In vivo experiments and hydrogen-deuterium exchange (HDX) with tandem mass spectrometry (MS) studies showed that NT-UCH-L1 is readily aggregated and degraded, and has more flexible structure than UCH-L1. Post-translational modifications including monoubiquitination and disulfide crosslinking regulate the stability and cellular localization of NT-UCH-L1, as confirmed by mutational and proteomic studies. Stable expression of NT-UCH-L1 decreases cellular ROS levels and protects cells from H2O2, rotenone and CCCP-induced cell death. NT-UCH-L1-expressing transgenic mice are less susceptible to degeneration of nigrostriatal dopaminergic neurons seen in the MPTP mouse model of PD, in comparison to control animals. These results suggest that NT-UCH-L1 may have the potential to prevent neural damage in diseases like PD.Ubiquitin C-terminal hydrolase-L1 (UCH-L1) has been proposed as one of the Parkinson's disease (PD) related genes, but the possible molecular connection between UCH-L1 and PD is not well understood. In this study, we discovered an N-terminal 11 amino acid truncated variant UCH-L1 that we called NT-UCH-L1, in mouse brain tissue as well as in NCI-H157 lung cancer and SH-SY5Y neuroblastoma cell lines.  Ubiquitin C-terminal hydrolase-L1 (UCH-L1) catalyzes the hydrolysis of C-terminal ubiquitin esters and amides. UCH-L1 is highly expressed in metastatic lung cancer Mutations, environmental stresses, and aging cause protein denaturation, rendering them aggregation-prone forms Post-translational modifications of proteins and alternative splicing can change biochemical properties including solubility of a protein. Phosphorylation, ubiquitination, and truncation affect aggregation behavior of α-synuclein The discovery that 1-methyl-4-phenyl-1,2,3,6-tetrahydrodropyridine (MPTP) infusion causes Parkinsonism by selective inhibition of mitochondrial complex-1, raised the possibility that mitochondrial dysfunction is at the heart of PD. Mitochondrial dysfunction has been commonly observed in autopsied PD brain tissues Oxidative stress has been shown to be the cause of nigrostriatal dopaminergic neuron loss in PD patients and in the MPTP mice model of PD in vitro and in vivo.In this study we identified a variant of UCH-L1 lacking N-terminal 11 amino acids designated as NT-UCH-L1, and compared it to UCH-L1 by physical, chemical, and proteomic approaches with the goal of understanding their possible relation to PD. We found that NT-UCH-L1 is aggregation prone and is localized in mitochondria, which are regulated by monoubiquitination. Furthermore, NT-UCH-L1 was found to have a protective role in the PD model Human NCI-H157, SH-SY5Y, and HeLa cells were maintained respectively in RPMI 1640, dulbecco's modified eagle medium and minimum essential medium supplemented with 10% fetal bovine serum, 100 µM/mL of streptomycin and 100 units/mL of penicillin G .The sources of antibodies used in this study were as follows: Monoclonal anti-myc antibody from Millipore, polyclonal anti-UCH-L1 and anti-ubiquitin antibody from Chemicon, anti-flag antibody (M2) from Sigma, anti-tubulin antibody from Santa Cruz Biotechnology, Alexa Fluor 488 goat anti-mouse antibody from Molecular Probes. Polyclonal antibodies against N-terminal peptide of UCH-L1 were generated in rabbits and characterized by Abclon (Korea).This was performed as described previously The lentiviral vector for expressing wild type UCH-L1 and NT-UCH-L1 was constructed by inserting PCR gene fragment into the XbaI–EcoRV site of LentiM1.4 vector. Pseudotyped lentiviruses were produced and used for stable expression of UCH-L1s as described earlier Ubiquitin C-terminal hydrolase activity was measured using the Ub-AMC (AG scientific) as substrate 2O and maintained at 25°C for several time scales. The labeling reaction was quenched by 5 mM tris(2-carboxyethyl)phosphine (TCEP), pH 2.3. For peptic digestion, porcine pepsin (1 µg/µL) was added to each quenched protein sample and incubated at 0°C for 3 min before injection m/z 300–1500 with scan time of 1 s.UCH-L1 and NT-UCH-L1 (about 1 µg/µL) were diluted 10-fold with DThe gel spots of proteins were destained and digested with trypsin and the resulting peptides were analyzed by nanoAcquity UPLC/ESI/MS  as described previously The individual MS/MS spectra acquired from each of the precursors within a single LC run were combined, smoothed, deisotoped and centroided using the Micromass ProteinLynx Global Server (PLGSTM) 2.1 data processing software and output, as a single MASCOT-searchable peak list (.pkl) file. The peak list files were used to query the SwissProt database using the MASCOT , with the following parameters: peptide mass tolerance, 0.2 Da; MS/MS ion mass tolerance, 0.2 Da; allowing up to 1 missed trypsin cleavage site, considering variable modifications, such as acetylation, deamidation, pyro-glu , oxidation, formylation, phosphorylation, carbamidomethyl and cysteine propionamide but not fixed modifications; enzyme limited to trypsin; and toxonomy limited to mouse. All reported assignments were verified by automatic and manual interpretations of spectra from MASCOT.Soluble and insoluble fractions of cell lysates were prepared as previously described 3VO4, 0.5% NP-40). Lysates were centrifuged at 14,000 rpm for 10 min and the supernatant was incubated for 3 h at 4°C with anti-flag or anti-myc antibody cross-linked to protein G sepharose beads. Beads were washed 3 times with immunoprecipitation buffer, bound proteins were eluted by gel sample buffer and separated on SDS-PAGE.Cells or mouse brain tissues were lysed with immunoprecipitation buffer . Cells were then incubated with 250 nM of MitoTracker Red 580 in HBSS at 37°C for 30 min and fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilization was done with 0.1% Triton X-100 in HBSS for 15 min at room temperature. The cells were treated with blocking solution  for 1 h at room temperature and incubated with monoclonal anti-myc antibody, diluted 1∶250 in HBSS containing 1% BSA and 1% sucrose for 1 h at 37°C. After three washes with PBS, the cells were incubated with Alexa Fluor 488 conjugated goat anti mouse IgG diluted 1∶50 for 1 h at 37°C. After three washes, the cells were analyzed under Zeiss LSM 510 META confocal microscope.Mitochondrial fractions of the cells were prepared as described previously 2DCFDA (Invitrogen) following manufacturer's instructions. Briefly, trypsinized cells were washed and resuspended in 0.5 mL of HBSS; 3 µL of 0.5 mM CM-H2DCFDA was added and incubated at 37°C for 15 min. After brief wash with HBSS, the cells were analyzed with FACS Calibur flow cytometer (BD Biosciences).Cellular ROS were measured using CM-H2O2 treatment, cells were treated with H2O2 for 1 h before plating in the E-plate. After 24 h, the cells were treated with rotenone or CCCP and cell growth was monitored by measuring electrical impedance every 30 min.Five thousand HeLa cells were plated in a 96 well E-plate for the real-time cell analyzer, xCELLigence (Roche Applied Science). For H5′-TGCTGAACAAAGTGCTGTCC-3′; reverse: 5′-AGCCCAGAGACTCCTCTTCC-3′) were used. The offsprings were screened with specific primers . All experiments were performed in accordance with approved animal protocols and guidelines established by Ewha Womans University [2011-01-025] and Kyung Hee University [KHUASP(SE)-10-030].We generated transgenes by cloning the hNT-UCH-L1-myc DNA under the control of CAG promoter in pCAGEN vector (Addgene). The plasmid was linearized by digestion with ScaI and BamHI and microinjected into the pronuclei of newly fertilized C57BL/6N mouse eggs (Macrogen). Germline transmission of NT-UCH-L1 was obtained in five independent NT-Tg lines and the levels of transgenic mRNA were assessed by quantitative RT-PCR. cDNAs from mouse whole brain were prepared using RNA extraction kit (Qiagen) and SuperScript III (Life Technologies) reverse transcriptase. For quantitative RT-PCR, specific primers for the transgene  maintained in a room at 20–22°C on 12 h light/dark cycle with food and water available As described previously As described previously 1MQLKPMEINPEMLNK15 (1815.9144 Da) of UCH-L1, but this was replaced by 12MLNK15 (505.2905 Da)   . The pep3, and 4 s and mou3, and 4 . The del (PARK5) . The exoNT-UCH-L1 did not exhibit the ubiquitin hydrolase activity of UCH-L1 1C. Also,82MKQTIGNSCGTIGL95) in NT-UCH-L1 exchanged less deuterium than in UCH-L1. These results indicate that truncation of N-terminal 11 amino acids induces remarkable structural changes.In order to understand the structural basis of these differences, we employed mass spectrometry with hydrogen/deuterium exchange (HDX) in which residues exposed to surface more readily exchange deuterium allowing their mass increases to be detected. HDX occurred slowly in UCH-L1, taking up to 16 h to complete, while in NT-UCH-L1, this exchange was completed in just about 2 min . This inUCH-L1 recruited free ubiquitin resulting in high cellular free ubiquitin levels We identified the sites of monoubiquitinated lysine residue(s) in NT-UCH-L1, employing both peptide sequencing with MS/MS and mutation studies. We analyzed peptides of immunoprecipitated and trypsinized NT-UCH-L1 by MS/MS and detected Lys15 having a mass increase 114 Da which is equivalent to C-terminal Gly-Gly of attached ubiquitin. This indicates that the candidate site of ubiquitination is Lys15 . We consUCH-L1 gene The focus of PD research has been on mitochondrial functions and its respiratory activities. The presence of PD-related gene products in autopsied PD brain tissues and in mitochondria, underscores their effect on mitochondrial functions To further dissect the location of NT-UCH-L1 in mitochondria, we incubated the mitochondrial fractions with varying concentrations of proteinase K and performed Western blot analysis with antibodies specific for Tom20, cytochrome C, and COX-4, markers of the outer membrane, intermembrane space, and inner membrane of mitochondria, respectively . The prom-chlorophenylhydrazone (CCCP) induced mitochondrial damage. We did not detect any increase of NT-UCH-L1 in mitochondria after CCCP treatment  in HeLa cells expressing UCH-L1-myc, NT-UCH-L1-myc, and K15/157R NT-UCH-L1-myc, and assessing the time course of disappearance of the protein bands by immunoblotting. NT-UCH-L1-myc and K15/157R NT-UCH-L1-myc disappeared faster than UCH-L1 . NT-UCH-4Cl, and 3MA  2-ME) and non-reducing ((−) 2-ME) conditions . Some of2O2 and the proteins were separated under both reducing and non-reducing conditions . We used a CAG promoter to drive expression of NT-UCH-L1 in transgenic mice  to specifically detect DA neurons. As we reported previously In this study, we found that a variant of UCH-L1 lacking 11 N-terminal amino acids, that we designated NT-UCH-L1, exists in lung cancer and neuronal cells, and in brain tissue. NT-UCH-L1 differs from UCH-L1 in its tertiary structure, in its lack of deubiquitinating activity, lack of ability to increase cell migration, in its greater tendency to be monoubiquitinated and readily aggregated.UCH-L1 gene (PARK5). The exon 1-deleted mRNA has been reported to be expressed originally in a protein given a different name, PGP9.5 (NCBI accession number X04741) th methionine may also possible. Several examples including human insulin-degrading enzyme AtLIG1 gene in Arabidopsis thalianaProtein isoforms from one gene are produced by alternative splicing, alternative promoters, or alternative translation initiation sites. The 11 N-terminal amino acids missing in NT-UCH-L1, corresponds to exon 1 of the To compare the amounts of UCH-L1 and NT-UCH-L1 in mouse brain tissue and cell lines, we separated them in 2D-PAGE and probed with two different kinds of antibodies; one for both UCH-L1 and NT-UCH-L1 and the other only for UCH-L1. Significant amounts of NT-UCH-L1 exist in mouse brain tissue, neuronal cells, and lung cancer cells .We further compared UCH-L1 and NT-UCH-L1 with regard to their structures, stability, modifications, localizations, and biological functions. NT-UCH-L1 did not exhibit the ubiquitin hydrolase activity of UCH-L1 1C. In HDProtein monoubiquitination plays roles in DNA repair, histone regulation, gene expression, and receptor endocytosis 2O2 treatments, forming inter-molecular disulfide bonds and cells expressing NT-UCH-L1 resist H2O2 induced cell death. This is similar to the action of DJ-1 under oxidizing environment. Cysteines in DJ-1 are easily oxidized in response to ROS and play antioxidant roles NT-UCH-L1 was easily oxidized by HBased on our results, we suggest a molecular mechanism involving ubiqitination for the role of NT-UCH-L1 in protecting cells against stresses. This suggested mechanism is summarized as follows . NT-UCH-This study suggests that NT-UCH-L1 has the potential for preventing neurotoxicity in PD. However, approaches to increasing NT-UCH-L1 levels in neurons to protect against mitochondrial or other damage might be hampered by thresholds for NT-UCH-L1 solubility, because NT-UCH-L1 forms insoluble protein aggregates once the concentration threshold is exceeded. This suggests that a fine control of the synthesis and degradation of NT-UCH-L1, is necessary for the maintenance of optimum levels of NT-UCH-L1. Currently, we do not know which factor(s) regulates the expression of NT-UCH-L1 and which E3 ubiquitin ligase promotes its monoubiquitination and degradation. Further studies are needed to shed light on qualitative and quantitative aspects of NT-UCH-L1's function in PD.Figure S1N-terminal 11 amino acid truncated UCH-L1, NT-UCH-L1, doesn't have ubiquitin hydrolase enzyme activity. (A) Isoforms and mutants of human UCH-L1 registered in NCBI. (B) UCH-L1 spots on the corresponding silver stained 2D gel were subjected to tryptic digestion and analyzed with MALDI-TOF MS. 505.2905 Da peak of 12MLNK15 peptide of NT-UCH-L1, instead of a peptide corresponding to the first 15 amino acids peak (1815.9144 Da) of UCH-L1, was detected and schematically represented. (c) Flag-UCH-L1 and Flag-NT-UCH-L1 transiently expressd in NCI-H157 cells were immunoprecipitated using anti-Flag antibody. Ubiquitn C-terminal hydrolase activities of the immunoprecipitates were measured using Ub-AMC as a substrate. pFlag-CMV-2 empty vector transfected cells (closed circle) were compared to Flag-UCH-L1 or Flag-NT-UCH-L1 expressing cells (open circle). Fluorescence of released free AMC was monitored at 460 nm. (D) HeLa cells transiently expressing Flag-NT-UCH-L1 or Flag-UCH-L1 cells were subjected to migration assay using transwell coated with Matrigel™. After 24 h, the number of migrated cells in the lower chamber was counted. The expression of Flag-NT-UCH-L1 and Flag-UCH-L1 in HeLa cells were shown by Western blot analysis of same number of cells using anti-Flag antibody. The mean ± s.d. of three independent experiments is shown. *p<0.05.(TIF)Click here for additional data file.Figure S21MQLKPMEINPE11, b) antibodies (lower panels).NCI-H157 cells were analyzed using 2D-PAGE and visualized by Western blot analysis using anti-UCH-L1 (a), anti-N-terminal peptide Click here for additional data file.Figure S3(A) HeLa cells transiently expressing UCH-L1-myc, K15/157R UCH-L1-myc, and NT-UCH-L1-myc were stained with anti-myc and Alexa Fluor 488 secondary antibody (green). Mitochondria and nucleus were stained with Mitotracker (red) and DAPI (blue), respectively. Cells were visualized by confocal microscopy. Lower pannel is enlarged figure of the dotted rectangle region of each upper pannel. (B) HeLa cells transiently expressing UCH-L1-myc, K15/157R UCH-L1-myc, and NT-UCH-L1-myc were treated with 10 µg/mL cycloheximide for the indicated times and immunoblotted using anti-myc antibody. (c) SN4741 cells were transiently transfected with pcDNA3.1 UCH-L1-myc or NT-UCH-L1-myc expressing plasmid. Cells were divided into soluble and insoluble fractions and analyzed in non-reducing ((-) 2-ME) and reducing ((+) 2-ME) gels. Proteins were immunoblotted using anti-myc antibody.(TIF)Click here for additional data file.Figure S466QIEELKGQEVSPK78.  Mice in each group , NT-Tg ) were sacrificed 7 d after the last MPTP injection  or PBS as controls . Brain tissues were processed for Nissl staining (blue) (B) and Nissl (blue) and TH (brown) double staining (C). Dotted lines indicate substantia nigra pars compacta. Insets, pictures with higher magnifications. Scale bars, 100 µm.(A) Immunoprecipitation analysis of hNT-UCH-L1-myc in control and NT-Tg mouse brain using anti-myc antibody. Immunoprecipitants were analyzed by silver staining. The band only detected in NT-Tg sample (arrow head) was cut and identified by peptide finger printing and MS spectrometry. We observed one peptide peak with M.W. 742.9290 which matches with the UCH-L1 sequence, (TIF)Click here for additional data file.


Recent proxy measurements reveal that subglacial lakes beneath modern ice sheets periodically store and release large volumes of water, providing an important but poorly understood influence on contemporary ice dynamics and mass balance. This is because direct observations of how lake drainage initiates and proceeds are lacking. Here we present physical evidence of the mechanism and geometry of lake drainage from the discovery of relict subglacial lakes formed during the last glaciation in Canada. These palaeo-subglacial lakes comprised shallow (<10 m) lenses of water perched behind ridges orientated transverse to ice flow. We show that lakes periodically drained through channels incised into bed substrate . Canals sometimes trend into eskers that represent the depositional imprint of the last high-magnitude lake outburst. The subglacial lakes and channels are preserved on top of glacial lineations, indicating long-term re-organization of the subglacial drainage system and coupling to ice flow. Our understanding of subglacial lake drainage events is limited by a lack of direct observations. Here, the authors use ground penetrating radar to identify a relict subglacial lake and infer drainage mechanisms based on geomorphological features. There are hundreds of reported and thousands of predicted subglacial lakes beneath the Antarctic and Greenland ice sheets135689512Palaeo-subglacial lakes are widely predicted beneath the former Laurentide Ice Sheet111617192 study area where channels emanate from flat spots of which 11 connect to another flat spot downstream. In eight localities, meltwater channels show a downstream transition into 10–40 m wide by 1–10 m high eskers , reveal linear clusters of <5 km diameter flat spots connected by subglacial meltwater channels and eskers , 2. ThesWe acquired ground-penetrating radar (GPR) across two flat spots and a sediment core (see the Methods for details) at the edge of the flat spot at Site 2. The coring site was chosen where we could penetrate through the lacustrine sediments into the underlying glacial lineation, based on GPR investigation of thick sediment infills. The results revealed that the flat spots are composed of 5- to 10-m-thick glaciolacustrine sediments overlying glacial lineations . The 3-mAt Site 1, GPR data were collected across a round-crested esker deposited downstream of a meltwater channel . The eskIn summary, mapping of glacial landforms from high-resolution digital elevation models (DEMs) revealed the presence of flat spots associated with channels and eskers , 2, 3. IGiven the geometry of the flat spots (hundreds of metres to few kilometres in diameter), the thickness of the sediment fill (<∼10 m) and the subdued relief of the glacially lineated topography, the lakes must have existed as shallow (few m deep), sediment-floored water lenses. Modelling of the hydraulic potential surfaceN (ice overburden pressure—water pressure). An increase in N will cause ice viscosity to decrease and sediment stiffness to increase such that canals will tend to form when N is high, whereas R-channels are favoured when N is low. Critically, the flood theory predicts a decrease in N over a fairly short distance adjacent to the lake and the length of this region is about a kilometre, which is roughly consistent with the observed length scale of the meltwater channels (<2 km). Thus, the observed transition from canals to R-channels is consistent with theory and may occur if the ratio of sediment stiffness to ice viscosity passes through one.The drainage of subglacial lakes is typically assumed to occur through Röthlisberger-channels incised in ice (R-channels)5143) was derived from the upstream canal , then at least four similar-sized lake drainage events are required to erode the composite canal. Thus, we interpret this as evidence for periodic jökulhlaup-style floods beneath contemporary ice sheets, capable of eroding and depositing large volumes of subglacial sediment. This is consistent with work in NE Alberta that invoke periodic high-magnitude outbursts from subglacial lakes to explain the evolution of large  tunnel valleys cut into sedimentGPR investigation of an esker downstream of a subglacial lake and meltwater channel  has allo2425The depositional environment of the middle unit (2) in the sediment core is interpreted as a low-energy ice-proximal water body dominated by fine-grained outwash and rain-out sedimentation, which is consistent with a subglacial lake origin. The organic carbon content of the lake sediments (0.4–2.8%) is within the range of sediments sampled from small subglacial lakes emerging from beneath the Antarctic Ice SheetDevelopment of palaeo-subglacial lakes and the preservation of their associated deposits above glacial lineations  and 3 deTo our knowledge, this is the first time former subglacial lakes and their drainage architecture have been directly observed beneath the North American ice sheets, and therefore provides a unique glimpse into one of the least explored and understood environments on Earth. We are able to provide new constraints on the mechanism and geometry of subglacial lake drainage that are consistent with our current theoretical understanding. We also show the potential for long-term hydro-dynamic re-organizations, demonstrated by the association of subglacial lakes with the cessation of fast ice-flow in the region. Our results provide constraints for the modelling of similar subglacial lake drainages beneath the Antarctic and Greenland ice sheets.−1 was calculated across the flat spots, in accordance with silt and sand dominated sediments, and a velocity of 0.1 m ns−1 was calculated for the eskers, which is in accordance with other GPR surveys of eskers in AlbertaA MALA Ramac system was deployed, comprising a 100-MHz rough terrain antenna array (RTA), linked by fibre optic cables to a backpack communication unit and thence an XV11 control monitor. The RTA is a 4-m-long cable with transmitting and receiving antennas in an in-line endfire orientation. This antenna is ideal for the extremely rugged environments surveyed. Lines of roughly 1 km length were located and heights above ordnance datum (OD) recorded using a Garmin E30 GLONASS and global positioning system (GPS) enabled hand-held Global Navigation Satellite System device, with 2–4 m lateral accuracy. A total of 45 km line length was gathered, of which we show 2.7 km here. The remaining data are available as images upon request to the first author. Data were gathered in continual trigger mode, with 16 stacks at a 0.5-s delay at a constant walking speed, checked by Global Navigation Satellite System positions. Data were processed using Sandmeier's proprietary software ReflexW (Queen's University Licence number 401). This comprised de-wow, trace averaging, application of gain, migration, topographic correction and depth conversion. A limitation of the RTA is these antennas are inseparable, which precludes wide-angle reflection experiments to determine subsurface velocities. Instead, the hyperbola-fitting function within ReflexW was used to check the ground-truthed sediment type and depth control afforded by our 3-m-deep core. A velocity of 0.085 m nsA surface sediment core was collected using a percussion hammer corer to a total sediment depth of 3 m. Some sediment was extruded from the core upon bringing to the surface leading to a gap in the data from 200 to 235 cm. Physical characteristics were manually described and logged. The core was subsampled every 1 cm to determine organic and carbonate content based on % weight loss on ignition after combustion at 430 °C for 24 h. Particle sizes of the <2 mm fraction (sand–silt–clay) were determined using a Horiba laser defraction system. Samples were dated from the base of the herbaceous peat unit (120–121 cm) together with four bulk samples from relatively ‘organic-rich' sections of the core . The fouLiDAR data were collected and processed by Airborne Imaging and are used under licence from Clean Harbors Exploration Services Ltd. The data were collected from a fixed wing aircraft between 23 May and 20 August 2006, using an Optech ALTM 3100. Data were classified using the programme Terrascan to generate a bare earth digital elevation model, which has an horizontal accuracy 45 cm root mean square (RMS) and a vertical accuracy 30 cm RMS. We manually digitized glacial lineations, transverse ridges (moraines), eskers and meltwater channels from 2 m LiDAR data. Flat spots were calculated using the standard deviation of the elevation in a 100-m window. By calibrating to known palaeo-subglacial lakes in The data that support the findings of this study are available from the corresponding author upon request.How to cite this article: Livingstone, S. J. et al. Discovery of relict subglacial lakes and their geometry and mechanism of drainage. Nat. Commun. 7:11767 doi: 10.1038/ncomms11767 (2016).Supplementary Figures 1-8, Supplementary Discussion and Supplementary References


TMPRSS2:ERG gene fusion is common in androgen receptor (AR) positive prostate cancers, yet its function remains poorly understood. From a screen for functionally relevant ERG interactors, we identify the arginine methyltransferase PRMT5. ERG recruits PRMT5 to AR-target genes, where PRMT5 methylates AR on arginine 761. This attenuates AR recruitment and transcription of genes expressed in differentiated prostate epithelium. The AR-inhibitory function of PRMT5 is restricted to TMPRSS2:ERG-positive prostate cancer cells. Mutation of this methylation site on AR results in a transcriptionally hyperactive AR, suggesting that the proliferative effects of ERG and PRMT5 are mediated through attenuating AR’s ability to induce genes normally involved in lineage differentiation. This provides a rationale for targeting PRMT5 in TMPRSS2:ERG positive prostate cancers. Moreover, methylation of AR at arginine 761 highlights a mechanism for how the ERG oncogene may coax AR towards inducing proliferation versus differentiation.The DOI:http://dx.doi.org/10.7554/eLife.13964.001 Prostate cancers are among the most common types of cancer in men, which, like other cancers, are driven by genetic mutations. Roughly half of all prostate cancers contain a genetic change that incorrectly fuses two genes together, causing the cells to produce abnormally high levels of a protein called ERG.ERG is a transcription factor, a protein that binds to specific sequences of DNA to influence the activity of nearby genes. ERG represses genes that help to prevent prostate cancers from growing, and so promotes prostate cancer development. Like most other transcription factors, ERG is difficult to target with drugs and no therapies that directly prevent the activity of ERG currently exist.Mounir et al. wanted to find out whether ERG cooperates with other proteins to cause prostate cancer cells to grow, with the hope that these proteins could be more easily targeted with a drug. By using various biochemical techniques in human prostate cancer cell lines, Mounir et al. found that ERG interacts with an enzyme called PRMT5. This interaction enables PRMT5 to chemically modify other proteins to change their activity. In the case of prostate cancer cells, PRMT5 inappropriately modifies the androgen receptor, a protein that regulates the growth of normal prostate cells. This abnormal modification contributes to the excessive growth of the cancer cells.Although PRMT5 will be easier to target with drugs than ERG, it also has many other roles besides those described by Mounir et al. Much more work is therefore needed to investigate whether PRMT5 could be safely targeted to treat patients with prostate cancer.DOI:http://dx.doi.org/10.7554/eLife.13964.002 TMPRSS2:ERG fusion  is highly prevalent and lethal . Drugs tG fusion . TMPRSS2 in mice . ERG is  in mice , suggest in mice . We explTMPRSS2:ERG positive PC cells, we performed a pooled short hairpin RNA (shRNA) screen in TMPRSS2:ERG and AR-positive VCaP prostate cancer cells, using ERG-negative 22Rv1 cells as a control . The shRNA pool targets 648 genes involved in transcriptional and epigenetic regulation . Identified proteins  includedTMPRSS2:ERG positive NCI-H660) and one PRMT5 hairpin likely to have minimal off-target effects. This shRNA shows a trend of sensitivity in ERG-positive lines, in agreement with our findings -inducible shRNA vectors targeting PRMT5 and a non-targeting control shRNA (NTC). PRMT5 knockdown was robust in all cell lines . Robust findings .PRMT5 is a protein arginine methyltransferase that regulates multiple signaling pathways through the mono- and symmetric di-methylation of arginines on its target proteins .To deterPSA, NKX3-1 and SLC45A3  to investigate ERG, AR, and PRMT5 recruitment to the AR targets cterized  binding cterized . We alsocterized . In VCaPcterized . As expecterized , ERG expcterized , and ERGcterized . Like ERcterized .To extend these findings to a genome wide scale, we performed AR and ERG ChIP-seq experiments in androgen (DHT or R1881) stimulated VCaP cells upon PRMT5 knockdown . ERG and AR recruitment were robust in these experiments, as judged by recovery of the canonical DNA binding sites of these proteins . In agrePSA or NKX3-1 loci  and symmetric di-methylation (SDMA) levels compared to wild-type controls  , suggest3-1 loci . We ther3-1 loci . To confenotypes . Like VCcontrols . On the sus VCaP , suggestsus VCaP . In thessus VCaP , suggestTo further understand if the observed AR methylation was directly dependent on PRMT5, we performed biochemical assays using purified PRMT5 (complexed with its requisite binding partner MEP50/WDR77) and AR LBD. PRMT5 activity, as judged by production of SAH (the by-product of SAM-dependent substrate methylation), was observed in the presence of AR LBD as substrate, but not in the presence of ERG ETS DNA binding domain or pointed (PNT) domain. PRMT5 activity in the presence of AR LBD further increased with the addition of ERG ETS domain protein to the reaction, but not with PNT domain . Unlike To identify the arginine methylation site(s) on AR, we cloned AR cDNA and mutated all arginines in the LBD to lysine . We expressed each construct in AR-negative, ERG-negative RWPE-1 prostate cells. R1881 stimulates exogenous AR to induce target gene expression in these cells. ERG expression represses this effect  and induPSA locus as a model AR-regulated gene. Relative to WT AR, the AR R761K mutant showed enhanced recruitment to PSA and increased PSA expression. Moreover, R761K mutation prevented ERG-dependent attenuation of AR recruitment to PSA and PSA expression  and tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection kit (Lonza).Redundant siRNA Activity or RSA metric was used as described  were used for further analysis  targeting transcriptional and epigenetic regulators similar to a previously reported library . shRNA lescribed . Brieflyhttp://tools.proteomecenter.org/software.php]) using a false positive threshold of <1% for protein identifications (See Nuclear extracts of VCaP cells (~600 million cells) were pre-cleared using agarose beads  and used for pulldown with either an ERG antibody (Santa Cruz#353) or an anti-rabbit IgG antibody. All antibodies were pre-coupled to beads , washed then used for pulldown. Immunoprecipitations were eluted at pH2.5 followed by TCA protein precipitation, alkylated with iodoacetamide, and separated on a NuPage 4–12% Bis-Tris gradient gel (Invitrogen). Complete gel lanes were excised using a LEAP 2DiD robot and in-gel digested with trypsin (Tecan Freedom EVO 20). Peptide sequencing for the resulting resulting 16 digest samples was performed by liquid chromatography-tandem mass spectrometry using an Eksigent 1D+ high-pressure liquid chromatography system coupled to a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific). Peptide mass and fragmentation data were searched against a combined forward-reverse IPI database (v3.55) using Mascot 2.2 (Matrix Science). Peptide and protein validation were done using Transproteomic pipeline v3.3sqall  was previously described, as were the sequences of the nontargeting control and ERG shRNA inserts and stable cell line generation. PRMT5 sPRMT5 shRNA#1:AGGGACTGGAATACGCTAATTCTCGAGAATTAGCGTATTCCAGTCCCTPRMT5 shRNA#2:AGGGACTGGAATACGTTAATTGTTAATATTCATAGCAATTAGCGTATTCCAGTCCCTPRMT5 shRNA#3:GCGGATAAAGTTGTATGTTGTGTTAATATTCATAGCACAGCATACAGCTTTATCCGCAll shRNA were expressed from a puromycin resistant vector. Lentiviral production and cell transduction was as previously described .CGCGATTGGAACACGTTGATT .The Dox-inducible ERG constructs named ERG or ERG DNAx used for stable and inducible expression in 22Rv1 cells were generated as previously described . The cDNCAGAATCAAGCTCTACGCCGT .For shPRMT5-2: the GCGGATAAAGCTGTATGCTGT target sequence was converted to Full sequence of scPRMT5 below:scPRMT5 sequence: ATGTACCCCTATGACGTGCCAGATTACGCCATGGCGGCGATGGCGGTCGGGGGTGCTGGTGGGAGCCGCGTGTCCAGCGGGAGGGACCTGAATTGCGTCCCCGAAATAGCTGACACACTAGGGGCTGTGGCCAAGCAGGGGTTTGATTTCCTCTGCATGCCTGTCTTCCATCCCAGGTTCAAGCGCGAGTTTATTCAGGAACCTGCTAAGAATCGGCCCGGTCCCCAGACACGATCAGACCTACTGCTGTCAGGACGCGATTGGAACACGTTGATTGTGGGAAAGCTTTCTCCATGGATTCGTCCAGACTCAAAAGTGGAGAAGATTCGCAGGAACTCCGAGGCGGCCATGTTACAGGAGCTGAATTTTGGTGCATATTTGGGTCTTCCAGCTTTCCTGCTGCCCCTTAATCAGGAAGATAACACCAACCTGGCCAGAGTTTTGACCAACCACATCCACACTGGCCATCACTCTTCCATGTTCTGGATGCGGGTACCCTTGGTGGCACCAGAGGACCTGAGAGATGATATAATTGAGAATGCACCAACTACACACACAGAGGAGTACAGTGGGGAGGAGAAAACGTGGATGTGGTGGCACAACTTCCGGACTTTGTGTGACTATAGTAAGAGGATTGCAGTGGCTCTTGAAATTGGGGCTGATTTGCCCTCTAATCACGTCATTGATCGCTGGCTTGGGGAGCCCATCAAAGCAGCCATTCTCCCCACTAGCATTTTCCTGACCAATAAGAAGGGATTTCCTGTTCTTTCTAAGATGCACCAGAGGCTCATCTTCCGGCTCCTCAAGTTGGAGGTGCAGTTCATCATCACAGGCACCAACCACCACTCAGAGAAGGAGTTTTGTAGCTACCTGCAGTACCTGGAATACTTAAGCCAGAACCGTCCTCCACCTAATGCCTATGAACTCTTTGCCAAGGGCTATGAAGACTATCTGCAGTCCCCGCTTCAGCCACTGATGGACAATCTGGAATCTCAGACATATGAAGTGTTTGAAAAGGACCCCATCAAATACTCTCAGTACCAGCAGGCCATCTATAAATGTCTGCTAGACCGAGTACCAGAAGAGGAGAAGGATACCAATGTCCAGGTACTGATGGTGCTGGGAGCAGGACGGGGACCCCTGGTGAACGCTTCCCTGCGGGCAGCCAAGCAGGCCGACCGCAGAATCAAGCTCTACGCCGTGGAGAAAAACCCAAATGCCGTGGTGACGCTAGAGAACTGGCAGTTTGAAGAATGGGGATCCCAGGTCACGGTAGTCAGCTCAGACATGAGGGAATGGGTGGCTCCAGAGAAAGCAGACATCATTGTCAGTGAGCTTCTGGGCTCATTTGCTGACAATGAATTGTCGCCTGAGTGCCTGGATGGAGCCCAGCACTTCCTAAAAGATGATGGTGTGAGCATCCCCGGGGAGTACACTTCCTTTCTGGCTCCCATCTCTTCCTCCAAGCTGTACAATGAGGTCCGAGCCTGTAGGGAGAAGGACCGTGACCCTGAGGCCCAGTTTGAGATGCCTTATGTGGTACGGCTGCACAACTTCCACCAGCTCTCTGCACCCCAGCCCTGTTTCACCTTCAGTCACCCTAATCGCGACCCCATGATTGACAACAACCGCTATTGCACCTTGGAATTTCCTGTGGAGGTGAACACAGTACTACATGGCTTTGCCGGCTACTTTGAGACTGTGCTTTATCAGGACATCACTCTGAGTATCCGTCCAGAGACTCACTCTCCTGGGATGTTCTCATGGTTTCCTATTCTGTTTCCCATCAAGCAGCCCATAACGGTACGTGAAGGCCAAACCATCTGTGTGCGTTTCTGGCGATGCAGCAATTCCAAGAAGGTGTGGTATGAGTGGGCTGTGACAGCACCAGTCTGTTCTGCTATTCATAACCCCACAGGCCGCTCATATACCATTGGCCTCTGA.Generation of the PRMT5 catalytic dead mutant was performed by site-directed mutagenesis  through mutation of G365 to A and R368 to A .2 incubator at 37°C. Doxycycline  was used at 100 ng/ml. Cell proliferation was measured in 6-well plates (Corning) using automated confluence readings . R1881 (Sigma) and charcoal-stripped serum (Omega Scientific) were used where indicated.VCaP, 22Rv1, LNCaP and RWPE-1 cell lines (ATCC) were grown in vendor-recommended media and maintained in a humidified 5% CO2–5 ug of AR, ERG or IgG control antibody was coupled to 1mg of magnetic beads according to manufacturer’s protocol (Invitrogen Dynabeads Antibody Coupling kit#143.11D). After coupling, 1 mg of the antibody/bead mixture was incubated with 1–5 mg of protein lysate overnight under rotation at 4°C. IP samples were then washed with RIPA buffer containing protease/phosphatase inhibitor cocktail for 3–4 washes and resuspended in non-reducing loading buffer, boiled and loaded on a gel for western blot analysis. Immunoprecipitations were performed using the following antibodies: anti-ERG antibody (Epitomics# 2805–1), anti-HA antibody (Roche#11815016001), anti-AR antibody (Thermo Scientific# MA5-13426) or anti-IgG antibody .Procedures were previously described  and the Generation of labeled cDNA, hybridization to Affymetrix U133plus2 human arrays, and data normalization were performed as described . For theBlack line  represents expressed probe set position and is ranked by average fold-change. Blue, green, and red lines indicate where the probe sets mapping to genes in the androgen receptor activation signatures appear in our data set and show the cumulative sum of the probe sets in the androgen receptor activation signatures that overlap with our gene list (only the highest expressing probe set was used per gene). The dashed line represents the hypothetical cumulative sum for a random list of genes that are unenriched.PSA, NKX3-1 and SLC45A3 and VIC-labeled probe for Beta-2-macroglobulin (B2M) as a normalization control. Samples were run on a 7900HT Real-Time PCR machine (Applied Biosystems) and data was analyzed and normalized according to manufacturer’s instructions (2-ΔCt method).RNA isolation was performed as previously described . Taqman VCaP, 22Rv1 and RWPE-1 cells were treated as specified followed by cross-linking with 1% formaldehyde for 10 min. Cells were next lysed in 1% SDS and sonicated until DNA ladder is below 1 kb (Diagenode). Sheared chromatin was then used for IP with specific primary antibodies  pre-complexed with Protein A/G Dynabeads and incubated overnight under rotation at 4°C. The next day, ChIP samples were washed with RIPA buffer and TE followed by reverse crosslinking using 1%SDS and 30 ug/ml proteinase K (Invitrogen) at 65°C for 6 hr with beads. The eluates were then purified using the QIAquick PCR purification kit (Qiagen) and used for qPCR with the following primer sets:PSA -4100: acctgctcagcctttgtctc AND ttgtttactgtcaaggacaatcgPSA -3800: agaattgcctcccaacactg AND cagtcgatcgggacctagaaPSA -100: cttccacagctctgggtgt AND aaaccttcattccccaggacPSA +700: agccccagactcttcattca AND atgcagatttggggaatcagNKX3-1 -2800: gagagcagctgttcctccac AND acgagccttttccacctttcNKX3-1 -200: agggaggagagctggagaag AND tcctccctaggggattcctNKX3-1 +2150: accaggatgaggatgtcacc AND cagggacagagagagccttgNKX3-1 -+10800: tctctcgttggctcctgatt AND ccagcttttgttccttcctgNKX3-1 +62100: cggtttattgcccatgaaga AND aacagggctcacagtgctttVCaP cells harboring Dox inducible shRNAs targeting PRMT5 where grown to 80% confluency and re-seeded (day 0) into full media containing 100 ng/ml Doxycycline. On day 3 media was replaced with ‘hormone reduced’ media, containing 100 ng/ml Doxycycline. Cells were stimulated with adding indicated ligands (DHT (Sigma), R1881 [Sigma]) or vehicle on day 4 and harvesting of cells was performed on day 5. Cells were harvested by fixation using 1% methanol free formaldedyde  in PBS at room temperature. Fixation was stopped after 8 min by replacing fixation buffer with ice cold PBS containing 125 mM glycine and 5 mg/ml BSA. Cells were further washed once using ice-cold PBS and re-suspended into 500 ul of PBS containing Complete Protease Inhibitors (Roche). Cells where then pelleted and supernatant removed, and the resulting pellet either snap frozen in liquid Nitrogen or immediately re-suspended in lysis buffer for further processing.3 and 1% (w/v) SDS. Crosslink reversal was done at 65C°C for 6 hr and ChIP DNA were isolated using DNA purification beads (MagBio). ChIP-seq libraries were generated using the KAPA HTP library preparation kit (Kapa biosciences). All handling of samples after sonication was done on using a Sciclone NGS Workstation . Sequencing was performed on an Illumina NextSeq500 instrument.For ChIP the resulting cell lysate was sonicated using a Covaris E210 instrument according to manufacturers recommendations. Each ChIP reaction was performed using soluble fraction chromatin corresponding to 7.5 ug purifed DNA and 4 ug of antibodies. Antibodies were allowed to bind overnight before capture on protein A magnetic beads . Bound beads where washed 4 times in RIPA buffer containing 500 mM LiCl, and 2 times with TE buffer before being re-suspended in containing 100 mM NaHCOReads passing Illumina standard QC were mapped to genome version Hg19 using BWA, and binding sites (‘peaks’) were identified using MACS2, evolutionary conservation scores at peak locations was calculated using Phastcons and enriched DNA motifs using MDscan and Seqpos. These were performed using the ChiLin QC pipeline (liulab.dfci.harvard.edu/WEBSITE/software). Peaks from AR ChIP-sequencing samples with a MACS2 enrichment score higher than 10 were extended to a uniform 400 bp across all samples and overlapping peaks where collapsed to generate a union of all peaks. This resulted in a Cistrome of 25,593 peaks that were used in all genome wide ChIP analyses. Using the features of this Cistrome as GTF, read counts from BWA mapped Bam files were processed using the Qlucore 3.1.19 software. Heatmaps and statistical test (two-sided t-test using correction for multiple hypothesis testing) of differential binding scores on the 25,593 features were performed in Qlucore v3.1.19. The AR and ERG ChIPseq datasets are available at the NCBI Gene Expression Omnibus (Accession number GSE79128).RWPE-1 cells were treated as specified, fixed with 4% paraformaldehyde for 45 min at room temperature (Electron Microscopy Sciences), blocked with 5% goat serum, 0.5% Triton X-100 in PBS for 2 hr and incubated with 1:50 dilutions of AR (LSBIO #LS-C87494) and symmetric di-methyl arginine antibodies (CST#13222) in 5% goat serum and 0.05% Triton X-100. Fixed samples were incubated overnight at 4°C in primary antibody before incubations with proximity ligation assay (PLA) secondary antibodies .The secondary antibody incubation, ligation, amplification and final wash steps were performed according to the manufacturer’s specifications. Confocal microscopy was performed using an LSM 510 META  with a 40x C-Apochromat objective, NA 1.2. Images were collected and processed using Zen software .Homo sapiens androgen receptor (AR) sequence  was synthesized to include a 5’ NotI and 3’ BamHI sites and used as a template for the mutagenesis of each arginine in the ligand binding domain of AR into lysine . Following sequence verification to ensure mutation incorporation, each AR mutant sequence was cloned into the pLVX vector via the 5’ NotI and 3’ BamHI as previously described  and incubated with 1:50 dilution of AR antibody (LSBIO #LS-C87494). Confocal microscopy was performed using an LSM 510 META  with a 40x C-Apochromat objective, NA 1.2. Images were collected and processed using Zen software .The heterodimeric structure of PPARγ-RXRα (PDB Code: 3DZY) was used as a template to overlay individual domain structures of AR including the DBD in complex with DNA (PDB Code: 1R4I) and the LBD in complex with coactivator peptide TIF2(iii) and ligand R1881 (PDB Code: 2AO6). A superposition was achieved using secondary structure matching in COOT . The AR °C. Cells were resuspended in buffer A containing 50 mM Tris-HCl (pH 7.3), 150 mM NaCl, 10% glycerol, 0.25 mM TCEP, and 10 µM DHT, to which 50 µg/ml DNase I and protease inhibitor cocktail (Roche) were added. Cells were lysed using an M-110L Microfluidizer at 18,000 psi, followed by the addition of 0.5% CHAPS to the lysate prior to high speed centrifugation. For one-step batch purification, the soluble extract was incubated with 2 ml of glutathione sepharose 4 fast flow medium  for 1 hr at 4°C with rotational mixing. The sepharose medium was washed in buffer A with the addition of 0.5% CHAPS. Elution was accomplished by resuspending and incubating the media for 10 min in the wash buffer plus 10–20 mM reduced glutathione. The eluted fractions were then combined and concentrated to 0.3 mg/ml.The gene encoding human AR LBD (residues 663–919), was inserted into a pGEX-6P-1 vector and expressed as a GST-tagged fusion protein in BL21 Star (DE3) cells. Cells were grown in TB2 medium containing 10 µM dihydrotestosterone (DHT) and induced with 1 mM IPTG for 14 –16 hr at 16S-adenosyl-L-homocysteine (SAH) product formation utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). 0.5–1 uM of PRMT5/MEP50 recombinant enzyme  was incubated with 50 uM SAM, 2 uM GST-AR LBD and/or 5 uM ETS or PNT ERG protein for 2 hr at 37°C. Reactions were quenched to 0.1% HCOOH followed by addition of -SAH (SAH-D4) in 20% DMSO as an internal standard for MS quantification. Samples were sonicated with a Hendrix SM-100 sonicator (Microsonics Systems) and centrifuged. SAH was separated from the reaction mixture by reversed phase chromatography using polar endcapped C18 reversed phase columns  and detected using a 4000 QTRAP Hybrid Triple Quadrupole/Linear Ion Trap LC-MS/MS system (AB Sciex).PRMT5 enzymatic activity was assessed by monitoring  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kevin Struhl as the Senior Editor.Thank you for submitting your work entitled "ERG signaling in prostate cancer is driven through PRMT5-dependent methylation of the Androgen Receptor" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. As you will see below, the reviewers have some concerns that will require quite an additional effort to address. Ordinarily, we allow just two months to return a revised version but we are concerned that this required experiments may take longer to complete. To help us assess the likelihood of a successful completion of the required additional work, please send us a note indicating how you plan to proceed and an estimate of the time it will take to complete the work.Summary:In this manuscript, the authors present two major findings: 1) PRMT5 methylates AR at R761 and these methylation results in decreased AR recruitment at target genes and decreased AR function and 2) ERG targets AR to be methylated. Overall, the findings address long-standing questions of ERG function in prostate tumorigenesis and possibly posit new therapies to ERG translocated prostate cancers. This would be certainly relevant to the prostate field. In addition, it is appropriate for a general audience for implications of a general enzyme that affects specific programs. The experiments are well done and logical. The manuscript is well written. However, at this point, there are several critical experiments that should be performed to solidify the conclusion.Essential revisions:1) Definition of the ERG and AR binding genome wide with and without PRMT5 and ERG knockdown and correlation with the gene expression is very important for the conclusion. Is AR binding specifically depleted in ERG co-bound sites? The changes genome wide may not reflect the selected sites, as highlighted by the effect of FOXA1 on AR. This does require time and effort. But AR and ERG ChIP-seq is well optimized and ChIP-seq today is cost effective so this is eminently doable.2) One weakness of the manuscript, intrinsic to prostate cancer research, is the use of a single ERG positive cell line. So any hit of the screen could be ERG specific, or even more likely, VCAP specific. Since there are no other available ERG dependent cell lines, the authors should note in the manuscript this major caveat. It would be very helpful if the authors could examine xenografts or perform some validation of the mechanisms in primary human tissue samples. For example, authors can IP AR from several ERG positive and ERG negative prostatectomy specimens to assess for methylation.3) Mass spec evidence of methylation at R761 would be helpful. Since you know what you're looking for, IP of AR in VCAP and another ERG negative line and looking for this should not be hard and would provide definitive evidence. The mutagenesis is highly suggestive, but could still change conformation in a way such that R761 is not the direct methylation site. Essential revisions:1) Definition of the ERG and AR binding genome wide with and without PRMT5 and ERG knockdown and correlation with the gene expression is very important for the conclusion. Is AR binding specifically depleted in ERG co-bound sites? The changes genome wide may not reflect the selected sites, as highlighted by the effect of FOXA1 on AR. This does require time and effort. But AR and ERG ChIP-seq is well optimized and ChIP-seq today is cost effective so this is eminently doable.KLK3 (aka PSA) and NKX3-1, the ChIPseq dataset shows a significant increase of AR recruitment upon PRMT5 knockdown at 6% of the total AR-bound cistrome. Genome-wide recruitment of ERG was not significantly affected upon PRMT5 knockdown, including to the sites where AR recruitment was increased . This data agrees with our model that ERG recruits PRMT5 to attenuate AR activity to a significant subset of its target genes.We thank the reviewers for raising this important point. To extend our findings beyond individual genes, we performed ChIPseq for both ERG and AR in the presence of the androgen molecules R1881 and DHT, and in the absence or presence of PRMT5 knockdown. This data is provided in 2) One weakness of the manuscript, intrinsic to prostate cancer research is the use of a single ERG positive cell line. So any hit of the screen could be ERG specific, or even more likely, VCAP specific. Since there are no other available ERG dependent cell lines, the authors should note in the manuscript this major caveat. It would be very helpful if the authors could examine xenografts or perform some validation of the mechanisms in primary human tissue samples. For example, authors can IP AR from several ERG positive and ERG negative prostatectomy specimens to assess for methylation.TMPRSS2:ERG positive models, we have explored the ERG/PRMT5 interaction using additional model systems. First, in TMPRSS2:ERG transgenic mice vs. control mice ; AR immunoprecipitates from the TMPRSS2:ERG mouse prostates show increased SDMA and MMA signals at the correct size for AR. Fifth, we show in We thank the reviewers for acknowledging the challenges of obtaining appropriate prostate models and patient specimens. To address the general lack of We further attempted to confirm our findings in human tissue samples. In the absence of an AR-R761me2s-specific antibody for immunoblotting or IHC, we planned to use a chromogenic in situ proximity ligation assay (PLA)  with primary antibodies recognizing AR and symmetric di-methyl arginine (SDMA) to detect AR-R761me2s in human prostate cancer samples. First, we focused on optimizing PLA for formalin-fixed paraffin-embedded (FFPE) tissues, using FFPE RWPE-1 prostate cells, FFPE human prostate cancer samples, and two primary antibodies against Ki67 . While numerous individual spots representing positive signal were detected in the nuclei of RWPE-1 prostate cells, few, if any, spots were seen in the human tissues see , suggest3) Mass spec evidence of methylation at R761 would be helpful. Since you know what you're looking for, IP of AR in VCAP and another ERG negative line and looking for this should not be hard and would provide definitive evidence. The mutagenesis is highly suggestive, but could still change conformation in a way such that R761 is not the direct methylation site.We agree that mass spectrometry data would be an important support of our findings. We made several attempts at mass spec identification of the arginine site using AR immunoprecipitated from VCaP cells. Unfortunately, we could not identify this site by mass spectrometry. In our hands, arginine methylation has been generally difficult to detect, including on histones, which are 'classic' PRMT5 targets. We suspect that R761me2s does not occur on the entire population of AR within the cell lysate, which 'masks' the detection of methylated AR by mass spec. This may be in line with the observation that PRMT5 knockdown does not affect AR recruitment to all possible sites in our ChIPseq experiments.While not directly addressing the reviewers’ question, we provide additional supporting data that PRMT5 can methylate purified AR ligand binding domain in vitro. These biochemical assays demonstrate PRMT5 activity  on purified AR but not ERG, and that addition of purified ERG DNA binding domain can facilitate greater PRMT5 activity on AR. This data is provided in


Given that the literature suggests there are various (and often contradictory) interpretations of holistic practice in brain injury rehabilitation and multiple complexities in its implementation , this study aimed to examine the experiences of practitioners in their conceptualization and delivery of holistic practice in their respective settings. Nineteen health practitioners purposively sampled from an extensive Brain Injury Network in Queensland, Australia participated in individual interviews. A systematic text analysis process using Leximancer qualitative analysis program was undertaken, followed by manual thematic analysis to develop overarching themes. The findings from this study have identified several items for future inter-professional development that will not only benefit the practitioners working in brain injury rehabilitation settings, but the patients and their families as well. Many thousands of people sustain a traumatic brain injury (TBI) in Australia every year, with a similar representation of diagnoses worldwide –6. DefinThe overall goal of rehabilitation following TBI is to assist the person to achieve the highest degree of cognitive, functional and physical capacity to maximise an independent post-injury life ,16,17. Ainterrelationship of all aspects of functioning for improved rehabilitation outcomes. The application of this best-practice interactive perspective of the bio-psycho-social model remains highly relevant to the practicing clinician  personal practice framework” (ID: P3SW) in thinking about general rehabilitation, as well as international frameworks of practice (‘the ICF’ [n = 2]), discipline specific frameworks , client-base frameworks  or more formal legal frameworks . All practitioners were unclear as to which framework was of greatest importance, and believed they all contributed to some extent to their practice.For Cluster 1, the theme of Specialised Rehabilitation comprised a description of frameworks and practices tailored and targeted to brain injury rehabilitation. In relation to frameworks, it was evident that practitioners held multiple and competing frameworks in mind when they considered quality brain injury rehabilitation. For instance, practitioners identified the use of the ICF’ , for exa“It’s different injuries happening to different people in different contexts–you can’t have a one-size-fits-all approach” (ID: P9CM), and “there are often challenging behaviours that present themselves which may not be present in general rehab” (ID: P4NUR). Paediatric practitioners highlighted this issue further by discussing the unique circumstances of children with a brain injury;  The injury happens to a developing brain and so any skills that may not have been acquired yet, the future development of those skills may be affected by the injury. So in terms of minimizing the impact of that injury damage, we don’t know what we’re aiming for in terms of the development of those skills in the future (ID: P18PSY).Practitioners also commented that any framework they held had to be person-oriented and tailored to brain injury depending on the individual capacity or circumstances of the client, n = 8), how much the individual’s context was incorporated into the rehabilitation goals (n = 8), and how well individuals were encouraged to participate in their rehabilitation (n = 7). As highlighted by ID P1PSY, “[A person’s context] is also very important… if you don’t look at their context then you really will never get anywhere with rehab”. Further, participants emphasized system structures and their associated contexts  as largely mediating these factors, thereby influencing the likelihood of collaborative principles being implemented into practice.Teamwork, shared information and participation  were also important qualifiers to this theme. Although participants recognised that brain injury rehabilitation is best collaborative in its approach, this was dependent on a number of factors. These included how much the family were able to be involved in the rehabilitation (n = 4) using ‘client’-centred (adult sector) or ‘family’-centred (paediatric sector) (n = 8) and strength-based (n = 2) approaches that focus on re-engagement (n = 3) and identification of individual needs (n = 5) were identified as ideal. However, according to practitioners, the effectiveness of such approaches depended on the individual with the injury, as well as system structures. For example, the individual’s degree of self-awareness, insight, and ensuing capacity , ‘system time’ available to spend with each person, as well as the discharge destination of the individual are all illustrations of implementation factors that largely influence the efficacy of goal-driven principles.Goal-directed principles were also acknowledged by participants as necessary to the individual’s recovery. In particular, individualized goal setting .To elaborate on the ‘Person-in-Context’ theme, n = 8), “rather than just the components …. of his disability, it would be looking at whether he’s [re]engaging in his occupation, how he’s going about those occupations, how he’s interacting with his environment and looking at his goals in relation to that” (ID: P12OT); past, present, and future histories (n = 9), “it’s about recognizing that the person has a life that existed prior to the brain injury and post brain injury” (ID: P1PSY); and control and coordination of health services by the individual (n = 10), “the participation has to be very much directed by the person themselves” (ID: P9CM). While the importance of family support (and context) was reflected on by adult client-base practitioners, the support and active involvement of the family system was emphasized further by all paediatric practitioners in their definition of holistic practice: I think [holistic practice and family-centred practice] go hand in hand. I don’t think that you can treat a child holistically in terms of rehab without considering them in their family context. … [However], family-centred care is not necessarily the same as seeing a child in context either. You might be aware of the context but not necessarily modify your practice accordingly (ID: P18PSY).Specifically, participants defined Person-in-Context as the interaction of the person with their environment. This included concepts such as healing (“[the brain injury] is not the primary focus” (ID: P5PSY).Also important in defining holistic practice but to a lesser extent was an acknowledgment from practitioners of the physical functionality, medical treatment and core activity capacity of the person with the injury (Patient-in-Care). Subsequently, the person is viewed as a whole where the injury is part of the package, rather than the injury being the entirety of that package, n = 9), service capacity (n = 8) and the physicality or visibility of the injury (n = 4). As ID: P14OT describes, “It really depends on access–access to the client, access to their home situation, school, work environment and the different reasons why that access might not be provided”. Thus, although participants agreed on how holistic practice in brain injury rehabilitation should be defined, it was also clear that a number of factors influence the likelihood of these definitions being realized in brain injury rehabilitation.Additionally, the scope of holistic practice in brain injury rehabilitation depends on a number of influencing factors that subsequently determine how much the individual is viewed as a ‘Person-in-Context’ and/or a ‘Patient-in-Care’. These factors include extent to which the practitioner has physical access to, or only background knowledge of, the person’s home environment  greatly influenced Client Outcomes (refer to n = 12). Also identified as important in the assessment process was the need for practitioners to think outside of their own specialist skill set (n = 4), and promote ‘whole of person’ practice through teamwork mechanisms that encourage the practitioner to endorse early and comprehensive assessment practices (n = 11), “… no matter what our background, we all undertake the same holistic bio-psycho-social assessment process” (ID: P9CM). According to practitioners, the success of assessment processes and subsequent outcomes was dependent on the early relationship and timely referral processes established.Various Processes incorporating assessment processes, relationship processes and referral processes were identified by participants as relevant to holistic practice implementation. In relation to assessment processes, practitioners reported that assessment methods needed to be comprehensive in their scope and facilitate mutual understanding between all relevant parties (including the injured person and their families) in order to inform and educate (n = 3), trust (n = 6), respect (n = 5), engagement (n = 15), family involvement (n = 7) and information sharing (n = 17) were highly valued by practitioners in the delivery of holistic care. It was reported by participants that the degree to which these relationship processes were positively established subsequently affected assessment processes  and referral outcomes. For instance, Your relationship with the client and the willingness of the client to engage in a rehab program and their disclosure about particular things that are really important… [influences] whole of person practice. The client may not be willing to disclose that information [if a strong rapport is not developed] (ID: P8OT).Within relationship processes, rapport , access to resources (n = 12), as well as appropriate frameworks of practice (n = 10). According to ID: P5PSY, “There’s not a lot of understanding of some of the more complex issues associated with brain injury”, which in essence, can lead to fragmented service systems, lack of appropriate community-based resources, and frameworks that do not engage the person within their contexts. Although the majority of participants voiced concern over current system support structures, they felt that within the limits imposed by the system composition, their current processes underpinning holistic practice implementation as practitioners in brain injury rehabilitation was mostly adequate.Participants also recognized that the implementation of holistic practice in brain injury rehabilitation is fundamentally driven by the system supports available in brain injury service delivery. In particular, rehabilitation was thought to be influenced by the capacity of the system to provide adequate funding (n = 3), the system capacity to refer to culturally appropriate services that are sensitive to the person’s needs (n = 9), and whether issues of transition from hospital to home or between children’s services and adult’s services were being adequately addressed (n = 4). As highlighted by ID: P6NUR, It's very difficult for us to get that aftercare follow-up… [because] they don't fit into the mental health service quite often because they're not acutely unwell, the community mental health teams won't pick up most of our patients, even though we refer a lot of them to them, they don't see them as meeting their criteria. So they're just discharged and we hope for the best, you know?Individual outcomes as reported by participants related to how well the person’s learned skills during rehabilitation would transfer over to other environments (“big picture” (ID: P5PSY). That is, a person’s broader bio-psycho-social health needs may not be addressed once they have been referred out by the acute or subacute rehabilitation team.Referral processes, then, are at risk of not seeing the “… there’s nothing that brings it [brain injury rehabilitation] together over time, and there’s nothing that ensures that problems are picked up before they become a crisis”.Participants viewed individual outcomes as ultimately influenced by processes and system supports. In this way, implementation of holistic practice is not the sole responsibility of individual practitioners, but rather is a result of the systemic culture of the rehabilitation setting and its capacity to deliver. Implementation of holistic practice in brain injury rehabilitation was considered mostly adequate in terms of rehabilitation processes, reliant on the establishment of positive relationships between all stakeholders, and influenced greatly by structures and systems. As highlighted by ID P1PSY, The aim of this study was to qualitatively explore practitioner perceptions of the guiding principles, definition and scope, and implementation of holistic practice in the adult and child brain injury rehabilitation context. It was anticipated that most practitioners would describe an inclusive conceptualisation of holistic rehabilitation (based on well-established frameworks). The findings of this study supported this proposition, with participants confirming holistic practice was best defined by a collaborative bio-psycho-social approach (inclusive of patient- and family-centred philosophies) that was exceedingly tailored to brain injury rehabilitation. In this way, practitioners recognised the unique care and support needs of individuals with TBI. These needs included cognitive remediation, behaviour management, addressing self-awareness issues and facilitating adjustment over time. Indeed, teamwork, shared information, individualised goal setting, and participation of the person with brain injury (and their families) were also highlighted by practitioners as necessary to the individual’s recovery. These findings, unique to the perspective of brain injury rehabilitation practitioners, support previous literature investigating the perspective of patients, their families, and children’s school teachers ,77, obseIt was also proposed in this study that practitioners would find implementation of holistic practice challenging for multiple reasons. The current study found that multiple challenges exist in implementing holistic practice ranging from patient factors to system factors. Despite the perceived benefits of what appeared to be an overarching approach to TBI rehabilitation , guiding principles and subsequent health professional practices appeared to be driven by multiple and competing discipline specific frameworks  and client-base frameworks . These results are consistent with those reported by Kuipers and colleagues  where thParticipants also described other factors that contributed to holistic practice implementation success or failure, including available resources within individual rehabilitation settings, ‘system time’ provided to spend with each person, the capacity of the service system in supporting health professionals in referring clients to other services and settings, and how much the individual’s family were encouraged to participate in the rehabilitation program. The qualitative data indicated that despite a comprehensive approach  to holistic rehabilitation, the translation of holistic theory or frameworks into practice and related outcomes largely depended on the processes undertaken by practitioners and were only as successful as the system supports that were available.Although system level factors  appear to be beyond the control of the practitioner, there are important implications from this study for health practitioners working within brain injury rehabilitation. For instance, findings from this study highlight opportunities for practitioners to maximise their holistic practice within their own setting by maintaining a strong focus on relationship and assessment processes. Specifically, rehabilitation practitioners have the capacity to ensure the following actions remain part of their everyday practice so as to facilitate positive outcomes for individuals with traumatic brain injury:Build and maintain rapport, trust, and respect with patients and their families;combination of patient- (or family-) centred philosophy and the bio-psycho-social approach to brain injury rehabilitation may improve the person’s rehabilitation outcomes;Purposefully engage the patient and their families in the process of defining (and redefining) their rehabilitation goals over the recovery course , while prompting broader conceptualization of particular rehabilitation goals across all facets of the person’s life . In this way, the Practice inter-professional healthcare culture and communication;Undertake timely and comprehensive assessment processes that facilitate mutual understanding between all relevant parties (including the injured person and their families) in order to inform and educate. Assessment processes should be collaborative in nature and respectful of discipline speciality; andProvide appropriately detailed information to the person (and the person’s family) and to other team members, and maintain these open lines of communication across the rehabilitation trajectory.how multidisciplinary practitioners can apply each action in practice is missing. The development of such guidelines tailored to brain injury rehabilitation would be especially useful for junior practitioners, or those new to the field, given that brain injury rehabilitation is vastly different to general rehabilitation settings [Although these actions have been reported elsewhere ,82, specsettings .Overall, the findings from this exploratory study revealed that practitioners in the current sample held a comprehensive understanding of holistic practice regardless of setting, client-base, and for the most part, discipline. The findings also confirmed that the implementation of holistic practice was dependent on environmental factors; most of which appear to be beyond the control of the individual practitioner and as mentioned above, rely on systemic level influences surrounding ‘access’: access to resources within the system, physical access to the person’s home, school, or vocational environment, and service capacity, in particular. The interrelationship of these environmental aspects appears important for policy makers to consider for optimal rehabilitation outcomes . Indeed,Based on the findings of this exploratory study, the following items appear particularly important for future development in brain injury rehabilitation:The communication of a unified framework of holistic practice, tailored to brain injury, to practitioners within an inter-professional context. Specifically, this framework should not represent competing models and professional standpoints, but instead combine important philosophical concepts of human rights with practical application of coordinated practice such as timely assessment and referral, and continuity of care. With a unified framework in place, increased collaboration and practice support across disciplines regardless of discipline background could enhance patient outcomes.Further, patient-centred care was perceived by this sample of practitioners to be underlying one of the guiding principles of providing specialised rehabilitation, and is thought to be crucial to enhance patient outcome. This finding, together with the increasing expectation of practitioners to adopt a person-centred approach to healthcare , emphasihow multidisciplinary practitioners can enhance their relationship and assessment processes.Using the abovementioned unified framework, practitioners would likely benefit from specific guidelines tailored to brain injury rehabilitation that detail A review of current referral processes and system supports available in brain injury service delivery in South-East Queensland, Australia could be undertaken to better determine transitional care needs and rehabilitation planning. Findings from the current study and previous research undertaken –89 confiThese items are important to consider given that participants viewed client outcomes as ultimately influenced by processes and system supports.The findings of the current study must be contextualised in relation to the study limitations. First, there was insufficient information made available to the researchers to conduct a responders analysis to determine any potential bias between responders and non-responders. Second, given the unequal representation of practitioner disciplines and settings, the generalizability of the current findings must be interpreted with caution. Future investigations could pursue greater sample representation . It is particularly noted that the current sample was limited in its representation of medical professionals, and that the involvement of medical practitioners were restricted to inpatient rehabilitation . This suggests that the clinical experience and contexts of practice were somewhat different between the medical and allied health participants. Females also represented a large proportion of the current sample, although this is not uncharacteristic of the allied health workforce which has high female representation. Future research ought to explore whether the findings from the current study may generalise to a broader range of aetiologies for diffuse encephalopathy (both of traumatic and non-traumatic origin). Further research also ought to investigate the efficacy of holistic brain injury rehabilitation by comparing the costs incurred in providing holistic rehabilitation with a rational assessment of validated outcomes . Lastly, how practitioners might incorporate a phenomenological approach to holistic brain injury rehabilitation  is a noteworthy line of inquiry that could also be explored in future research studies.The present study extended our understanding of how brain injury rehabilitation practitioners conceptualise and subsequently implement holistic approaches in their everyday practice. Although untested, the study identified important key processes and influencing factors that might determine the eventual success of holistic practice. In line with previous research, the current study also highlighted the need for a systems-approach in achieving comprehensive rehabilitation in brain injury. Utilising a common framework could be the beginning of a changed system–multiple stakeholders  ought to arrive at a shared understanding of holistic care facilitated by a consistent language.Notwithstanding the reported limitations, and despite the potential usefulness and intuitive appeal of the holistic approach, practitioners have identified difficulties in implementing the holistic model in the brain injury rehabilitation of both children and adults. Indeed, researchers confirm that there is little guidance for how practitioners in Australia (and around the world) contemplate and then implement holistic theory into practice within the brain injury rehabilitation context . The finQ1Can you tell me what are the guiding principles of brain injury rehabilitation within your practice?Q2Are they different from general rehabilitation principles?Q3From your experience, how would you define holistic practice in brain injury rehabilitation? Please describe.Q4What do you think would be the opposite of ‘whole of person’ practice?Q5Does ‘whole of person’ practice look different for children and adults in brain injury rehabilitation?Q6How exactly might ‘whole of person’ practice change across practitioners and settings ? Your answer can be as broad or specific as you like…Q7How do you achieve ‘whole of person’ practice in your discipline and setting?Q8How do you see other practitioners implementing ‘whole of person’ practice? Good examples / bad examples.Q9What external factors do you think might influence the real world delivery of ‘whole of person’ practice?Q10Can you provide an example of when you feel you didn’t or couldn’t achieve ‘whole of person’ practice within your discipline or setting, and why?S1 File(DOC)Click here for additional data file.


Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 have demonstrated this direct conversion of carbon dioxide into FFAs; however, FFA yields in these hosts are limited by the negative impact of FFA production on the host cell physiology. This work investigates the use of Synechococcus sp. PCC 7002 as a cyanobacterial host for FFA production. In comparison to S. elongatus PCC 7942, Synechococcus sp. PCC 7002 strains produced and excreted FFAs at similar concentrations but without the detrimental effects on host physiology. The enhanced tolerance to FFA production with Synechococcus sp. PCC 7002 was found to be temperature-dependent, with physiological effects such as reduced photosynthetic yield and decreased photosynthetic pigments observed at higher temperatures. Additional genetic manipulations were targeted for increased FFA production, including thioesterases and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Overexpression of non-native RuBisCO subunits (rbcLS) from a psbAI promoter resulted in more than a threefold increase in FFA production, with excreted FFA concentrations reaching >130 mg/L. This work illustrates the importance of host strain selection for cyanobacterial biofuel production and demonstrates that the FFA tolerance of Synechococcus sp. PCC 7002 can allow for high yields of excreted FFA.Microbial free fatty acids (FFAs) have been proposed as a potential feedstock for renewable energy. The ability to directly convert carbon dioxide into FFAs makes cyanobacteria ideal hosts for renewable FFA production. Previous metabolic engineering efforts using the cyanobacterial hosts  Escherichia coli, which began nearly four decades ago  into FFAs. Cyanobacteria are photosynthetic prokaryotes, which are particularly amenable to genetic manipulation, and as such, several cyanobacterial strains have been engineered for FFA production. Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942, two model freshwater strains of cyanobacteria, were engineered for FFA production by gene knockout of the FFA-recycling acyl-acyl carrier protein (ACP) synthetase/long-chain-fatty-acid CoA ligase and expression of a thioesterase for fatty acid (FA) cleavage from the ACP, resulting in extracellular FFA concentrations of 83.6 and 32 mg/L, respectively , expression of a chloroplastic acetyl-CoA carboxylase, and improved gene expression using strong native promoters, yet these modifications did not improve the concentration of excreted FFAs in engineered S. elongatus PCC 7942  has recently garnered much attention as a potential feedstock for renewable energy production  , BD (agar), MP Biomedicals (CuSO4⋅5H2O), Fisher Scientific , and Sigma-Aldrich . Primers were synthesized by Integrated DNA Technologies (IDT). DNA purifications were performed using the Plasmid Miniprep, DNA Clean and Concentrator, and Gel DNA Recovery kits from Zymo Research. Restriction enzymes, DNA polymerases, and ligases were purchased from New England Biolabs. Suppliers of all other materials used in this study are described below.Chemicals used in this study were purchased from Acros Organics , responsible for FFA recycling  and BglII/XhoI (3′ fragment) restriction enzyme sites, generating the fadD knockout plasmid, pS12. Transformation of pS12 into Synechococcus sp. PCC 7002, followed by spectinomycin selection and PCR screening of fadD, resulted in the construction of strain S01. Transformation of Synechococcus sp. PCC 7002 was performed using a modified protocol based on that proposed by Stevens and Porter  in a 16 mm glass test tube with ventilated cap. The transformation culture was incubated at 30°C with 150 rpm and 60 μmol photons m−2 s−1. After 24 h of incubation, the transformation culture was concentrated to 100 μL using centrifugation  and spread on medium A+/agar plates with 1 mM of sodium thiosulfate and 40 μg/mL of spectinomycin dihydrochloride pentahydrate and 50 μg/mL of kanamycin monosulfate, as required. Colonies were re-streaked a minimum of three times to obtain complete segregants. After antibiotic selection and PCR screening, two positive transformants were tested for FFA production in 400 mL cultures (see Culture Conditions).Strains used and constructed in this study are listed in Table d Porter . SynechoE. coli thioesterase, ‘tesA, from pSE16  was constructed by redesigning the forward primer to eliminate the predicted chloroplast-targeting signal, determined using ChloroP 1.1  from S. elongatus PCC 7942 were cloned from pSE18  and insertion into pS20 at the BsrGI site produced pS21. Transformation of pS21 into Synechococcus sp. PCC 7002 yielded strain S07.To insert another restriction enzyme site for Ruffing,  . Agar plates of medium A+ contained 1 mM of sodium thiosulfate. Single transformed colonies were re-streaked at least three times or until full segregants were obtained, as confirmed by PCR. Synechococcus sp. PCC 7002 strains grown on solid media were inoculated into 4 mL of medium A+ with antibiotics as necessary and grown at 30 or 38°C and 150 rpm in a New Brunswick Innova 42R shaking incubator with photosynthetic light bank. Alternating cool white and plant fluorescent lights provided ~60 μmol photons m−2 s−1 of continuous illumination. After 5–7 days of growth, 1 mL of the test tube inoculum was transferred to a 500 mL baffled Erlenmeyer flask containing 100 mL of medium A+ with appropriate antibiotics. After 4 days of growth under the aforementioned conditions, the 100 mL culture was used to inoculate 400 mL of medium A+ in a 1 L glass media bottle with a three-port lid for sampling, bubbling of 1% CO2 in air, and ventilation through a 0.22 μm filter. The initial cell concentration of the 400 mL culture vessels was approximately an OD730 of 0.1. No antibiotics were added to the large culture vessels to eliminate any potential effects associated with antibiotic supplementation. The large cultures were sampled every 2 days for ~3 weeks. IPTG was added at 100 h to induce expression from the trc and LlacO-1 promoters.Wild-type and engineered strains of Fv) is the difference between the maximum fluorescence (Fm) under saturated light and the initial fluorescence (F0) under actinic light; the apostrophe indicates that the samples were not dark adapted prior to measurement. Photosynthetic yield Cell growth was measured using optical density (OD) readings at 730 nm from a PerkinElmer Lambda Bio spectrophotometer. Photosynthetic yield measurements were obtained using a Waltz mini-PAM photosynthetic yield analyzer; for each sample, triplicate technical measurements were averaged due to variability associated with these measurements. Photosynthetic yield or efficiency measurements Synechococcus sp. PCC 7002 was genetically engineered for FFA production and excretion by targeting the long-chain-fatty-acid CoA ligase  for gene knockout and overexpression of a truncated thioesterase from E. coli (‘tesA). The resulting engineered strains, S01 (ΔfadD) and S02 , are analogous to the previously constructed, FFA-producing strains of S. elongatus PCC 7942, SE01 (Δaas) and SE02 . A schematic of the engineered pathway for FFA production can be found in the Supplemental Material . The FFA concentrations, cell growth profiles, and photosynthetic yields of Synechococcus sp. PCC 7002, S01, and S02 are shown in Figure S. elongatus PCC 7942, SE01, and SE02  did not show improved FFA production in comparison to the S. elongatus PCC 7942 strains (SE01 and SE02), there were notable differences in the physiological responses of the hosts. The late exponential growth rates of S01 and S02 showed a slight decrease in comparison to the wild-type 7002 after induction at 100 h (4.17 days)  may also influence the optimal growth temperature, and a more rigorous, multi-factorial investigation of Synechococcus sp. PCC 7002 growth is required to evaluate the optimal growth temperature of this strain. In addition to this unexpected growth response, there were significant changes in photosynthetic yields at 38°C in comparison to 30°C. At the higher temperature, the wild-type (7002) had reduced photosynthetic yields with values up to 50% lower compared to the 30°C temperature condition  and the phycobiliproteins (635 nm) at 38°C . This suggests that DesB is not involved in FFA tolerance at this temperature, but it does not rule out a role for membrane saturation in the mechanism of temperature-dependent FFA tolerance in Synechococcus sp. PCC 7002.At high temperatures, bacteria have been shown to change the degree of saturation of their cell membrane lipids, with increasing saturation accompanying increasing temperatures Koga, . The incSynechococcus sp. PCC 7002 compared to S. elongatus PCC 7942, additional strategies for improving FFA biosynthesis were investigated. The acyl-ACP thioesterase from C. reinhardtii (fat1) was previously shown to have activity similar to ‘tesA in S. elongatus PCC 7942 . Expression of tfat1 in S05 led to a nearly threefold increase FFA production and excretion compared to the fat1-expressing S03 strain  did not yield an improvement in FFA production in the Synechococcus sp. PCC 7002 strain S06 [, overexpression of the truncated E. coli thioesterase (‘tesA), and overexpression of a heterologous RuBisCO (rbcLS), an engineered strain of Synechococcus sp. PCC 7002 was constructed, which produced FFAs at concentrations as high as 131 mg/L. The high levels of FFA achieved in this study demonstrate not only a potential for large-scale production applications, but also a potential separation mechanism, as the FFAs precipitated from the culture medium and floated to the surface upon reaching saturation  which are more desirable, as these temperatures are readily achieved in open outdoor cultivation systems without additional heating . The enhanced membrane viscosity associated with a lower degree of membrane saturation may allow for increased FFA diffusion across the cell membrane and therefore higher extracellular FFA concentrations. This proposed mechanism of FFA excretion also supports the aforementioned hypothesis for improved FFA tolerance at lower temperatures. The change in photosynthetic pigments in response to high FFA production also differed for the Synechococcus sp. PCC 7002 strains in comparison with the S. elongatus PCC 7942 strains. FFA-producing strains of S. elongatus PCC 7942 showed selective degradation of chlorophyll-a pigment in response to FFA production . When a cytotoxic unsaturated FFA, α-linolenic acid, was exogenously added to cultures of Synechococcus sp. PCC 7002 also displayed different responses to the genetic manipulations of aas/fadD knockout, thioesterase expression, and RuBisCO expression. While Aas from S. elongatus PCC 7942 and FadD from Synechococcus sp. PCC 7002 are homologs (58.4% amino acid identity), aas knockout in SE01 led to the accumulation of 43 mg/L of excreted FFAs, yet fadD knockout in S01 yielded only 5 mg/L of excreted FFAs. These results may indicate that S. elongatus PCC 7942 has a naturally higher rate of membrane degradation and FFA recycling compared to Synechococcus sp. PCC 7002. In the S. elongatus PCC 7942 strains SE02 and SE03, both ‘TesA’ and ‘Fat1’ thioesterases had similar activities, generating nearly equivalent levels of excreted FFA , suggesting that further improvement may be achieved with codon optimization. Regardless of the underlying mechanisms for improved FFA tolerance and production, Synechococcus sp. PCC 7002 was shown to be a superior host for FFA biosynthesis compared to S. elongatus PCC 7942. This work highlights the importance of host selection in metabolic engineering efforts, and since host responses are difficult to predict a priori, screening and testing of potential hosts remains a critical step in cyanobacterial strain development.In addition to differences in FFA stress response, Ruffing, . In the Synechococcus sp. PCC 7002 (this study), S. elongatus PCC 7942  may weaken the integrity of the host strain and its ability to survive under adverse conditions, which may be experienced in large-scale production systems. Moreover, as the genetic manipulations differ between the engineered strains of Synechocystis sp. PCC 6803 and those of Synechococcus sp. PCC 7002 and S. elongatus PCC 7942, this direct comparison must be viewed with caution. Synechocystis sp. PCC 6803 also has the highest tolerance of exogenous FFA, yet the results of this study clearly demonstrate that exogenous FFA tolerance does not directly correlate with tolerance to intracellular FFA biosynthesis. When comparing inherent strain properties of doubling time, high light tolerance, high salt tolerance, and temperature tolerance, Synechococcus sp. PCC 7002 is superior to the other two cyanobacteria. These basic strain traits, which can be difficult to engineer into a host, make Synechococcus sp. PCC 7002 a beneficial host for cyanobacterial biofuel production, and this study provides direct evidence of the advantages afforded by this host, encouraging further investigation and development of Synechococcus sp. PCC 7002 for biofuel production.To date, three model cyanobacterial strains have been genetically engineered for FFA production: Anne M. Ruffing conceived the study, conducted all experimental work and data analysis, and drafted the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fbioe.2014.00017/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.


Cytochrome b5 reductase 2 (CYB5R2) is a potential tumor suppressor that inhibits cell proliferation and motility in nasopharyngeal carcinoma (NPC). Inactivation of CYB5R2 is associated with lymph node metastasis in NPC. This study aimed to explore the mechanisms contributing to the anti-neoplastic effects of CYB5R2.Polymerase chain reaction (PCR) assays were used to analyze the transcription of 84 genes known to be involved in representative cancer pathways in the NPC cell line HONE1. NPC cell lines CNE2 and HONE1 were transiently transfected with CYB5R2, and data was validated by real-time PCR. A chick chorioallantoic membrane (CAM) embryo model was implanted with CYB5R2-expressing CNE2 and HONE1 cells to evaluate the effect of CYB5R2 on angiogenesis. An immunohistochemical assay of the CAM model was used to analyze the protein expression of vascular endothelial growth factor (VEGF).FAS), FBJ murine osteosarcoma viral oncogene homolog (FOS), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), integrin beta 3 (ITGB3), metastasis suppressor 1 (MTSS1), interferon beta 1 (IFNB1), and cyclin-dependent kinase inhibitor 2A (CDKN2A) and down-regulated levels of integrin beta 5 (ITGB5), insulin-like growth factor 1 (IGF1), TEK tyrosine kinase (TEK), transforming growth factor beta receptor 1 (TGFBR1), and VEGF. The angiogenesis in the CAM model implanted with CYB5R2-transfected NPC cells was inhibited. Down-regulation of VEGF by CYB5R2 in NPC cells was confirmed by immunohistochemical staining in the CAM model.In CYB5R2-transfected NPC cells, PCR assays revealed up-regulated mRNA levels of Fas cell surface death receptor (VEGF to reduce angiogenesis, thereby suppressing tumor formation.CYB5R2 up-regulates the expression of genes that negatively modulate angiogenesis in NPC cells and down-regulates the expression of  Nasopharyngeal carcinoma (NPC) is a major health problem in South China, with a morbidity of 25–30 per 100,000 people each year .Cytochrome b5 reductase 2 (CYB5R2), a nicotinamide adenine dinucleotide (NADH)-dependent flavin reductase, belongs to the cytochrome reductase family and has functions in oxidation reduction, drug metabolism, and methemoglobin reduction in erythrocytes . CYB5R mThe expression of CYB5R2 varies in different types of cancer. CYB5R2 is abnormally inactivated in prostate and breast cancers, and it regulates cell proliferation and differentiation, similar to other TSGs , 8, but In previous study, we showed that promoter DNA hypermethylation was responsible for inactivation of CYB5R2 and associated with lymph node metastasis in NPC patients . In addiIn the present study, we further explored the pathways affected by CYB5R2. Using polymerase chain reaction (PCR) assays of NPC cells transfected with CYB5R2, we investigated the changes in mRNA expression of 84 genes in 6 signaling pathways related to cancer. A chick chorioallantoic membrane (CAM) embryo model was used to validate the inhibitory effect of CYB5R2 on angiogenesis in NPC cell xenograft.2 at 37°C.EBV-negative NPC cell lines CNE2  and HONETotal RNA was isolated from HONE1 cells, in which the transcription of CYB5R2 is almost silence, using Trizol (Invitrogen). First-strand cDNA was synthesized using M-MLV reverse transcriptase . The PAHS-033A Human Cancer Pathway Finder Superarray and Microsoft Excel-based data analysis software  were used to identify changes in the mRNA expression of 84 genes that are representative of 6 signaling pathways: cell cycle control and DNA damage repair, apoptosis and cell senescence, signal transduction molecules and transcription factors, cell adhesion, angiogenesis and tumor invasion, and metastasis.5 cells/well) and transfected with 2 μg pCMV-Tag3A-CYB5R2 or pCMV-Tag3A (empty vector) plasmids using an X-treme GENE HP DNA Transfection Reagent  and incubated for 48 h. Each experiment was repeated three times.Full-length cDNA from the open reading frame of CYB5R2, purchased from Origen Co. , was subcloned into the pCMV-Tag3A vector . CNE2 or HONE1 cells were seeded in 6-well plates experimental − (CTtarget gene − CTGAPDH)control. Empty vector-transfected cells were used as the control. Independent experiments were performed in triplicate.Significant modification of gene transcription by ectopic expression of CYB5R2 was further confirmed by quantitative real-time PCR. Primer information is in Table 2 window was carefully opened. An amount of 8 × 105 CNE2 or HONE1 cells was resuspended in 10 μL of IMDM and seeded into a silicon ring , which was then placed on the CAM. The window was covered with parafilm , and the egg was placed back into the incubator. Silicone rings were removed 24 h later  were up-regulated. Some genes involved in angiogenesis   were up-regulated or down-regulated with CYB5R2 expression. These changes in the gene expression in CYB5R2-transfected NPC cells (CNE2 and HONE1) was verified by real-time PCR  after transfection with a CYB5R2 expression plasmid  ), insuliTo evaluate the influence of exogenously expressed CYB5R2 on the angiogenic capacity of NPC cells, we implanted CYB5R2-transfected CNE2 and HONE1 cells into a CAM model to analyze the formation of tumors and angiogenesis. CYB5R2-transfected CNE2 and HONE1 cells formed smaller tumors than empty vector- cells Figure a, b, andP < 0.001; Figure CYB5R2 was higher in the xenografts derived from CYB5R2-transfected CNE2 and HONE1 cells than from empty vector-transfected cells  are associated with angiogenesis, which provides clues to the mechanism of the association between epigenetic inactivation of CYB5R2 and lymph node metastasis [We found, using PCR assays, that the overexpression of tastasis . We focuThe presence of palpable cervical lymph nodes is usually the first presenting symptom in NPC, indicating that NPC has a high propensity to invade the lymphatic system and spread to the cervical lymph nodes. NPC cells may also spread to distant organs, including the lungs, liver, and bones, through the blood stream. Angiogenesis and lymphangiogenesis are essential for tumor growth and metastasis, which is primarily driven by VEGF. As a member of the platelet-derived growth factor family, VEGF is the most influential stimulator of vascular endothelial growth during tumor angiogenesis . VEGF isWe found that CYB5R2 expression in NPC cells altered the angiogenic balance by down-regulating a series of angiogenesis-promoting factors  and up-regulating an angiogenesis inhibitor, IFNB1. These data imply that CYB5R2 plays a role in angiogenic suppression. To further investigate this role, we used CAM, an in vivo model, to study tumor growth and angiogenesis and to evaluate the effect of CYB5R2 on angiogenesis , 33. CYBIn summary, CYB5R2 transfection in NPC cells affected mRNA levels of several factors involved in angiogenesis, including VEGF, IFNB1, IGF1, TEK, and TGFBR1. When we overexpressed CYB5R2 in NPC cells and implanted these cells into a CAM model, cell proliferation and angiogenesis were suppressed. The malignant phenotype may be inhibited by CYB5R2 through a down-regulation of VEGF or suppression of HIF-1α expression. Inactivation of CYB5R2 may contribute to angiogenesis in NPC. The results of this study provide insight into the mechanism by which CYB5R2 acts to suppress tumor function, and further support CYB5R2 as a novel TSG in NPC.