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Annex: HyLYZER Revision 120829 Hydrogenics Corporation reserves the right to make changes at any time without notice, in materials, equipment, specifications and models showns in this document. These are not necessarly showing the equipment that will be installed in your system
Annex: HyLYZER Revision 120829 1. INTRODUCTION The HyLYZER is a modular electrolyzer which uses deionized (DI) water and either AC or DC electricity to produce up to 1.1 and 2.2 normal cubic metre per hour (Nm3/h) of hydrogen. The electrolysis reaction takes place within a proton exchange membrane (PEM) cell stack. The PEM Electrolyzer features fully automatic hydrogen production and comes installed inside a practical and easy to install cabinet. Production of hydrogen using this method is a simple and emission-free process that is safe and reliable due to the minimal amount of moving...
Annex: HyLYZER Revision 120829 3. SCOPE OF SUPPLY Each HyLYZER features: Enclosure for indoor installation Control interface and HMI PEM electrolyzer stack Hydrogen Purification System (HPS) Optionally, the HyLYZER can include: Remote monitoring package Storage tank Water treatment system 3.1. Enclosure for Indoor Installation The Hylyzer comes installed inside an indoor enclosure that is mounted on wheels to simplify installation. It comes with clear interconnection points on the cabinet. All controls are located on the front side of the cabinet, as shown on the picture on the right. The...
Annex: HyLYZER Revision 120829 3.2. Human Machine Interface - HMI The HyLYZER comes equipped with an HMI that is visible from the from front side of the cabinet. The HMI allows for the operator to operate the HyLYZER, view the status, read alarms and guide you through the startup phase. This also includes a diagnostic and local monitoring software package with HMI and event log. 3.3. PEM Electrolyser Stack The advanced proprietary cell stack consists of circular electrolytic cells, each containing two electrodes, the PEM membrane assembly and bipolar plates. The bipolar plates separate the...
Annex: HyLYZER Revision 120829 4.3. Water Treatment System We can include a feed water treatment system that will convert incoming drinking water into deionized water required to produce hydrogen in the HyLYZER. The customer is responsible to provide water meeting or exceeding the following specifications to the water treatment system at all times. FEED WATER AND PROCESS WATER REQUIREMENTS FEED WATER REQUIREMENTS (BEFORE WATER TREATMENT SYSTEM) Operating Pressure Maximum Temperature Maximum Turbidity Maximum Silt Density 5.0 (based on 15 min. test time) Hydrogen Sulfide Langelier Saturation...
Annex: HyLYZER Revision 120829 6. TECHNICAL SPECIFICATIONS PROPERTY ELECTROLYSER Model Type System efficiency Proton Exchange Membrane Electrolyser 3 Net Production after Dryer Turndown Ratio Electrolyser Output Pressure Hydrogen Purity Nm /h (scfh) % barg (psig) % Atm. Dew Point After Dryer 200-260,1 phase,2 wire+gnd, 50/60 Hz Direct connection to DC possible upon request. Cooling Requirements Indoor, in a non-hazardous, non-classified area OPERATING CONDITIONS Site Ambient Temperature Range Relative Humidity 1520 (higher altitudes available on request) ENCLOSURE DIMENSIONS Cabinet (WxDxH)...
Annex: HyLYZER Revision 120829 7. STARTUP SERVICES Field services of one (1) supervisor for three (3) days on site is required for the verification of the installation (done by others), start-up, commissioning of the complete system and training of staff on the operation of the system. All Hydrogenics expenses for travel, accommodation and living are not included in this proposal. Delays beyond the control of Hydrogenics shall be billed at standard daily rates. Commissioning consumables are not included. 8. DOCUMENTATION Standard documentation in English language to be provided includes -...
Copyright: Oeggerli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Competing interests: LB has filed a US patent related to KCNMA1 (KCNMA1 as a therapeutic target in cancer treatment"). This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials. All other authors have no competing interests with regard to patenting.
In many diseases, defective regulation/or expression of BK channels have repeatedly been associated with altered cell cycle progression [9], cell proliferation [10], [11], [12], [13], [14], [15], [16], and cell migration [17], [18]. These factors are fundamental to the development of cancer [19], [20]. As demonstrated in electrophysiological studies on cervical and breast cancer cells, BK channels are directly activated by estrogens, which have an essential role in cancers of the uterus, breast and prostate [21], [22]. Specific blockade of BK channels leads to membrane depolarization, cell cycle progression and inhibition of cell proliferation [1], [23]. Early reports pointed to high levels of KCNMA1-expression in human glioblastomas [24]. A number of subsequent reports pointed to a more general role of BK channels in different types of cancer, although this not seems to apply to all (Cambien et al., 2008). We previously detected genomic amplification of the BK channel encoding gene KCNMA1 in 16% of late-stage prostate cancers, identifying KCNMA1 as one of the most common amplifications in prostate cancer [13]. We found that knockdown of KCNMA1 by siRNA and specific BK channel blockade by iberiotoxin inhibited cell proliferation of the prostate cancer cell line PC3, which carries an amplification of KCNMA1. This study suggested a specific role of KCNMA1 in the transition from hormone-sensitive to hormone-insensitive and castration-refractory prostate cancer [13].
T47D [27], SKBR3 [28] and PC3 [13] cell lines were obtained from American Type Culture Collection (ATCC, LGC Promochem, Molsheim Cedex, France) and grown under standard cell culturing conditions in Optimem cell culture medium (Invitrogen, Carlsbad, CA), supplemented with 1% penicillin/streptomycin (Amimed, Basel, Switzerland) and 10% FCS (Amimed) at 37C/5% CO2. Trypsin-EDTA (Amimed) was used as a transferring reagent. Cell lines MFM223 [29], MCF7 [30], LNCaP [13] and BPH1 [13] were obtained from German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and grown under the same conditions. In case of MCF7, the Optimem medium was replaced by RPMI 1640-cell culture medium. PC346 [31] and PC3456C [31] were obtained from G. Jenster (Department of Urology, Josephine Nefkens Institute, Erasmus Medical Center, P.O. Box 1738, 3000 DR, Rotterdam, The Netherlands) and grown under standard conditions.
Trypsinized cells were harvested and washed in PBS twice. Aliquots of 100 L were centrifuged for 2 minutes and dried with a ventilator. Frozen tissue samples and cytospins were incubated with an anti-KCNMA1 polyclonal antibody and prepared as previously described [13]. Images were obtained using a Zeiss Axioplan 2 fluorescence microscope (Zeiss, Jena, Germany), equipped with an appropriate filter set. The selection of target cells was based on clear visibility in DAPI-staining. An ISIS-digital camera (MetaSystems, Altlussheim, Germany) was set up to capture multi-stack images. The resultant images were resized, cropped and had their levels and contrast adjusted accordingly. PBS-washed slides were used as negative controls.
Total RNA from cell lines was extracted using RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the instructions of the manufacturer. RNA concentrations were determined with Nanodrop spectrophotometer (Witec AG, Littau, Switzerland). Real-time PCR was performed using the LightCycler system and LightCycler FastStart DNA Master Hybridization Probes kit (Roche, Hilden, Germany). Primers were obtained from TIB MolBiol (Berlin, Germany): KCNMA1-for: TggCCTCCTCCATggTgA, KCNMA1-rev: TTCTgggCCTCCTTCgTCT, GAPDH-for: gAAggTgAAggTCggAgTC, GAPDH-rev: gAAgATggTgATgggATTTC, KCNMA1-FL: AgCgTCCgCCAgAgCAAgAT-FL, KCNMA1-LC: LC640-ATgAAgAggCCCCCgAAgAAAgT-PH, GAPDH-FL: AggggTCATTgATggCAACAATATCCA-FL, GAPDH-LC: LC640-TTTACCAgAgTTAAAAgCAgCCCTggTg-PH. PCR conditions were: Activation 10 min 95C, annealing 5 s at 54C, elongation 15 s at 72C, 40 cycles. Relative levels of expression (normalized against GAPDH) were determined according to [33].
17β-estradiol (Sigma-Aldrich, Buchs, Switzerland) was dissolved in 100% ethanol to build a stock solution of 0.5 M, which was further diluted to a final concentration of 10 nM, using the appropriate cell culture medium. Paxilline (Sigma-Aldrich, Buchs, Switzerland) was dissolved in 100% DMSO to build a stock solution of 50 mM, and then further diluted in phenol red-free medium supplemented with 5% dextran-coated charcoal-stripped FCS [27] to a final concentration of 15 M. Prior to the experiments, standard growth curves were calculated to adjust optimal conditions for all cell lines and reagents, respectively. For all patch clamp experiments, Student's t-test P-values
Analysis frozen sections from nine human breast cancers by immunofluorescence revealed strong expression of BK channels in seven, and weak expression in two specimens. There was no correlation between amplification of KCNMA1 and BK expression in these specimens, suggesting that KCNMA1 can also be up regulated by mechanisms other than genomic amplification (Figure 2). The KCNMA1-amplified breast cancer (MFM223) and prostate cancer (PC3; positive control) cell lines were strongly positive for BK channels, while non-amplified breast cancer cell lines T47D and MCF7 were only weakly positive or negative as evaluated by immunofluorescence (Figure 3).
To explore the association between amplification of KCNMA1 and tumour cell proliferation, we constructed a TMA with 34 specimens enriched for KCNMA1 amplified breast cancers (9 with and 25 without KCNMA1 amplification) using two tissue cores per specimen. The specimens were also analyzed by IHC for expression of ER-α, AR and Ki67 (Mib1). KCNMA1 amplification was significantly associated with high tumour cell proliferation (defined as Ki67 LI>30%; p
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