Bacterial Community Legacy Effects Following the Agia Zoni II Oil-Spill, Greece

In September 2017 the Agia Zoni II sank in the Saronic Gulf, Greece, releasing approximately 500 tonnes of heavy fuel oil, contaminating the Salamina and Athens coastlines. Effects of the spill, and remediation efforts, on sediment microbial communities were quantified over the following 7 months. Five days post-spill, the concentration of measured hydrocarbons within surface sediments of contaminated beaches was 1,093–3,773 μg g–1 dry sediment (91% alkanes and 9% polycyclic aromatic hydrocarbons), but measured hydrocarbons decreased rapidly after extensive clean-up operations. Bacterial genera known to contain oil-degrading species increased in abundance, including Alcanivorax, Cycloclasticus, Oleibacter, Oleiphilus, and Thalassolituus, and the species Marinobacter hydrocarbonoclasticus from approximately 0.02 to >32% (collectively) of the total bacterial community. Abundance of genera with known hydrocarbon-degraders then decreased 1 month after clean-up. However, a legacy effect was observed within the bacterial community, whereby Alcanivorax and Cycloclasticus persisted for several months after the oil spill in formerly contaminated sites. This study is the first to evaluate the effect of the Agia Zoni II oil-spill on microbial communities in an oligotrophic sea, where in situ oil-spill studies are rare. The results aid the advancement of post-spill monitoring models, which can predict the capability of environments to naturally attenuate oil

Human α2β1HI CD133+VE epithelial prostate stem cells express low levels of active androgen receptor

Stem cells are thought to be the cell of origin in malignant transformation in many tissues, but their role in human prostate carcinogenesis continues to be debated. One of the conflicts with this model is that cancer stem cells have been described to lack androgen receptor (AR) expression, which is of established importance in prostate cancer initiation and progression. We re-examined the expression patterns of AR within adult prostate epithelial differentiation using an optimised sensitive and specific approach examining transcript, protein and AR regulated gene expression. Highly enriched populations were isolated consisting of stem (α(2)β(1)(HI) CD133(+VE)), transiently amplifying (α(2)β(1)(HI) CD133(-VE)) and terminally differentiated (α(2)β(1)(LOW) CD133(-VE)) cells. AR transcript and protein expression was confirmed in α(2)β(1)(HI) CD133(+VE) and CD133(-VE) progenitor cells. Flow cytometry confirmed that median (±SD) fraction of cells expressing AR were 77% (±6%) in α(2)β(1)(HI) CD133(+VE) stem cells and 68% (±12%) in α(2)β(1)(HI) CD133(-VE) transiently amplifying cells. However, 3-fold lower levels of total AR protein expression (peak and median immunofluorescence) were present in α(2)β(1)(HI) CD133(+VE) stem cells compared with differentiated cells. This finding was confirmed with dual immunostaining of prostate sections for AR and CD133, which again demonstrated low levels of AR within basal CD133(+VE) cells. Activity of the AR was confirmed in prostate progenitor cells by the expression of low levels of the AR regulated genes PSA, KLK2 and TMPRSS2. The confirmation of AR expression in prostate progenitor cells allows integration of the cancer stem cell theory with the established models of prostate cancer initiation based on a functional AR. Further study of specific AR functions in prostate stem and differentiated cells may highlight novel mechanisms of prostate homeostasis and insights into tumourigenesis

ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3Gal1) synthesis of Siglec ligands mediates anti-tumour immunity in prostate cancer

Immune checkpoint blockade has yet to produce robust anti-cancer responses for prostate cancer. Sialyltransferases have been shown across several solid tumours, including breast, melanoma, colorectal and prostate to promote immune suppression by synthesising sialoglycans, which act as ligands for Siglec receptors. We report that ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3Gal1) levels negatively correlate with androgen signalling in prostate tumours. We demonstrate that ST3Gal1 plays an important role in modulating tumour immune evasion through the synthesises of sialoglycans with the capacity to engage the Siglec-7 and Siglec-9 immunoreceptors preventing immune clearance of cancer cells. Here, we provide evidence of the expression of Siglec-7/9 ligands and their respective immunoreceptors in prostate tumours. These interactions can be modulated by enzalutamide and may maintain immune suppression in enzalutamide treated tumours. We conclude that the activity of ST3Gal1 is critical to prostate cancer anti-tumour immunity and provide rationale for the use of glyco-immune checkpoint targeting therapies in advanced prostate cancer

26th Annual Computational Neuroscience Meeting (CNS*2017): Part 3 - Meeting Abstracts - Antwerp, Belgium. 15–20 July 2017

This work was produced as part of the activities of FAPESP Research,\ud Disseminations and Innovation Center for Neuromathematics (grant\ud 2013/07699-0, S. Paulo Research Foundation). NLK is supported by a\ud FAPESP postdoctoral fellowship (grant 2016/03855-5). ACR is partially\ud supported by a CNPq fellowship (grant 306251/2014-0)

Identification of CNGB1 as a Predictor of Response to Neoadjuvant Chemotherapy in Muscle-Invasive Bladder Cancer

Cisplatin-based neoadjuvant chemotherapy (NAC) is recommended prior to radical cystectomy for muscle-invasive bladder cancer (MIBC) patients. Despite a 5–10% survival benefit, some patients do not respond and experience substantial toxicity and delay in surgery. To date, there are no clinically approved biomarkers predictive of response to NAC and their identification is urgently required for more precise delivery of care. To address this issue, a multi-methods analysis approach of machine learning and differential gene expression analysis was undertaken on a cohort of 30 MIBC cases highly selected for an exquisitely strong response to NAC or marked resistance and/or progression (discovery cohort). RGIFE (ranked guided iterative feature elimination) machine learning algorithm, previously demonstrated to have the ability to select biomarkers with high predictive power, identified a 9-gene signature (CNGB1, GGH, HIST1H4F, IDO1, KIF5A, MRPL4, NCDN, PRRT3, SLC35B3) able to select responders from non-responders with 100% predictive accuracy. This novel signature correlated with overall survival in meta-analysis performed using published NAC treated-MIBC microarray data (validation cohort 1, n = 26, Log rank test, p = 0.02). Corroboration with differential gene expression analysis revealed cyclic nucleotide-gated channel, CNGB1, as the top ranked upregulated gene in non-responders to NAC. A higher CNGB1 immunostaining score was seen in non-responders in tissue microarray analysis of the discovery cohort (n = 30, p = 0.02). Kaplan-Meier analysis of a further cohort of MIBC patients (validation cohort 2, n = 99) demonstrated that a high level of CNGB1 expression associated with shorter cancer specific survival (p < 0.001). Finally, in vitro studies showed siRNA-mediated CNGB1 knockdown enhanced cisplatin sensitivity of MIBC cell lines, J82 and 253JB-V. Overall, these data reveal a novel signature gene set and CNGB1 as a simpler proxy as a promising biomarker to predict chemoresponsiveness of MIBC patients

Side Population in Human Non-Muscle Invasive Bladder Cancer Enriches for Cancer Stem Cells That Are Maintained by MAPK Signalling

<div><p>Side population (SP) and ABC transporter expression enrich for stem cells in numerous tissues. We explored if this phenotype characterised human bladder cancer stem cells (CSCs) and attempted to identify regulatory mechanisms. Focusing on non-muscle invasive bladder cancer (NMIBC), multiple human cell lines were used to characterise SP and ABC transporter expression. <em>In vitro</em> and <em>in vivo</em> phenotypic and functional assessments of CSC behaviour were undertaken. Expression of putative CSC marker ABCG2 was assessed in clinical NMIBC samples (n = 148), and a role for MAPK signalling, a central mechanism of bladder tumourigenesis, was investigated. Results showed that the ABCG2 transporter was predominantly expressed and was up-regulated in the SP fraction by 3-fold (ABCG2^hi) relative to the non-SP (NSP) fraction (ABCG2^low). ABCG2^hi SP cells displayed enrichment of stem cell markers (Nanog, Notch1 and SOX2) and a three-fold increase in colony forming efficiency (CFE) in comparison to ABCG2^low NSP cells. <em>In vivo</em>, ABCG2^hi SP cells enriched for tumour growth compared with ABCG2^low NSP cells, consistent with CSCs. pERK was constitutively active in ABCG2^hi SP cells and MEK inhibition also inhibited the ABCG2^hi SP phenotype and significantly suppressed CFE. Furthermore, on examining clinical NMIBC samples, ABCG2 expression correlated with increased recurrence and decreased progression free survival. Additionally, pERK expression also correlated with decreased progression free survival, whilst a positive correlation was further demonstrated between ABCG2 and pERK expression. In conclusion, we confirm ABCG2^hi SP enriches for CSCs in human NMIBC and MAPK/ERK pathway is a suitable therapeutic target.</p> </div

AR expression and activity within the prostate epithelial hierarchy of differentiation.

<p>A) Representative images of expression of CD133 and AR counterstained with DRAQ5™ within prostate EpCAM^+VE α₂β₁^HI CD133^+VE (Left panel), α₂β₁^HI CD133^–VE (central panel) and α₂β₁^LOW CD133^–VE cells (right panel). <b>B)</b> Expression of the AR regulated genes PSA, KLK2 and TMPRSS2 normalised to GAPDH (n = 10) (p<0.001 comparing α₂β₁^HI and α₂β₁^LOW). Error bars represent standard error of the mean.</p

Strategy of enrichment for required cell types.

<p><b>A)</b> Schematic work flow for enrichment of epithelial cells for assessment of AR expression. <b>B)</b> Purity of selection by expression of the lineage specific markers CD24 (epithelial), CD146 (endothelial) and CD45 (haematopoietic) normalised to GAPDH following real-time PCR for unsorted prostate epithelia and EpCAM/HEA sorted epithelia, error bars represent standard error of the mean for n = 3. <b>C)</b> CD133/1 Sorted samples were assessed for purity by co-expression of the CD133/2 epitope, confirming that these two epitopes are co-expressed in the prostate and that our CD133 selection efficiently enriches for CD133^+VE cells: Upper dotplot representative of CD133/2 staining for unsorted α₂β₁^HI epithelial cells; lower left dotplot representative of CD133/2 staining for α₂β₁^HI CD133/1^–VE cells; lower right dotplot representative of CD133/2 staining for α₂β₁^HI CD133/1^+VE cells. Gates are set according to appropriate isotype controls. <b>D)</b> Confirmation of CD133 enrichment with real-time PCR. CD133 expression is shown normalised to GAPDH, error bars represent standard error of the mean n = 10.</p

Expression of the AR within the prostate epithelial hierarchy of differentiation.

<p>Error bars represent standard error of the mean. <b>A)</b> Expression of AR transcript relative to GAPDH. Error bars represent standard error of the mean for n = 10. <b>B)</b> Upper dotplot representative of CD133 staining for progenitor α₂β₁^HI cells. Lower left dotplot representative of AR expression in CD133^–VE gated α₂β₁^HI cells. Lower right dotplot representative of AR expression in CD133^+VE gated α₂β₁^HI cells. Gates were set according to appropriate isotype controls. <b>C)</b> Mean percentage of cells expressing the AR in CD133^+VE and CD133^–VE α₂β₁^HI cells (n = 6). <b>D)</b> Representative histograms for fluorescence of α₂β₁^HI and α₂β₁^LOW isotype controls and AR detection. <b>E)</b> Mean fold change in median staining relative to isotype control for AR stained α₂β₁^HI cells and α₂β₁^LOW cells (n = 6).</p

Validation of AR detection with flow cytometry.

<p><b>A)</b> Percentage of cells staining above a no antibody control for either isotype antibody (hollow points) or PG-21 AR antibody (solid points) in LNCaP (circles) or PC3 (triangles) across a dilution series. <b>B)</b> Representative staining patterns for PC3 (upper dotplots) and LNCaP (lower dotplots) for either 1∶200 isotype antibody (left dotplots) or 1∶200 PG-21 AR antibody (right dotplots) of equivalent concentrations. Gates set according to isotype control. <b>C)</b> Left dotplot representative of staining of LNCaP with isotype control, right dotplots representative of AR staining in non-transfected LNCaP (upper), LNCaP transfected with scrambled siRNA (middle dotplot) and LNCaP transfected with AR siRNA (lower). Gates were set according to an appropriate isotype control. <b>D)</b> Percentage of cells staining positive for AR relative to an isotype control in non-transfected LNCaP, LNCaP transfected with scrambled siRNA and LNCaP transfected with AR siRNA. Error bars represent standard error of the mean for n = 3. <b>E)</b> Western blots of AR expression for non-transfected LNCaP, LNCaP transfected with scrambled siRNA and LNCaP transfected with AR siRNA are shown using a different AR antibody (G122-434, BD Pharmingen).</p