diazotrophic cyanobacterium Trichodesmium erythraeum(IMS101) showed that increasing CO2 partial pressure (pCO2) enhances N2 fixation and growth. Significant uncertainties remain as to the degree of the sensitivity to pCO2, its modification by other environmental factors, and underlying processes causing these responses. To address these questions, we examined the responses ofTrichodesmium IMS101 grown under a matrix of low and high levels of pCO2 (150 and 900 µatm) and irradiance (50 and 200 µmol photons m-2 s-1). Growth rates as well as cellular carbon and nitrogen contents increased with increasing pCO2 and light levels in the cultures. The pCO2-dependent stimulation in organic carbon and nitrogen production was highest under low light. High pCO2 stimulated rates of N2fixation and prolonged the duration, while high light affected maximum rates only. Gross photosynthesis increased with light but did not change with pCO2. HCO3- was identified as the predominant carbon source taken up in all treatments. Inorganic carbon uptake increased with light, but only gross CO2 uptake was enhanced under high pCO2. A comparison between carbon fluxes in vivo and those derived from 13C fractionation indicates high internal carbon cycling, especially in the low-pCO2treatment under high light. Light-dependent oxygen uptake was only detected underlow pCO2 combined with high light or when low-light-acclimated cells were exposed to high light, indicating that the Mehler reaction functions also as a photoprotective mechanism in Trichodesmium. Our data confirm the pronounced pCO2 effect on N2fixation and growth in Trichodesmium and further show a strong modulation of these effects by light intensity. We attribute these responses to changes in the allocation of photosynthetic energy between carbon acquisition and the assimilation of carbon and nitrogen under elevated pCO2. These findings are supported by a complementarystudy looking at photosynthetic fluorescence parameters of photosystem II, photosynthetic unit stoichiometry (photosystem I:photosystem II), and pool sizes of key proteins in carbon and nitrogen acquisition.

Combined effects of ocean acidification and light or nitrogen availabilities on 13C fractionation in marine dinoflagellates

Along with increasing oceanic CO2 concentrations, enhanced stratification constrains phytoplankton to shallower upper mixed layers with altered light regimes and nutrient concentrations. Here, we investigate the effects of elevated pCO2 in combination with light or nitrogen-limitation on 13C fractionation (epsilon p) in four dinoflagellate species. We cultured Gonyaulax spinifera and Protoceratium reticulatum in dilute batches under low-light (LL) and high-light (HL) conditions, and grew Alexandrium fundyense and Scrippsiella trochoidea in nitrogen-limited continuous cultures (LN) and nitrogen-replete batches (HN). The observed CO2-dependency of epsilon p remained unaffected by the availability of light for both G. spinifera and P. reticulatum, though at HL epsilon p was consistently lower by about 2.7 per mil over the tested CO2 range for P. reticulatum. This may reflect increased uptake of (13C-enriched) bicarbonate fueled by increased ATP production under HL conditions. The observed CO2-dependency of epsilon p disappeared under LN conditions in both A. fundyense and S. trochoidea. The generally higher epsilon p under LN may be associated with lower organic carbon production rates and/or higher ATP:NADPH ratios. CO2-dependent epsilon p under non-limiting conditions has been observed in several dinoflagellate species, showing potential for a new CO2-proxy. Our results however demonstrate that light- and nitrogen-limitation also affect epsilon p, thereby illustrating the need to carefully consider prevailing environmental conditions.

The larvae of congeneric gastropods showed differential responses to the combined effects of ocean acidification, temperature and salinity

The tolerance and physiological responses of the larvae of two congeneric gastropods, the intertidal Nassarius festivus and subtidal Nassarius conoidalis, to the combined effects of ocean acidification (PCO2 at 380, 950, 1250 ppm), temperature (15, 30 degrees C) and salinity (10, 30 psu) were compared. Results of three-way ANOVA on cumulative mortality after 72-h exposure showed significant interactive effects in which mortality increased with pCO(2) and temperature, but reduced at higher salinity for both species, with higher mortality being obtained for N. conoidalis. Similarly, respiration rate of the larvae increased with temperature and pCO(2) level for both species, with a larger percentage increase for N. conoidalis. Larval swimming speed increased with temperature and salinity for both species whereas higher pCO(2) reduced swimming speed in N. conoidalis but not N. festivus. The present findings indicated that subtidal congeneric species are more sensitive than their intertidal counterparts to the combined effects of these stressors. (c) 2014 Elsevier Ltd. All rights reserved.

CT Image Electron Density Quantification in Regions with Metal Implants: Implications for Radiotherapy Treatment Planning

In radiotherapy planning, computed tomography (CT) images are used to quantify the electron density of tissues and provide spatial anatomical information. Treatment planning systems use these data to calculate the expected spatial distribution of absorbed dose in a patient. CT imaging is complicated by the presence of metal implants which cause increased image noise, produce artifacts throughout the image and can exceed the available range of CT number values within the implant, perturbing electron density estimates in the image. Furthermore, current dose calculation algorithms do not accurately model radiation transport at metal-tissue interfaces. Combined, these issues adversely affect the accuracy of dose calculations in the vicinity of metal implants. As the number of patients with orthopedic and dental implants grows, so does the need to deliver safe and effective radiotherapy treatments in the presence of implants. The Medical Physics group at the Cancer Centre of Southeastern Ontario and Queen's University has developed a Cobalt-60 CT system that is relatively insensitive to metal artifacts due to the high energy, nearly monoenergetic Cobalt-60 photon beam. Kilovoltage CT (kVCT) images, including images corrected using a commercial metal artifact reduction tool, were compared to Cobalt-60 CT images throughout the treatment planning process, from initial imaging through to dose calculation. An effective metal artifact reduction algorithm was also implemented for the Cobalt-60 CT system. Electron density maps derived from the same kVCT and Cobalt-60 CT images indicated the impact of image artifacts on estimates of photon attenuation for treatment planning applications. Measurements showed that truncation of CT number data in kVCT images produced significant mischaracterization of the electron density of metals. Dose measurements downstream of metal inserts in a water phantom were compared to dose data calculated using CT images from kVCT and Cobalt-60 systems with and without artifact correction. The superior accuracy of electron density data derived from Cobalt-60 images compared to kVCT images produced calculated dose with far better agreement with measured results. These results indicated that dose calculation errors from metal image artifacts are primarily due to misrepresentation of electron density within metals rather than artifacts surrounding the implants.

ARB: Phase II window of opportunity study of short-term preoperative treatment with enzalutamide in ER-positive and triple-negative breast cancer

The androgen receptor (AR) is expressed in 60-80% of breast cancers (BC) across all molecular phenotypes, with a higher incidence in oestrogen receptor positive (ER+) BC compared to ER negative tumours. In ER+ disease, AR-expression has been linked to endocrine resistance which might be reversed with combined treatment targeting ER and AR. In triple negative BCs (TNBC), preclinical and clinical investigations have described a subset of patients that express the AR and are sensitive to androgen blockade, providing a novel therapeutic target. Enzalutamide, a potent 2nd generation anti-androgen, has demonstrated substantial preclinical and clinical anti-tumour activity in AR+ breast cancer. Short-term preoperative window of opportunity studies are a validated strategy for novel treatments to provide proof-of-concept and define the most appropriate patient population by directly assessing treatment effects in tumour tissue before and after treatment. The ARB study aims to assess the anti-tumour effects of enzalutamide in early ER+ breast cancer and TNBC, to identify the optimal target population for further studies and to directly explore the biologic effects of enzalutamide on BC and stromal cells. Methods: ARB is an international, investigator sponsored WOO phase II study in women with newly diagnosed primary ER+ BC or AR+ TNBC of ≥ 1cm. The study has two cohorts. In the ER+ cohort, postmenopausal patients will be randomised 2:1 to receive either enzalutamide (160mg OD) plus exemestane (50mg OD) or exemestane (25mg OD). In the TNBC cohort, AR+ will receive single agent treatment with enzalutamide (160mg OD). Study treatment is planned for 15–29 days, followed by surgery or neo-adjuvant therapy. Tissue and blood samples are collected before treatment and on the last day of study treatment. The primary endpoint is inhibition of tumour-cell proliferation, as measured by change in Ki67 expression, determined centrally by 2 investigators. Secondary endpoints include induction of apoptosis (Caspase3), circulating hormone levels and safety. ARB aims to recruit ≈235 patients from ≈40 sites in the UK, Germany, Spain and USA. The study is open to recruitment.

The role of anaerobic bacteria in the cystic fibrosis airway

PURPOSE OF REVIEW: Anaerobic bacteria are not only normal commensals, but are also considered opportunistic pathogens and have been identified as persistent members of the lower airway community in people with cystic fibrosis of all ages and stages of disease. Currently, the role of anaerobic bacteria in cystic fibrosis lower airway disease is not well understood. Therefore, this review describes the recent studies relating to the potential pathophysiological role(s) of anaerobes within the cystic fibrosis lungs.

RECENT FINDINGS: The most frequently identified anaerobic bacteria in the lower airways are common to both cystic fibrosis and healthy lungs. Studies have shown that in cystic fibrosis, the relative abundance of anaerobes fluctuates in the lower airways with reduced lung function and increased inflammation associated with a decreased anaerobic load. However, anaerobes found within the lower airways also produce virulence factors, may cause a host inflammatory response and interact synergistically with recognized pathogens.

SUMMARY: Anaerobic bacteria are potentially members of the airway microbiota in health but could also contribute to the pathogenesis of lower airway disease in cystic fibrosis via both direct and indirect mechanisms. A personalized treatment strategy that maintains a normal microbial community may be possible in the future.

Targeting treatment resistant breast cancer stem cells with FKBPL and its peptide derivative, AD-01

FKBPL and its peptide derivative, AD-01, have already demonstrated well-established inhibitory effects on breast cancer growth and CD44 dependent anti-angiogenic activity1, 2, 3. Since breast cancer stem cells (BCSCs) are CD44 positive, we wanted to explore if AD-01 could specifically target BCSCs. FKBPL stable overexpression or AD-01 treatment were highly effective at reducing the BCSC population measured by inhibiting mammosphere forming efficiency (MFE) in cell lines and primary breast cancer samples from both solid breast tumours and pleural effusions. Flow cytometry, to assess the ESA+/CD44+/CD24- subpopulation, validated these results. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed across three generations of mammospheres, where mammospheres were completely eradicated by the third generation (p<0.001). Clonogenic assays suggested that AD-01 mediated BCSC differentiation, with a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones. In support of this, the stem cell markers, Nanog and Oct4 were significantly reduced following AD-01 treatment, whilst transfection of FKBPL-targeted siRNAs led to an increase in these markers and in mammosphere forming potential, highlighting the endogenous role of FKBPL in stem cell signalling. The clinical relevance of this was confirmed using a publically available microarray data set (GSE7390), where, high FKBPL and low Nanog expression were independently associated with improved overall survival in breast cancer patients (log rank test p=0.03; hazard ratio=3.01). When AD-01 was combined with other agents, we observed synergistic activity with the Notch inhibitor, DAPT and AD-01 was also able to abrogate a chemo- and radiotherapy induced enrichment in BCSCs. Importantly, using ‘gold standard’ in vivo limiting dilution assays we demonstrated a delay in tumour initiation and reoccurrence in AD-01 treated xenografts. In summary, AD-01 appears to have dual anti-angiogenic and anti-BCSC activity which will be advantageous as this agent enters clinical trial.

Targeting treatment resistant cancer stem cells with FKBPL and its peptide therapeutics

FKBPL and its peptide derivatives have already demonstrated well-established inhibitory effects on cancer growth and CD44-dependent anti-angiogenic activity. Since cancer stem cells (CSCs) are CD44 positive, we wanted to explore if these therapeutics could specifically target CSCs in breast and ovarian cancer. In a tumoursphere assay, FKBPL stable overexpression or FKBPL-based peptide (AD-01, preclinical peptide or ALM201, clinical peptide candidate) treatment were highly effective at reducing the CSC population measured by inhibiting tumoursphere forming efficiency in breast and ovarian cancer cell lines and primary breast cancer samples from both solid breast tumours and pleural effusions. Flow cytometry, to assess the ESA+/CD44+/CD24- and ALDH+ cell subpopulations representative of CSCs, validated these results. The ability of AD-01 and ALM201 to inhibit the self-renewal capacity of CSCs was confirmed across three generations, eradicating CSC completely by the third generation (p<0.001). Furthermore, clonogenic assay demonstrated that FKBPL-based peptides mediated CSC differentiation, with a significant decrease in the number of CSCs or holoclones and an associated increase in differentiated cancer cells or meroclones/paraclones. In addition, AD-01 treatment in vitro and in vivo led to a significant reduction in the stem cell markers, Nanog, Sox2 and Oct4 protein and mRNA levels; whilst transfection of FKBPL-targeted siRNAs led to an increase in these markers and in tumoursphere forming potential, highlighting the endogenous role of FKBPL in stem cell signalling. The clinical relevance of this was confirmed using a publically available microarray data set (GSE7390), where, high FKBPL and low Nanog expression were independently associated with improved overall survival in breast cancer patients (log rank test p=0.03; hazard ratio=3.01). Additionally, when AD-01 was combined with other agents, we observed additive activity with the Notch inhibitor, DAPT and AD-01 was also able to abrogate a chemo- and radiotherapy induced enrichment in CSCs. Importantly, using gold standard in vivo limiting dilution assays we demonstrated a delay in tumour initiation and reoccurrence in AD-01 treated xenografts. In summary, FKBPL-based peptides appear to have dual anti-angiogenic and anti-CSC activity which will be advantageous as this agent enters clinical trial.