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Conceived and designed the experiments: TK SY YS. Performed the experiments: TK SY NH. Analyzed the data: TK SY S. Kusakawa NH YK KS MT SN S. Kawamata YS. Contributed reagents/materials/analysis tools: YK MT S. Kawamata. Wrote the paper: TK SY YS. Acquired the funding: SN S. Kawamata KS YS.
Human induced pluripotent stem cells (hiPSCs) possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells (hESCs). These characteristics make hiPSCs a promising choice for future regenerative medicine research. There are significant obstacles, however, preventing the clinical use of hiPSCs. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. To locate residual undifferentiated cells, in vivo teratoma formation assays have been performed with immunodeficient animals, which is both costly and time-consuming. Here, we examined three in vitro assay methods to detect undifferentiated cells (designated an in vitro tumorigenicity assay): soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR). Although the soft agar colony formation assay was unable to detect hiPSCs even in the presence of a ROCK inhibitor that permits survival of dissociated hiPSCs/hESCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE) cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of Lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×104 RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived products for future regenerative medicine research.
Pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells have two capabilities: 1) pluripotency: the ability to differentiate into a variety of cells and 2) self-renewal: the ability to undergo numerous cycles of cell division while maintaining their cellular identity. Because of these two characteristics, it has been expected that they would provide new sources for robust and continuous production of a variety of cells and tissues for regenerative medicine/cell therapy. Additionally, hiPSCs offer us a possible solution to the ethical problems and the immune rejection of hESC-derived cells, thus raising novel avenues for patient-specific cell therapy. As previously reported [1], [2], many attempts are currently underway to differentiate hESCs and hiPSCs into various tissues: cardiomyocytes [2], [3], neurons [2], [4], and hepatocytes [5], [6]. It is noteworthy that clinical trials have been conducted with retinal pigment epithelial (RPE) cells derived from hESCs to treat patients with dry age-related macular degeneration and Stargardt's macular dystrophy by Advanced Cell Technology. hiPSCs have also been shown to differentiate into RPE cells, which display functionality both in vitro and in vivo [7], [8]. Thus, autologous transplant of hiPSC-derived RPE cells holds great promise in the clinical therapy of macular degeneration.
Although hiPSCs overcome immunogenic and ethical barriers, the translation of hiPSCs into the clinical setting faces the same significant problems as those of hESCs. One of the most important issues in the development of a safe pharmaceutical or medical device derived from human pluripotent stem cells is ensuring that the final product does not form tumors after implantation [9]. There are two primary concerns. First, the cell-based product may be unstable and transform to produce a tumor, which is a common problem for any cell-based products, regardless of the cell types of the raw materials. Second, the product derived from human pluripotent stem cells might contain residual undifferentiated stem cells that would eventually proliferate and form a teratoma [10]. Previous reports have shown that several hundred hESCs were sufficient for generating tumors in immunodeficient mice [11], [12]. Hence, to adress the second concern above, it is critical to develop highly sensitive assays for detection of residual undifferentiated stem cells in the final products, and to determine their lower limit of detection (LLOD). An evaluation study of the in vivo tumorigenicity assay using severe combined immunodeficiency (SCID) mice has shown that 245 undifferentiated hESCs spiked into 106 feeder fibroblasts produce a teratoma [11]. On the other hand, some in vitro assays, such as quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry and immunohistochemistry, have been used to indicate the undifferentiated state of stem cells with various markers (such as Oct-3/4, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4) [13]–[15]. However, it has not been determined how many residual undifferentiated hiPSCs can be detected by these in vitro assays.
In this study, to establish a high sensitivity assay for detection of residual undifferentiated hiPSCs in the final product, we evaluated three in vitro assays: soft agar colony formation assay, flow cytometry and qRT-PCR. To achieve this goal, these assays were used on cell mixtures that contained defined numbers of undifferentiated hiPSCs in primary RPE cells, and we also tried to determine the LLOD of each assay by using multiple lots of primary RPE cells as backgrounds. Through this process, we revealed that one-step qRT-PCR using probes and primers targeting Lin28 transcripts can detect levels as low as 0.002% residual undifferentiated cells in hiPSC-derived RPE cells.
Minimizing contamination of undifferentiated pluripotent stem cells in cell therapy products is crucial because of the risk of tumorigenesis. To evaluate residual undifferentiated hiPSCs in differentiated cells, it is necessary to determine the LLOD of the hiPSC content in RPE cells. First, we differentiated hiPSCs into RPE cells using the in vitro differentiation protocol previously described (Fig. 1A) [7]. The hiPSC-derived RPE cells exhibited polygonal, cobblestone-like morphology, an indication of RPE maturation, which is similar to that of the primary RPE cells (Fig. 1B). Immunocytochemical staining revealed that N-cadherin, the major cadherin expressed in RPE cells [16], showed a distribution to the tight junction of the hiPSC-derived RPE cells, which is consistent with primary RPE cells (Fig. 1C). Moreover, in flow cytometry experiments, a strong expression of the visual cycle protein CRALBP and the melanosomal matrix protein GP-100 was detected in both primary RPE and hiPSC-derived RPE cells compared to in undifferentiated hiPSCs (Fig. 1D). To characterize developmental stages during RPE differentiation, a qRT-PCR assay was used to identify transcript levels of CRALBP and the visual cycle protein RPE65, indicating that CRALBP and RPE65 increased as differentiation progresses and were equally well expressed both in the mature hiPSC-derived RPE cells and primary RPE cells (Fig. 1E). Together, these data showed that mature RPE cells differentiated from hiPSCs possess similar properties to primary RPE cells.
The soft agar colony formation assay is a general method to monitor anchorage-independent growth, which is considered the most appropriate in vitro assay for detecting the malignant transformation of cells [17]. To measure cell transformation quantitatively, we used the CytoSelect 96-well Cell Transformation Assay, as described in the Materials and Methods section. Previous reports have shown that human pluripotent stem cells undergo apoptosis when dissociated into single cells [18]. However, preparation of single cell suspension is quite important because the presence of cell clumps or adjacent cells is critical for growth in an agar medium. Thus, we first sought to clarify whether single hiPSCs grow in the soft agar medium. The soft agar colony formation assay showed that single-hiPSCs could not proliferate in the agar medium. In addition, the ROCK inhibitor Y-27632, which has been reported to inhibit apoptosis [19], did not improve survival of hiPSCs under our conditions (Fig. 2A and S1A). These results demonstrated that the soft agar colony formation assay is not appropriate for detection of undifferentiated hiPSCs in single cell suspension.
Next, we tested whether colony formation of the human ovarian teratocarcinoma cell line PA-1 [20] occurs in an agar medium, since PA-1 cells could mimic possible malignant cells derived from hiPSC. PA-1 cells efficiently formed colonies in soft agar depending on the number of cells (Fig. S1B). On the other hand, primary RPE cells did not grow in a soft agar media with 1.0×104 cells/well, even when cultured for 30 days (Fig. S1C). Based on these results, we aimed to examine the sensitivity of the soft agar colony formation assay to detect PA-1 cells. We spiked 100 (1%), 50 (0.5%), and 25 (0.25%) PA-1 cells into 1×104 primary RPE cells in order to define the minimum number of PA-1 cells required to grow in a soft agar media. The largest number of PA-1 cells (1%) gave rise to detectable colonies within 20 days, whereas lesser numbers of PA-1 cells (0.5% and 0.25%) required 30 days until the colonies become detectable (Fig. 2A and B). We next tried to determine the LLOD of the soft agar colony formation assay. The LLOD of the assay signal was calculated as the mean plus 3.3 fold the standard deviation of the measurement of negative controls [21]. Based on signals from three lots of primary RPE cells as a negative control (1.71±0.40 [fold over the background signal]), the LLOD of the soft agar transformation assay was calculated as 3.03 (Fig. 2C). These results indicate that at least 1% spiked PA-1 cells are necessary for detecting colonies in primary RPE cells using the soft agar transformation assay (4.4±1.1). As shown in Fig. 2C, the hiPSC-derived RPE cells showed no colony formation in the soft agar medium, and the assay signal was lower than the LLOD (1.21±0.19). These results suggest that the contamination of transformed cells, assuming that their anchorage independency is comparable to PA-1, is less than 1% in the hiPSC-derived RPE cells.
In the second set of the experiments, we tried to detect residual undifferentiated cells via flow cytometry. Using five antibodies which recognize stem cell marker antigens (Oct3/4, Nanog, Sox2, TRA-1-60, TRA-1-81), we first attempted to identify highly selective markers that distinguish a small population of hiPSCs from primary RPE cells. To minimize nonspecific staining, we used fluorescent conjugated monoclonal antibodies for the flow cytometry. All of the stem cell markers were detected in undifferentiated hiPSCs, but the levels of cell staining differed between the antibodies, presumably attributable to the protein expression in the cells and/or the avidity of the antibodies. The fluorescence histograms of hiPSCs and primary RPE cells showed that anti-Oct3/4, anti-Sox2 and anti-TRA-1-60 antibodies clearly distinguished hiPSCs from RPE cells (Fig. 3A). Because TRA1-60 is not only a marker of undifferentiated hiPSCs but also of embryonal carcinoma [22], we employed anti-TRA-1-60 antibody in further experiments. The TRA-1-60+ gate was defined as including at most 0.05% of the primary RPE cells with the highest fluorescence (Fig. 3B). In the flow cytometry, the mean and standard deviation of TRA-1-60+ cells in the primary RPE cells (the negative controls) were 26.6 and 15.6 cells/105 cells, respectively, producing an LLOD of 78.1 cells/105 cells (Fig. 3B). To analyze the performance of the system for detection of residual hiPSCs, we spiked 2.5×103 (0.1%) and 2.5×102 (0.01%) hiPSCs into 2.5×106 primary RPE cells and analyzed 1.0×105 cells via flow cytometry using anti-TRA-1-60 antibody. We also confirmed the number of the applied hiPSCs by spiking CFDA-stained hiPSCs (Fig. S2). In the experiment shown in Fig. 3C, 130 and 19 cells were identified as TRA-1-60+ cells in 0.1% and 0.01% hiPSCs spiked samples, respectively. These results indicated that at least 0.1% of residual undifferentiated hiPSCs (2.5×103 cells out of 2.5×106 cells) can be detected via the flow cytomerty. Finally, to detect residual undifferentiated cells in hiPSC-derived RPE cells, we tested 1×105 cells and detected six TRA-1-60+ cells, suggesting that the population of undifferentiated hiPSCs in the hiPSC-derived RPE cells was no more than 0.1% (Fig. 3D).
We next tested the ability of qRT-PCRs to detect a trace amount of stem cell-specific mRNA. To identify highly selective markers for undifferentiated hiPSCs, we compared the mRNA levels of OCT3/4, KLF4, c-MYC, SOX2, NANOG, LIN28 and REX1 in hiPSCs and primary RPE cells (Fig. 4A). Primary RPE cells were found to endogenously express c-Myc at 25.49% of the levels observed in hiPSC, which was consistent with the previous finding that MEF expressed c-Myc at approximately 20% of levels observed in mouse ES cells [23]. The expression levels of Klf4 and Rex1 in primary RPE cells were 3.51% and 2.23% compared with hiPSCs, respectively. On the other hand, more than a 1000-fold difference between primary RPE cells and hiPSCs was observed in the gene expression of Nanog (0.07%), Sox2 (0.06%), Oct3/4 (0.01%) and Lin28 (not detected). These results suggested that the latter four genes are useful in detecting hiPSCs in RPE cells.
We then conducted two sets of qRT-PCR assays to evaluate the utility of these marker genes to detect spiked hiPSCs in primary RPE cells. We first measured mRNA levels of Nanog, Oct3/4 and Lin28 in the five lots of primary RPE cells (the negative control) to determine the LLOD. The LLODs for Nanog and Oct3/4 mRNAs were 0.45% and 0.04% those of hiPSCs, respectively (Fig. 4B). The LLOD of Lin28 could not be calculated because no fluorescent signal for Lin28 expression was detectable in the primary RPE cells. These results along with experiments spiking 2.5×104 (1%), 2.5×103 (0.1%) and 2.5×102 (0.01%) hiPSCs into 2.5×106 primary RPE cells (Fig. 4B) indicated that measurements of Nanog, Oct3/4 and Lin28 expression by qRT-PCRs could detect at least 1%, 0.1% and 0.01% contamination of residual undifferentiated hiPSCs in RPE cells, respectively.
Finally, we examined whether qRT-PCR for Lin28 was applicable in the detection of residual undifferentiated cells in differentiated RPE cells from hiPSCs. Total RNA (250 ng) extracted from the differentiating cells (day 5, 20, and 40) and purified hiPSC-derived RPE cells (the passage number 3 and 4) were subjected to qRT-PCR analysis. The mRNA level of Lin28 was continuously down-regulated during the differentiation process, being 0.02% that of hiPSCs at day 40. In early passage culture (passage number 3) after purification, Lin28 was still significantly expressed at a level 0.002% that of hiPSCs. From passage 4 onward, however, no detectable level of Lin28 expression was observed in hiPSC-derived RPE cells (Fig. 4C). These results suggest that qRT-PCR analysis for Lin28 detects 0.002% of residual undifferentiated hiPSCs in hiPSC-induced PRE cells, namely, that a single hiPSC in 5.0×104 RPE cells is detectable.
For the clinical use of products derived from human pluripotent stem cells, it is essential to improve both the efficacy and safety of the final product. In order to develop safe hiPSC-based treatments, the hurdle of tumorigenicity arising from undifferentiated cells must be overcome [9]. To address the issue of tumorigenicity, some recent publications have advocated the development of protocols for the derivation of hiPSC [23]–[25] and have outlined methods for the elimination of residual hESCs [26]. However, to date, more than several hundred cells are necessary for human pluripotent stem cells to form a tumor in immunocompromised mice [11]. Therefore, highly sensitive tumorigenicity assays and their standardization are necessary for detecting a small population of residual undifferentiated cells in products derived from human pluripotent stem cells.
In the present study, we evaluated three methods for detection of residual undifferentiated hiPSCs in hiPSCs-derived differentiated cells: soft agar colony formation assay, flow cytometry and qRT-PCR. Table 1 summarizes the advantages and disadvantages of the assays associated with product tumorigenicity. The soft agar colony formation assay is known to be more sensitive in the detection of certain types of tumorigenic cells, compared to in vivo methods using immunocompromised mice [27]. However, we found the soft agar colony formation assay unsuitable for detecting residual undifferentiated hiPSCs, presumably attributable to the dissociation-induced apoptosis of hiPSCs [18]. On the other hand, flow cytometry and qRT-PCR assays were found to be able to detect a trace amount of undifferentiated cells. These two assays have been exploited for characterization of stem cell-based products, as well as undifferentiated pluripotent stem cells, but the present study is the first analytical and quantitative approach designed to evaluate the detection of residual undifferentiated cells in products derived from human pluripotent stem cells. The advantage of the flow cytometry assay is that it is able to identify undifferentiated cells. Unfortunately, the results are greatly affected by gating, and only the cells expressing the marker protein are detectable. On the other hand, the advantages of qRT-PCR are its rapidity, quantitativity and high sensitivity, whereas its disadvantage is that only the cells expressing the marker gene are detectable. Although the in vivo tumorigenicity assay is costly and time-consuming, it can directly analyze tumor formation in a specific microenvironment where the product is implanted (eg. retina). Therefore, a combination of relevant in vitro and in vivo assays would be necessary to ensure the safety of a product derived from human pluripotent stem cells. The rationale for the choice of specific assays would be justified, based on their characteristics shown in Table 1.
We have demonstrated that the qRT-PCR assay can successfully detect 0.002% residual undifferentiated hiPSCs in hiPSC-induced RPE cells using Lin28 as a target gene (Fig. 4E). To the best of our knowledge, this qRT-PCR assay, using solely 250 ng of total RNA obtained from approximately 5×104 cells, is the most sensitive of the previously reported methods in detecting undifferentiated pluripotent stem cells. Lin28 is known to specifically inhibit the processing of let-7 miRNAs, which are involved in cell-fate decisions [28]. Interestingly, the aberrant expression of Lin28 transcripts has been recently reported in human germ-cell tumors [29], suggesting that Lin28 is a useful marker of germ-cell malignancy as well as of pluripotency of hiPSCs. Lin28 mRNA gradually decreased in the differentiation process and was completely diminished by passage 4 (Fig. 4C). These observations suggest that Lin28 transcripts are also available for presenting degree of differentiation in hiPSC-derived products because detection of residual Lin28 confirms the contamination of undifferentiated cells even at a late stage of differentiation. Needless to say, the distinct expression of Lin28 could possibly be observed in other normal somatic cells. However, Lin28 is, at least in part, one of the potent markers for detecting incompletely differentiated cells contained in RPE cells derived from pluripotent stem cells.
A great deal of international research is currently being directed at developing regenerative medicine using pluripotent stem cells. Until now, however, little attention has been paid to developing methods to detect undifferentiated cells in pluripotent stem cell-based products. Here, we have revealed that a qRT-PCR method targeting Lin28 can effectively detect a trace amount of hiPSCs in hiPSC-induced RPE cells and shows potential as an in vitro tumorigenecity assay of hiPSC-derived cells. We expect our findings to contribute to the process of validation and quality control of hiPSCs-based cell therapy products and to promote the application of regenerative medicine in the treatment of a wide variety of diseases in the near future.
HiPSC line 201B7 induced by transducing Oct3/4, Sox2, Klf4 and c-Myc [2] was obtained from the RIKEN Cell Bank. Undifferentiated hiPSCs were maintained on mitomycin C-treated SNL cells (a mouse fibroblast STO cell line expressing the neomycin-resistance gene cassette and LIF) in human ES cell culture medium (ReproCell, Japan) supplemented with 4 ng/ml human basic fibroblast growth factor (bFGF; WAKO, Japan). Undifferentiated colonies were passaged as small clumps once in every 5–6 days using CTK solution (ReproCell) and STEMPRO EZPassage (Invitrogen, Carlsbad, CA). Human primary RPE cells were obtained from the Lonza and ScienCell Research Laboratories. The primary RPE cells were maintained in Retinal Pigment Epithelial Cell Basal Medium (Lonza Biologics, Basel, Switzerland) containing supplements (l-glutamine, GA-1000 and bFGF; Lonza). PA-1 cells derived from human ovarian teratocarcinoma (ATCC, Manassas, VA) were maintained on Minimum Essential Medium Eagle medium (Sigma–Aldrich, St. Louis) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Paisley, UK). All cell lines and differentiated cells were grown in a humidified atmosphere of 5% CO2 and 95% air at 37°C.
The procedure for differentiating hiPSCs into RPE cells was performed according to the previously described protocol [7] as shown in Figure 1A. The hiPSC clumps were first incubated on poly-d-lysine/gelatin-coated dishes in human ES cell culture medium supplemented with 10 µM Y-27632 (WAKO), 5 µM SB431542 (Sigma–Aldrich) and 3 µM CKI-7 (Sigma–Aldrich) for 1 day. The cells were incubated in a differentiation medium (Glasgow minimum essential medium [GMEM; Invitrogen], 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, and 0.1 mM 2-mercaptoethanol) containing 20% knockout serum replacement (KSR; Invitrogen) for 4 days, then in 15% KSR-containing differentiation medium for 6 days, and finally in 10% KSR-containing differentiation medium for 11–30 days. Y-27632 (10 µM), SB431542 (5 µM) and CKI-7 (3 µM) were added to the differentiation medium for the first 13, 19 and 19 days, respectively. Partially differentiated cells were dissociated with the CTK solution and incubated on non-adhesive dishes (Corning, Corning, NY) in RPE maintenance medium (DMEM:F12 [73] supplemented with B-27 supplement [Invitrogen] and 2 mM l-glutamine [Invitrogen]) for 10 days. The resulting RPE cell aggregates were isolated and replated on CELLstart- (Invitrogen) coated dishes in RPE maintenance medium supplemented with 0.5 µM SB431542 and 10 ng/ml bFGF and we defined this stage as passage 1. The medium was changed every 2–3 days.
A soft agar colony formation assay was performed using CytoSelect 96-well Cell Transformation Assay kit (Cell Biolabs, San Diego, CA) according to the manufacturer's instructions with slight modification. Prewarmed 25 µl of 2×DMEM medium containing 20% FBS and 25 µl of 1.2% agar solution were mixed and transferred onto a well of 96-well plates, and then incubated at 4°C for 30 min to allow the bottom agar layer to solidify. Single cell suspensions were prepared as described below: 201B7 cells were dissociated with CTK solution to form cell clumps and incubated on gelatin-coated dishes in the presence of 10 µM Y-27632, a ROCK inhibitor, at 37°C for 1 h to separate with feeder cells. After centrifugation, cell pellets were dissociated into single cells with Accutase (Millipore). Primary RPE cells, hiPSC-derived RPE cells, and PA-1 cells were treated with 0.25% trypsin-EDTA solution (Invitrogen) to dissociate. Cells were passed through 40 µm nylon cell strainers (BD Falcon).
Next, 25 µl of single cell suspensions containing the defined number of cells were mixed with 25 µl of 2×DMEM medium containing 20% FBS and 25 µl of 1.2% agar, and placed on the bottom agar layer. The top agar layers were immediately solidified at 4°C for 10 min to avoid false-positive signals derived from gravity-induced adjacent cells in the agar medium. After the addition of 100 µl of DMEM containing 10% FBS to each well, the plates were incubate for 10, 20 and 30 days at 37°C and 5% CO2. The medium was changed every 2–3 days. Colonies were lysed and quantified with CyQuant GR dye using a fluorometer equipped with a 485/520 nm filter set (Wallac 1420 ARVOsx mutilabel counter, PerkinElmer, Boston, MA).
Total RNA was isolated from cell cultures using an RNeasy Mini Kit (Qiagen, Hilden, Germany) and treated with DNase I according to the manufacturer's instructions. In the spike study, 201B7 cells and RPE cells were mixed at a defined cell number, before total RNA isolation. qRT-PCR was performed with the QuantiTect Probe one-step RT-PCR Kit (Qiagen) on a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA). The expression levels of target genes were normalized to those of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) transcript, which were quantified using TaqMan human GAPDH control reagents (Applied Biosystems). Probes and primers were obtained from Sigma–Aldrich. The sequences of primers and probes used in the present study are listed in Table S1. All qRT-PCR reactions were run at 45 cycles.
201B7 cells and RPE cells were dissociated into single cells as described above. Cells were fixed with the BD Cytofix fixation buffer (BD Biosciences, Bedford, MA) for 20 min and permeabilized with BD Perm/Wash buffer (BD Biosciences) for 10 min at room temperature. Cells were incubated for 1 h at room temperature with the following primary antibodies and fluorochrome-conjugated antibodies: mouse anti-CRALBP monoclonal 11000 (B2, Thermo Scientific, Roskilde, Denmark); rabbit anti-GP-100 monoclonal 11000 (P14-V, Enzo Life Sciences, Lausen, Switzerland); FITC mouse anti-TRA-1-60 monoclonal 15 (TRA-1-60, BD Pharmingen); PE mouse anti-TRA-1-81 monoclonal 15 (TRA-1-81, BD Pharmingen); PerCP-Cy5.5 mouse anti-Oct3/4 monoclonal 15 (40/Oct-3, BD Pharmingen); Alexa Fluor 647 mouse anti-Sox2 monoclonal 15 (245610, BD Pharmingen); PE mouse anti-Nanog monoclonal 15 (N31–355, BD Pharmingen). Indirect immunostaining was then completed with either donkey-anti-mouse or donkey-anti-rabbit Alexa Fluor 647-conjugated secondary antibodies 11000 (Molecular Probes) for 1 h. Appropriate antibodies were used as a negative control. To obtain fluorescein-labeled hiPSCs, 201B7 cells were incubated with 10 µM carboxyfluorescein diacetate succinimidyl ester (CFDA; Invitrogen) in phosphate buffered saline (PBS) for 8 min, dissociated into a single cell suspension, and then fixed as described above. Stained cells were analyzed with a BD FACSAria II (BD Biosciences). Data retrieved from the sorting was analyzed with Flowjo software 9.3.3 (Tree Star, Ashland, OR).
All manipulations were performed at room temperature. Cultured primary and hiPSC-derived RPE cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. After washing with PBS, the cells were permeabilized with 0.2% Triton-X100 in PBS for 15 min and blocked with 2% bovine serum albumin in PBS for 30 min. Samples were incubated for 1 h with mouse anti-N-cadherin monoclonal antibody 11000 (GC-4, Sigma–Aldrich). The cells were washed with PBS and incubated with 11000 Alexa Fluor 488 F(ab′)2 fragment of goat anti-mouse IgG 11000 (Molecular Probes) for 1 h. The samples were mounted with a Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA) and examined with a Biozero-8000 fluorescence microscope (Keyence, Japan).
Soft agar colony formation assay of hiPSCs, teratocarcinoma PA-1 cells and primary RPE cells. (A) hiPSCs (10000 cells, 6000cells and 3000 cells/well) were grown in soft agar for 10, 20 and 30 days with 10 µM Y-27632. (B) PA-1 cells (1000, 500, 300, 200, 100, 50, 30 cells/well) were grown in soft agar for 20 days. (C) Primary RPE cells (lot. A, 100,000, 60,000, 30,000 and 10,000 cells/well) were grown in soft agar for 30 days. (A–C) Cell growth was quantified using a CytoSelect kit and the results expressed as a relative fold change of the value of a blank well. Error bars represent the standard deviation of the measurements (n=3).
Flow cytometry analysis of spiked hiPSCs cells in primary RPE. CFDA-stained hiPSCs (1%, 2,500 cells; 0.1%, 250 cells; 0.01%, 25 cells) were spiked into primary RPE (2.5×105 cells) and 1×105 cells were analyzed by flow cytometry. The numbers indicate the quantity of cells contained in the gate.
Probes and primers for qRT-PCR.
We would like to thank Masahiro Go, Hoshimi Kanemura and Akifumi Matsuyama for their valuable discussion on this paper. We also thank Satoshi Okamoto and Noriko Sakai for technical advice of RPE cell differentiation.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by a Strategic Fund for the Promotion of Science and Technology from Japan Science and Technology Agency, and Research Grants H21-SAISEI-IPPAN-021, H23-SAISEI-IPPAN-004&005 and H23-IYAKU-IPPAN-001 from the Japanese Ministry of Health, Labour and Welfare. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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About Fully Qualified Domain Name (FQDN)
In order to let the DNS server work efficiently, all the DNS client computers must have a Fully Qualified Domain Name or FQDN. A Fully Qualified Domain Name comprises of the name of the computer a.k.a. the hostname, and the name of the domain. The hostname and the domain name are written together but are separated by a period (.). An example of a complete Fully Qualified Domain Name can be C-01.mydomain.com where C-01 is the name of the computer (hostname), and mydomain.com is the name of the domain to which the computer is added.
In case the computer is not added to the domain, or no domain exists in the network at all, the computer name is not embedded with the domain name, hence it does not become eligible to communicate with the DNS server.
When the DNS server is deployed in an organization, all the DNS compliant client computers automatically register their hostnames and their corresponding IP addresses to the DNS database. Since the DNS server becomes the repository of IP addresses and hostnames of all the client computers in the network, when any computer tries to communicate with any other computer in the network through its IP address, it sends a DNS query to the DNS server requesting for the IP address of the target computer. The DNS query is sent on the basis of the computer name of the target computer.
For example, if computer A whose IP address is 192.168.0.2 wants to communicate with the computer B whose IP address is 192.168.0.24, since the DNS server database contains the records of both the computers, the source computer (computer A) sends a DNS query to the DNS server requesting for the corresponding IP address of the computer B. The DNS server, at its part, looks for the requested computer name and its corresponding IP address in its database, and as soon as it finds the requested information, it replies back to the requesting computer (computer A) with the IP address of the computer B, which in this case is 192.168.0.24.
Once the computer A knows the IP address of the computer B, it then establishes a direct connection with the target computer and starts communicating with it.
DNS Server Types
In Microsoft's context, the DNS server is a dedicated hardware computer running any network operating system such as Windows Server 2003, Windows Server 2008, or Windows Server 2012. However, the DNS server deployment is not only limited or restricted to Microsoft platform itself. There are many other ways through which a DNS server can be deployed in the network. A simple example can be a wireless or wired router that is present in your homes. The router that you use in your home to gain access to the Internet itself works as a DNS server.
Related resources
- How long does DNS name server take to propagate to the Int.. Forum
- SolvedHow does the isp's dns work? Forum
- How to Find the Fastest DNS Server Near You and Configure It In Windows Tutorial
- Internet works only with Fixed DNS server -how to change to automatic? Forum
- SolvedDoes anyone know how well an Intel 1007u would work in a plex server for transcoding? Forum


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200 Objects of Waterloo
Explore a Waterloo story through 200 fascinating objects from across the world.
Waterloo 200 Schools Programme
The Waterloo 200 schools programme is still open, with a goal of recruiting 2000 schools by 2020. Find out more.
George Rose: A black soldier at the Battle of Waterloo
Discover the story of George Rose, a soldier from the Caribbean who served with distinction at Waterloo.
Napoleon returns - Abel Gance's 1927 epic digitally restored.
Abel Gance's 1927 epic Napoleon is digitally restored by the BFI.
Chairman's blog - Napoleon's Horse to be 'restored'
Napoleon's horse is to be restored after years in the basement of the Louvre.
Waterloo200 Descendants Book
Discover the stories of the soldiers who fought at the Battle of Waterloo.
Blood Soaked Fields: Waterloo and the First Day of the Somme compared
A temporary exhibition comparing and contrasting the two battles28/06/2016 to 11/09/2016
Nelson, Navy, Nation: The story of the Royal Navy and the British people, 1688–1815
A major new permanent gallery exploring the story of the Royal Navy.21/10/2013 to 21/10/2020
Warfare Through the Ages 2016
The history of warfare in action from Medieval to Modern day with living history encampments, drills, weaponry displays, firearms evolution and period battles.06/08/2016 to 07/08/2016


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From The Print Section :: Page 121
Snow Shoo!
Village council member Gerry Simms cleared the sidewalks along West South College Street Monday afternoon to provide safe passage for local students walking to and from school.
A lesson in black and white
Last Friday the Mills Lawn fourth, fifth and sixth grade students performed a varied program of songs, readings, poems and a skit to celebrate the end of Black History Month.
Ethics group says no conflict for Wintrow
The Ohio Board of Ethics determined last week that Council President Karen Wintrow does not have a conflict of interest regarding the Center for Business and Education, and is free to discuss and vote on CBE-related issues.
SIDEBAR: Focus on mental health in the community
This spring, the Human Relations Commission, or HRC, will launch a series of programs aimed at educating villagers on mental health issues and finding better ways to help those in the community who deal with these concerns.
Schenck incident prompts concerns— Crisis training for police supported
In recent years, area police officers have noticed a change in their work, as their calls more frequently involve people with mental health issues.
SIDEBAR: Yellow Springs Water Rate Hike
Vilage Council members agreed on a 15 percent increase in Village water rates.
Yellow Springs Utility funds show deficits
At their Feb. 3 meeting, Village Council members heard that, in the projected 2014 Village budget, most of the Village enterprise (or utility) funds — the water fund, sewer fund and solid waste funds — show deficit spending.
Feb. 27, 2014 Bulldog sports round-up
Feb. 27, 2014 Bulldog sports round-up
Sweet and sappy
Last Sunday afternoon, Feb. 22, the Tecumseh Land Trust sponsored a tour of Flying Mouse Farm for the annual tapping of the sugar maples.
Folksinger Bob Lucas to play at Mills Lawn School
Folk singer Bob Lucas will perform at 7 p.m. on Saturday, Feb. 22, in the Mills Lawn auditorium for the Mills Lawn PTO Family Fun Night, an annual event to lighten the dreary winter months.


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Buddy Powell
Published: Sunday, January 19, 2003 at 3:15 a.m.
Last Modified: Sunday, January 19, 2003 at 12:00 a.m.
STARTEX -- Ernest "Buddy" Eugene Powell, 66, of 6 Jackson Drive died Jan. 17, 2003, at Spartanburg Regional Restorative Care.
A native of Spartanburg County, he was a son of the late Charlie Lee and Nettie Sue Rogers Powell and the husband of Earline Calvert Powell.
He was a veteran of the Army, having served during the Korean War, and was retired from Calvin Klein. He was a member of Duncan United Methodist Church.
Surviving in addition to his wife are a son, Dr. Rodney K. Powell of Aiken; three brothers, Charles Edward Powell of Startex, Herman Geddis Powell of Spartanburg and Jimmy Gerald Powell of Eustis, Fla.; and two grandchildren.
Visitation will be from 1:30 to 3:30 p.m. tonight at The Wood Mortuary.
Funeral services will follow at 3:30 at the mortuary, conducted by the Revs. Elizabeth Thompson and David Ervin. Burial will follow in Wood Memorial Park.
The family is at the home.
In lieu of flowers, memorials may be made to the M.J. Hendricks Family Life Center, c/o Duncan United Methodist Church, Main St., Duncan, SC, 29334.
Reader comments posted to this article may be published in our print edition. All rights reserved. This copyrighted material may not be re-published without permission. Links are encouraged.
Comments are currently unavailable on this article


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Posted| Updated
It's been 20 years since the launch of Microsoft's Windows 95 operating system. But what was so special about the software and why all the upgrades over the years?
Topics: software, science-and-technology, computers-and-technology, personal-computers, personal-computers-desktops, personal-computers-laptops, personal-computers-netbooks
Posted| Updated
Apple says it will issue a software update "very soon" to cut off the ability of spies and hackers to grab email, financial information and other sensitive data from Mac computers.
Topics: internet-technology, computers-and-technology, security-intelligence, personal-computers, personal-computers-laptops, personal-computers-desktops, mobile-phones, united-states
Posted| Updated
It was a year dominated by drought, floods and political upheaval in Queensland.
Topics: 20th-century, aboriginal, torres-strait-islander, men, unemployment, urban-development-and-planning, work, drought, floods, educational-resources, personal-computers-desktops, brisbane-4000


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delicious | beautiful | Italian | inspiration
new subscribers receive a FREE, PRIVATE, never-before published family recipe.
Patti says
Crispy Critters this looks good!
Marina says
Roz, what a beautiful meal! Corn and peas lok so bright, and fish is excellent! Bravo!
Ciao Chow Linda says
I made perch the other night, but it was nowhere near as scrumptious as your version.
Kitchen Belleicious says
the fish just looks perfectly crusted. I love a good crust on fish and this one beats all I have seen so far. I will definitely be trying this soon!
anniebakes says
oh my mouth is watering, the Oldest will love this!! anne
Big Dude says
Wow Roz – this looks awesome and I have some crappie in the freezer just dying to be treated this way.
Kim says
Your perch looks beautifully coated and crusted in yumminess. Gorgeous!
LindyLouMac in Italy says
I know what you mean about the breadcrumbs but I had never heard them called panko. The meal looks delicious.
Dujour Whitespawn says
Delicious blog, è proprio il caso di dirlo, complimenti e congratulations! Io bloggo di cinema e benessere e di viaggi di crescita più che altro in italiano ma a volte in inglese, and now also in english about life and maybe cooking which is becoming a brand new passion and hobbie..vieni a trovarmi e unisciti alla conversazione, mi farebbe davvero piacere!
Claudia says
This is now on my list Roz. So scrumptious and such a nice change from the lemon and herbs I always do.
Lizzy says
This looks so fabulous, Roz! I'm running out of good Friday entrees for Lent…and this just moved to the top of the list. Hoping the hubby will add perch to the seafood he likes…this could do it!
Rebekah says
Oh Yum! This looks delicious! So many wonderful recipes here.
yousef says
Its Looks Yummy
I like it
Roz, this looks amazing! I love the addition of lemon pepper in the mix. Thank you so much for sharing at Mix it up Monday, I hope you'll stop back next week and share some more 🙂
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スーパーコピー時計専売店当店は海外安心と信頼のスーパーコピーブライトリング、代引き店です.正規品と同等品質のシャネル コピー代引き,品質が秀逸,値段が激安!ブライトリングコピー,代引きなどの商品や情報が満載!全商品写真は100%実物撮影です! お客様の満足度は業界No.1です!スーパーコピー時計,時計コピー ,ブランド時計コピー販売(n級品)店舗 ブランド腕時計(ロレックス,ブライトリング,タグホイヤー,オメガ,ガガミラノなど)の最新 情報やイベントを紹介する正規販売店と腕時計コピーの専門サイトです。当店はロレックスやパテックフィリップなどの新品スーパーコピー時計の販売と。 http://www.gowatchs.com/brand-154.html


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The best Cyber Monday deals
Don't Waste Your Money
If you couldn't stomach the thought of fighting the Thanksgiving night or Black Friday crowds, we have good news and bad news.
The good: You now have another chance to bargain shop on Cyber Monday. The bad: The bargains are nowhere near as good as they were Thursday night, according to the website DealNews.com.
Don't Expect Black Friday Round Two
Don't expect another chance to get that 50 inch Panasonic HDTV for $199. That was a doorbuster "loss leader," to get you in the store to shop. Stores don't need to give away products on Cyber Monday.
They know people will shop anyway.
That said, here are some of the biggest markdowns you will find:
Amazon is advertising a new deal every 10 minutes, so you may want to check in on the website frequently (just have another page ready for when your boss walks by and asks what you are doing.)
Amazon has slashed the price of its slow selling Fire phone from $649 to just $199 without a contract. (They have been selling it for $1 with two-year contract)
It may still not be enough to move many units, though, as the phone has performed well under expectations.
The Fire HD tablet is marked down from $130 to $109.
Amazon also has Leapfrog kid's tablets at 50 percent off, and $50 off the popular Nest Thermostat.
Gamestop has some of the best Cyber Monday deals, with $70 off XBox One bundles, and big markdowns on used PS3's, Nintendo Wii consoles, and DS handheld devices.
You will also find discounts on most used games.
Sears and Kmart, stores that pretty much got lost in the Black Friday hype, are rolling out a whole week of deals starting Monday.
They will have a RCA 46 inch HDTV marked down $100 to $349. Remember, this is NOT Black Friday, so don't expect the same type of TV deals.
Sears also has big markdowns on Craftsman and Dewalt tools all week, so it's a great time to buy for that DIY-er.
Like Sears, Target is making it a "Cyber Week" of deals, so it pays to check their site daily.
Target is also offering store gift cards with several TV's and electronic items, but don't expect the $140 gift card offer that came with iPads on Black Friday. That deal is long gone.
Remember that Black Friday Asus laptop for just $99 at Staples?
If your store was sold out, you can get a similar budget Asus for $149 on Cyber Monday online from Staples. Not as good a deal, but not bad.
Walmart is offering deals of "up to 50% off," mostly on clothing and small kitchen appliances, according to CNN/Money.
It also has a "pay with cash" feature for shoppers who don't have credit cards, where you can pick up the item in store and pay with cash.
Bottom line: look around: every web retailer is offering discounts, but some may not be that great. And be sure to look for free shipping wherever you buy, so you don't waste your money.
WCPO Insider
In addition to deals offered by online retailers, WCPO is offering a Cyber Monday deal for readers of our website. For $19.99, 75 percent off the normal price, new subscribers will get access to rich, local Insider content for 52 weeks. In addition, you'll receive a free digital subscription to The Washington Post.
Tap here for more info on the Insider deal.
Don't Waste Your Money is a registered trademark of the EW Scripps Co.
Like John Matarese on Twitter @JohnMatarese
Follow John on Facebook


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Newnan's live theatre—for & by the community
Zombie Makeup Workshop!
Want to get into the spirit of things for Carnival of the Dead Murder Mystery Dinner Theatre? Come learn how to do your own zombie makeup! Get tickets here.
Carnival of the Dead: Murder Mystery Dinner Theatre
When the Zombie Apocalypse hits, the safest place to be is in Newtopia, the richest and safest real estate development in the world. But is it safe? The walking dead threaten outside, but inside it's just as dangerous!
Come see if you can solve the crimes as Newnan Theatre Company presents Carnival of the Dead, the annual interactive murder mystery on Sat, Aug 13. Check-in starts at 7:15, and the show starts at 8:00.
Tickets are $30 per person and are available online or by calling the theatre at 770-683-6282. Contact artistic director Tony Daniel at firstname.lastname@example.com with any questions.
Moonlight and Magnolias
Newnan Theatre Company announces their 2106-2017 season opener, MOONLIGHT AND MAGNOLIAS, running Sep 1–4 and 8–11.
THE STORY: 1939 Hollywood is abuzz. Legendary producer David O. Selznick has shut down production of his new epic, Gone with the Wind, a film adaptation of Margaret Mitchell's novel. The screenplay, you see, just doesn't work. So what's an all-powerful movie mogul to do? While fending off the film's stars, gossip columnists and his own father-in-law, Selznick sends a car for famed screenwriter Ben Hecht and pulls formidable director Victor Fleming from the set of The Wizard of Oz. Summoning both to his office, he locks the doors, closes the shades, and on a diet of bananas and peanuts, the three men labor over five days to fashion a screenplay that will become the blueprint for one of the most successful and beloved films of all time.
Newnan Theatre Company proudly announces auditions for the musical comedy, The Addams Family: a New Musical by Marshall Brickman and Rick Elice, Music and Lyrics by Andrew Lippa. Andrew Miller will direct on NTC's Mainstage.
The Addams Family features an original story, and it's every father's nightmare. Wednesday Addams, the ultimate princess of darkness, has grown up and fallen in love with a sweet, smart young man from a respectable family – a man her parents have never met. And if that weren't upsetting enough, Wednesday confides in her father and begs him not to tell her mother. Now Gomez Addams must do something he's never done before – keep a secret from his beloved wife, Morticia. Everything will change for the whole family on the fateful night they host a dinner for Wednesday's "normal" boyfriend and his parents.
Auditions will be Sunday and Monday, August 7th and 8th at 7:30. Callbacks will be Tuesday, August 9th at 7:30. Everyone auditioning needs to prepare 32 bars of a musical theatre song NOT from the show with a track on either CD or a music-playing device (phone, mP3, iPod, etc.). A dance combination will be taught, so wear clothes that you can move in. The rest of the audition will consist of cold readings from the script.
Academy of Theatre Arts begins classes
Newnan Theatre Company's Academy of Theatre Arts offers musical theatre classes for middle school and elementary students. Classes focus on staging a performance. This performance-based class welcomes all levels of experience. Students will learn many facets of theatre, including stage directions, vocal technique, choreography, character development, set design, costuming, and more. Each student will be able to perform. The class will run for 14 weeks and will end with performances for friends, family, and the general public.
This fall the performance is Disney's The Jungle Book. Banished by the ferocious tiger, Shere Khan, a human boy named Mowgli and his panther friend, Bagheera, are on the run in the deepest parts of the jungle. On their journey, the two meet a sinister snake named Kaa, a herd of elephants and a giant bear named Baloo, who teaches them the swingin' musical rhythms of the jungle. After surviving a dangerous encounter with a band of monkeys led by King Louie, Mowgli and Bagheera are forced to run for their lives. When Shere Khan returns, our heroes must rally their fellow animals into battle and restore peace throughout the jungle.
Classes begin Mon, Aug 8 for middle school and Tue, Aug 9 for elementary. Class times are 4:30-6:30. The performance dates are Fri, Nov 4, and Sat, Nov 5. You can register online here or by calling 770-683-6282.
Auditions: The Pillowman
Newnan Theatre Company announces auditions for the drama, The Pillowman, by Martin McDonagh on Aug 28 and 29. Directed by Bert Lyons, The Pillowman will be presented on Nov 10–13 and 17–20, 2016, in the NTC Black Box Theatre.
The Pillowman was written by Irish playwright Martin McDonagh in 2003. It tells the tale of Katurian, a writer of short stories that often depict violence against children who has been arrested by two detectives, Ariel and Tupolski, because some of his stories resemble recent child murders. When he hears that his brother Michal has confessed to the murders and implicated Katurian, he resigns himself to his execution but attempts to save his stories from destruction. The play includes both narrations and reenactments of several of Katurian's stories, most notably the autobiographical "The Writer and the Writer's Brother", which tells how Katurian developed his disturbed imagination by hearing the sounds of Michal being tortured by their parents.
Auditions will be Sun, Aug 28, from 6:30–9:00pm and Mon, Aug 29, from 6:30–8:30pm in the Newnan Theatre Company Black Box Theatre. For more details, click here.
2016-17 Season tickets
2016-17 Season Tickets are on sale now!
More than just a ticket to every show...
At Newnan Theatre Company, our season tickets are a little different. When you purchase our season ticket, you actually purchase a set number of admissions. For example, our regular season ticket is good for 10 admissions - however you wish to use them. You could attend every show in our season once and use 10 admissions, or attend five shows and bring a friend to each one (5 x 2 = 10). This system offers you total flexibility in how you use your admissions, and you never feel like you have to see every show in our season lineup, just the ones that appeal to you!
Season ticket holders enjoy discounted admission, special reserved seating (with a chocolate on your seat!), and invitations to "members only" events and performances.
As an NTC season ticket holder, you also enjoy knowing you are supporting live theatre in your community. Season Tickets are a great gift for the theatre lover in your life!
REGULAR SEASON 10 Admission Ticket - $150.00
SENIOR CITIZEN (65+) OR STUDENT (with valid ID)
REGULAR SEASON 10 Admission Ticket - $110
Purchase your 2016-17 season tickets HERE!
ALREADY HAVE YOUR SEASON TICKET? Go here for a brief tutorial on how to redeem your season ticket benefits! (Remember - your season ticket for 2016-17 doesn't become valid until August 2016!)
If you have questions about the content of any of these shows, please email NTC's Artistic Director, Tony Daniel, at email@example.com.


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When will Danica Patrick win a race?
February 15, 2013
NASCAR media day was on Valentine's Day with two of the sport's highest-profile drivers, Danica Patrick and Ricky Stenhouse Jr., answering questions about their recently announced relationship. Perhaps the questions were exactly what Patrick needed to take some of the media spotlight off her continued quest to win her first race. The 30-year-old with seven Nationwide top-10s to her name will look to come out at the end of the season with her relationship intact and a first-place trophy on her mantel. Will she succeed at both?
Will it last?
It's not uncommon for two athletes in the same sport to be dating, but competing against each other in the same event is.


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This a was a great trip!!! I went there with my mom and dad.We had the all exclusive package which made it great and included the best activities. It also got us the best food from there own restaurants!!!
- Official Description (provided by the hotel):
- With its exclusive ambiance and AAA four Diamond rating, Villa La Estancia in Riviera Nayarit is redefining luxury and sophistication. Nestled on the shores of Banderas Bay in between the popular destinations of Puerto Vallarta and Riviera Nayarit, the stunning beachfront property is an idyllic hideaway for indulging in the glory of Mexico. Its 250 Hacienda-style suites, stunning property, world-class Tatewari Spa and gourmet restaurants offer guests the ultimate beachside escape, where relaxation and indulgence are inevitable. Welcome to Villa La Estancia, welcome to magnificence. ... more less
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- TripAdvisor is proud to partner with BookIt.com, Booking.com, Hotels.com, Hotwire, Expedia, Orbitz, Priceline, Travelocity, Odigeo, Despegar.com, Cheap Tickets and Jetsetter so you can book your Villa La Estancia reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
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text: Curling Video: Howard, Koe, Gushue the ones to watch at Brier Add to ... CP Video Published Saturday, Mar. 03, 2012 8:45PM EST Last updated Saturday, Mar. 03, 2012 8:45PM EST Ontario's Glenn Howard, Alberta's Kevin Koe and Newfoundland's Brad Gushue helm the teams to watch at this year's Canadian men's curling championship, which began Saturday. Howard agrees that expectations for his team are high. Watch More Video: Video: Meet some of the athletes on the Refugee Olympic Team New Blue Jays outfielder 'complements' team: GM Canadian teen says defeating Kyrgios at Rogers Cup was a 'miracle' Don Cherry, Ron MacLean make mark on Walk of Fame A quick look at Rio flagbearer Rosie MacLennan Usain Bolt says 'good to send strong message' against Russian doping Canada sending biggest beach volleyball team yet to Rio Sidney Crosby shows off Stanley Cup to hometown fans Sidney Crosby senses 'excitement' in hometown over Cup arrival James Hinchcliffe looks for season's first victory at Indy Toronto 'Rogue Tenor' Romigio Pereira breaks his silence 'Lone wolf' member of the Tenors inserts 'All Lives Matter' into O Canada China seeks private approach to gymnastics Golfer Alena Sharp on joining Brooke Henderson at Rio Games Fans celebrate arrival of team Portugal and Euro trophy 'Wait till next year': Fans react to Raonic's Wimbledon loss Faces of battle: A look at the contorted faces of tennis titans Raonic and Federer Maple Leafs legends on hand for centennial season launch Track and field stars inspiring each other at trials for Rio Olympics Auston Matthews on working with skating coach Barb Underhill Comments AA Follow us on Twitter: @Globe_Sports


text:
|Publication number
|Publication type
|Publication date
|Mar 16, 1971
|Filing date
|Dec 26, 1968
|Priority date
|Dec 26, 1968
|Publication number
|Doremus R Ellsworth, Doremus Richard E
|Original Assignee
|Golden Gate Mfg Co
|Export Citation
|BiBTeX, EndNote, RefMan
|Referenced by (3), Classifications (11)
|External Links: USPTO, USPTO Assignment, Espacenet
March 16, 1971 R E DOREMUS ET AL 3,570,091
METHOD FOR CONVERTING VALVES 2 Sheets-Sheet 1 Filed Dec. 26. 196E Min -k I u. n w
INVENTORS March 16 1971 R. E. DOREMUS ETAL Filed Des. 26. 1968 METHOD FOR CONVERTING VALVES 2 Sheets-Sheet 2 INVENTORS B. ELLSWORTH DOREMUS RICHARD E. DO EMUS ATTORNEYS United States Patent 3,570,091 METHOD FOR CONVERTING VALVES Richard E. Doremus, Washington Valley, and R Ellsworth Doremus, Clifton, N.J., assignors to Golden Gate Manufacturing Company, Delawanna, NJ. Continuation-impart of application Ser. No. 669,111, Sept. 20, 1967. This application Dec. 26, 1968, Ser. No. 787,133
Int. Cl. B21d 53/00; B211; 29/00; B22g 15/26 U.S. Cl. 29--157.1 Claims ABSTRACT OF THE DISCLOSURE A method and apparatus for converting a valve used in tapping fluid from kegs from one having a tapping port on its end wall to one having an extending member with a tapping port therein and a washout passage in the end wall by attaching an insert piece with the washout passage therein and a valve member having an extending portion to the body of the original valve.
This application is a continuation-in-part of our copending application Ser. No. 669,111, filed Sept. 20, 1967, entitled Method and Apparatus for Converting Valves, which is assigned to the same assignee.
In our prior Pat. No. 2,992,654, which is assigned to the same assignee, a valve is disclosed which has particular utility in tapping fluids, such as beer, from a keg. The valve of that patent has a single tapping port in its end wall which is opened and closed by a rotatable, disc type, valve member. It is usually located at the bottom of the keg and the fluid is withdrawn through it by pressurizing the keg, usually through another valve of the same general type.
There have recently come into greater use valves which are capable of drawing more beverage from the keg. These valves have a valve member with a portion which extends beyond the confines of the body and has a tapping passage therein. Since the extending portion protrudes further down into the keg, more of the beverage can be withdrawn. As is known, valves with such extending portions are also usually provided with an arrangement for washing out the passage of the extending portion when the valve is closed. Several forms of washout configurations are disclosed in the prior Pat. No. 3,115,149 to Tonna et al. and in our co-pending applications 'Ser. Nos. 545,364 and 603,519, these applications being assigned to the same assignee.
Valves with extending portions cost substantially more to manufacture than a prior art valve of the type shown in our aforesaid patent where no such extending portion is used. A large part of the cost, however, is not in making the valve member with the extending portion but in forming the body and providing it with a washout arrangement. Because of the cost factor it would of course be desirable, if it is more economical, to convert valves Without extending portions into valves having such extending portions. The present invention is directed to a method and apparatus for accomplishing this.
In accordance with our invention an insert piece having a washout passage is attached to the top of the original valve body and a valve member having an extending portion is fastened for rotation thereto.
The insert pieces are of a configuration to provide the necessary functions of tapping the fluid and valve washout and they can be attached to the body of the original valve in one of several ways. In one way, the end wall of the original valve body is cut oif and the insert piece is attached by a metal joining process to the side wall of the body. In another way the end wall of the body is left on the valve, although a portion thereof may be cut or ground ice oif, and the insert piece is attached directly to the end wall. The latter can be accomplished either by welding or some other suitable metal joining technique or by the use of an adhesive.
It is therefore an object of the present invention to provide a method and apparatus for converting valves to have an extending portion and a washout passage.
A further object is to provide a method for converting valves having a tapping port on the end wall of the body to a valve having an extending portion, which is used for the tapping port, and a washout passage in the body.
An additional object is to provide an insert piece having a tapping port and washout passage which is used for converting valves.
Other objects and advantages of the present invention will become more apparent upon reference to the following specification and annexed drawings in which:
FIG. 1 is an exploded perspective view of a converted valve;
FIG. 2 is an elevational view taken in section of an assembled converted valve;
FIG. 3 is a sectional view of a portion of the prior art valve which has been cut away to receive the insert member;
FIGS. 4 and 5 are cross-sectional views of the valve of FIG. 2 taken along lines 44 and 55, respectively;
FIG. '6 is a perspective view of a portion of a prior art valve to be converted;
FIG. 7 is a side elevational exploded view showing another type of insert piece and method for converting the valve; and
FIG. 8 is a top cross-sectional view of the valve of FIG. 7 taken in cross-section through a position the same as lines 44 of FIG. 2.
Referring first to FIG. 6, a prior art valve 1 having a body 10 is shown. The body 1 has a flange 12 which fits within a fixture on the keg (not shown) with which the valve is to be used. The valve 1 has a valve member 14 fastened to the circular upper wall which has a port 15. A sealing disc 16 is located between the valve member 14 and the end wall of the valve body. This sealing disc may be of Teflon and it is secured to the valve member for rotation therewith, preferably in accordance with our aforesaid patent. As shown, the valve 1 has only a single tapping port 18 which lies within the confines of the valve body. Thus, only fluid above the level of the port 18 in the keg can be extracted. Port 18 is opened and closed as the valve member 14 is rotated by a suitable tapping device which is inserted within the valve body from the outside of the keg.
In accordance with the subject invention the original, prior art valve 1 of FIG. 6 is to be converted into a valve having a valve member with an extended tapping portion, and a passage for washing out this extending portion when the valve is closed. One such type of valve is shown in FIGS. 1 and 2 wherein the valve member is designated as 40, the extended tapping portion of the valve member as 44 and the washout passage as 30.
One method for converting the prior art valve 1 of FIG. 1 is illustrated in FIGS. 2L5. Here, as a first step, the end wall of the valve 1 in which the port 18- is formed is removed at points 20-20 leaving only the valve side wall and exposing the interior of the body, as shown in FIG. 3. This is accomplished by any suitable cutting member such as a saw, grinding wheel, etc.
After the end wall is removed an insert piece 24 is attached to the side wall at the open end of the body 10 at the point 20 such as by welding, soldering, etc. This is shown in greater detail in FIG. 2 where the welding or solder fillet is indicated at 26. The insert piece 24 is of generally circular shape with a diameter approximately equal to that of the body at point 20 and has a central opening 27 through which passes a headed bolt 29. Bolt 29 has a pair of opposing flats 29a.
As shown in FIGS. 2-4 the insert piece 24 has an arcuate washout passage or slot 30 formed therein between its top and bottom walls and a drain hole 32 in the top wall which communicates with slot 30. An elliptically shaped tapping port 34 is also formed through the insert piece 24 and, as shown in FIG. 4, a depressed track 36 is provided around a portion of its periphery. The top wall of the insert piece is otherwise smooth to provide a bearing surface for the sealing disc of the valve member. The purpose of the track 36 is described in greater detail below. Insert piece 24 can be made by any suitable process, such as by casting it with the various passages 27, and 34 and track 36. Alternatively, the piece can be cast with passages 27 and 34 and track 36 and the washout passage 30 cut or milled into it. This can be done before or after the insert piece is attached to the body.
To complete the conversion a valve member 40 is provided which has an extending tapping portion 42 with a passage 44 therethrough which communicates with a right angle outlet 46. A sealing disc 50, preferably of Teflon, is also provided which has ribs 52 thereon which interlock with corresponding grooves 41 in the bottom wall of the valve member 40. This interlocking arrangement is described in detail in our aforesaid patent.
The valve member 40 and the disc 50 are positioned so that an outlet port 56 on the disc is aligned with the outlet passage 46 of the valve member. The threaded end 62 of bolt 29 passes through the aperture 27 of the insert piece and central apertures and 55 in the valve member 40 and sealing disc 50. A washer 63 is positioned between the head of the bolt 29 and a boss 28 the inner face of the insert piece 24. A packing washer 65 is provided between the sealing disc and the valve member 40, this washer 6S resting in an annular depression 47 in the inner face of the wall of the valve member and a similar depression (not shown) in the sealing disc. The assembly is completed by a packing washer 66a which fits within a raised boss 66 on the outer face of the valve member, two washers '67 and 68 and a pair of nuts 69 and 70. The nuts 69 and 70 are tightened down to secure the desired pressure between the valve member 40 and the end wall 25.
A downwardly extending post 43, which rides in the track 36 of the valve body insert piece 24, is also provided on the valve member 40. The sealing disc 50 has a peripheral cutout portion 57 to accommodate the post 43, this cutout portion 57 corresponding to a similar peripheral cutout 48 on the valve member. The sealing disc 50 rotates with the valve member 40.
In operation, with the valve assembled as shown in FIG. 2, a tapping faucet is moved into the valve body to engage the head of the bolt 29. When the valve member 40 is rotated by the bolt 29 to the open position shown in FIG. 2 the outlet passage 46 of the valve member, and the corresponding passage 56 of the sealing disc which is aligned therewith, are brought into alignment with the tapping port 34 of the insert piece 24. When the valve member is rotated by about 90 to the closed position outlet passage 46 of the extending portion 42 is brought into alignment with the drain hole 32 so that any cleaning fluid pumped into the keg enters the passage 44 and drains out through the hole 32 and the washout passage 30'.
It should be apparent from considering FIG. 4 that the post 43 on the valve member and track 36 on the insert piece cooperate to prevent the valve member 40 from being assembled to the body in an improper position. That is, the valve member 40 cannot be installed 180 turned from the position shown. Therefore, the outlet passage 46 is always aligned with the outlet port 34 when the valve is open. The post and track also limit the turning of the valve member to approximately 90, that is, the arc of the slot.
When the valve is open the peripheral cutout portions 48 and 57 of the valve member and disc lie on the top wall 25 of the insert piece out to the inner edge of the track 36. Since there is no overhang above the edge of the track, the seat material is not scored or skived when the valve member is rotated.
FIGS. 7 and 8 show another embodiment of the invention. Here, the end wall of the valve body is not completely cut off as in FIGS. 2-5. Instead all or a portion thereof is left on the body leaving the original valve port 18 and the central opening for the bolt 29. It should be understood that in some cases a portion of the end wall of the valve body will have to be ground or cut away to satisfy a particular overall height requirement for the completed valve.
The insert piece 84 to be used with the conversion method described with respect to FIGS. 7-8 is of somewhat simpler construction than the insert piece 24. Since the remaining portion of the end wall of the original valve can be utilized as a seating surface, the arcuate slot for the washout can be formed directly in the bottom wall of the insert piece. This means that it is less expensive to make and can be produced, for example, by an investment casting process. As with the insert piece 24, a tapping port 34 and a central opening are formed in the insert piece 84.
The insert piece 84 can be fastened to the end wall of the body in one of several ways. First of all it can be attached by any suitable metal joining process, such as welding or soldering, on the outside of the valve body as shown, for example, at the fillet 26 on FIG. 2. The insert piece 84 also can be fastened to the end wall of the valve body by a suitable adhesive, such as an epoxy cement, which is placed on the end wall of the body and the lower facing surface of insert piece 84. Where an epoxy cement is used, the two pieces forming the converted valve body are clamped together and put into a drying unit to accelerate the curing and setting of the epoxy.
In utilizing the method of FIGS. 7-8, the mating surfaces of the valve end wall and the insert piece 84 must be finished to provide parallelism and a good fit. In addition, the port 34 of the insert piece must be properly aligned with the original port 18 in the valve body end wall. To facilitate this alignment a locating and holding pin 92 can be formed on the lower face of the insert piece and a complementary hole 94 formed in the end wall of the valve body. Where an adhesive is used, the mating pin and hole also provide a firmer grip by having adhesive placed therebetween.
The valve member 40 and seat used with the converted valve of FIGS. 7-8 is the same as that used for the valve of FIGS. 2-5.
It should be apparent that the present invention provides an economical way of converting valves without an extending portion on the valve member to one having such an extending portion and a washout passage. All that is required are the operations of cutting off the end of the old valve member, fastening the insert piece thereto and then attaching the valve member. It has been found that this can be done at a cost which is less than the cost of making a complete new valve.
It should be understood that other types of washout arrangements can be used, for example, those shown in the aforesaid Tonna Pat. 3,115,149 and in Pat. 3,279,493 to Zurit et a1. It is only necessary to modify the insert piece and valve member accordingly to obtain the same washout configuration and/ or valve seat holding arrangement. In the case of both of these patents, and also with the embodiment described herein, it is not always necessary to remove the entire end wall of the wall. Instead, only a portion can be sliced away leaving a reduced height section to which is attached the insert piece.
While preferred embodiments of the invention have been described above it will be understood that these embodiment are illustrative only and the invention is to be limited solely by the appended claims.
What we claim is:
1. A method of converting a valve having a hollow metal body with an end wall and only one fluid flow port therein which port communicates with the hollow interior of the valve body, comprising the steps of forming a metal insert piece having the same general overall peripheral shape as the portion of the valve body adjacent said end wall and also forming said insert piece with a fluid flow port which extends through said insert piece and both of its faces and a washout passage which communicates with the upper face of the insert piece, fixedly fastening said insert piece to said valve body with the lower face of the insert piece facing the valve body and the port of said insert piece in fluid flow communication with the hollow interior of the valve body, and attaching a rotatable valve member with one face opposing the upper face of the insert piece and having an extending portion with an outlet which communicates with an inlet on said one face of the valve member so that when said valve member is rotated to a first position where its inlet is in communication with the port of the insert piece the valve is open and when the inlet is rotated to a second position where it is in communication with the washout passage the valve is closed.
2. The method of claim 1 further comprising the step of cutting away the end wall of the body to expose its hollow interior and before fastening the insert piece to the body.
3. The method of claim 1 wherein at least a portion of the end wall of the body is left further comprising the step of aligning the port of the insert piece with the port of the end wall and fastening said insert piece to said end wall.
4. The method of claim 3 further comprising the step of forming mating aligning means on the opposing faces of said insert piece and said end wall for aligning the ports of the end wall and the insert piece.
5. The method of claim 3 wherein the fastening is accomplished by providing adhesive between the opposing faces of the insert piece and the end wall.
6. The method of claim 4 wherein the fastening is accomplished by providing adhesive between the opposing faces of the insert piece and the end wall.
7. The method of claim 1 wherein the washout passage area is formed as a slot between the upper and lower faces of the insert piece and further comprising the step of providing a communicating hole between the upper face of the insert piece and said slot.
8. The method of claim 1, wherein the washout passage area is formed on the lower face of the insert piece which faces the end wall of the valve body and further comprising the step of providing a communicating hole between the upper face of the insert piece and the washout passage area.
9. The method of claim 3 wherein the washout passage area is formed on the lower face of the insert piece which faces the end wall of the valve body and further comprising the step of providing a communicating hole between the upper face of the insert piece and the washout passage area.
10. The method of claim 9 further comprising the step of forming mating aligning means on the opposing faces of said insert piece and said end wall for aligning the ports of the end wall and the insert piece.
References Cited UNITED STATES PATENTS 2,992,654 7/1961 Doremus et al. 137323 3,115,149 12/1963 Tonna et al. 1317-323X 3,279,493 10/1966 Zurit et al 137-323 3,411,525 11/1968 Auger 137-625,46X 3,431,936 3/1969 Doremus et al. 137-323 3,476,138 11/1969 Doremus et al. 25 1-368X JOHN F. CAMPBELL, Primary Examiner V. A. DI PALMA, Assistant Examiner US. Cl. X.R. 137-323
|Citing Patent
|Filing date
|Publication date
|Nov 17, 1989
|Jan 1, 1991
|Masco Corporation Of Indiana
|Temperature and volume control valve assembly
|Sep 13, 2011
|Global Vintners Inc.
|Wine fermentation vessel with lees containment apparatus
|Dec 17, 2007
|Jun 19, 2008
|Poissant Philip L
|Wine fermentation vessel with lees containment apparatus
|U.S. Classification
|29/890.121, 137/323, 29/401.1
|International Classification
|Cooperative Classification
|European Classification


text:
BULVERDE, TX—Presidian Hotels & Resorts held a groundbreaking ceremony on July 26 for a new Hampton Inn by Hilton hotel and Bulverde Visitors & Convention Center in the Singing Hills development here.
Wednesday July 27th, 2016 - 10:19AM
NEW YORK—W Hotels Worldwide, part of Starwood Hotels & Resorts Worldwide, Inc., has completed its $18-million renovation of W Seattle.
Wednesday July 27th, 2016 - 9:48AM
BARCELONA, SPAIN—Room Mate Hotels launched two new boutique properties in Barcelona, bringing the collection's presence in Catalonia to four hotels. The hotels are Room Mate Anna, a new flagship in Passeig de Grácia and Room Mate Carla, located on Calle Mallorca.
Tuesday July 26th, 2016 - 12:35PM
BELLEVUE, NE—Courtyard by Marriott Omaha South–Bellevue at Event Center is scheduled to open its doors here on July 27. Located at 3730 Raynor Parkway, the 121-room hotel will operate as a Marriott franchise, owned and managed by Kinseth Hospitality.
Monday July 25th, 2016 - 9:19AM
CHICAGO—Waldorf Astoria Chicago has added 7,914 sq. ft. of meeting and event space on the hotel's third floor. Included in the renovation are four new venues: Hawthorne, Alcott, and Thoreau meeting rooms, along with a private dining room. The new meeting and event spaces complement the pre-existing 4,448 sq. ft. of meeting space on the hotel's fifth floor.
Friday July 22nd, 2016 - 11:12AM
PLAINFIELD, IL—Cicero's Development Corp., a general contractor specializing in the hotel and hospitality sector, has completed a property-wide refresh of the Fairfield Inn & Suites hotel in Melbourne, FL for its client ONE Hospitality Group.
Friday July 22nd, 2016 - 11:03AM
BETHESDA, MD—Marriott International, Inc., celebrated the groundbreaking of its first-ever hotel to contain three of its brands in a single building. The landmark project will rise in the SoBro neighborhood in Nashville, TN. The 470-room, $137-million hotel is expected to open mid-year in 2018.
Friday July 15th, 2016 - 10:19AM
OTTAWA, ON—What is the life cycle of a used bar of hotel soap? This frequently discarded item could end up in a landfill, or it could go on a life-saving mission to underserved areas around the world. The unlikely hero is playing a starring role in an initiative that aims to change lives by providing education to people at risk for hygiene-related illnesses.
Friday July 15th, 2016 - 8:25AM
PORTLAND, OR—Provenance Hotels has hired Ashley Stempler as director of interior design for its portfolio of hotels.
Wednesday July 13th, 2016 - 10:10AM
MIAMI—Meliá Hotels International has opened its first U.S. destination with ME by Meliá brand in Downtown Miami. The hotel has 129 luxury-appointed rooms and more than 1,000 sq. ft. of meeting space. It also has a 14th floor sun deck featuring open air sunset and sunrise pools and Bella's Bar, a poolside bar and lounge set to open later this season.
Wednesday July 13th, 2016 - 9:49AM
HONOLULU, HI—Pacific Beach Hotel plans a $115-million redevelopment that will transform the Waikiki hotel.
Wednesday July 13th, 2016 - 8:54AM
NEW YORK—Alfa Development will open its first boutique hotel project, HGU New York, following an $18 million renovation within a 111-year-old building.
Tuesday July 12th, 2016 - 9:34AM
PLATTSBURGH, NY—The 91-room Fairfield Inn & Suites by Marriott is scheduled to open here on July 14. Located at 579 State Route 3, the Fairfield Inn & Suites Plattsburgh will operate as a Marriott franchise, owned and managed by Ambema Corporation.
Tuesday July 12th, 2016 - 9:14AM
The 109-room Fairfield Inn & Suites by Marriott opened here Friday, July 8. Located at 7718 Front Beach Road, the Fairfield Inn & Suites Panama City Beach will operate as a Marriott franchise, owned and managed by BN Management Company of Harvey, Louisiana. This is the first Fairfield Inn & Suites in the area to feature the brand's new design and decor.
Monday July 11th, 2016 - 9:06AM
LOS ANGELES—The Sheraton Gateway Los Angeles Hotel has completed the second phase of their multimillion-dollar renovations led by design firm Gensler, with upgrades to their 802 guestrooms and suites as well as the hotel lobby area and Sheraton Club.
Friday July 8th, 2016 - 11:09AM
LAKE MARY, FL—Peachtree Hotel Group has completed a $3.5-million renovation of the 130-room Hampton Inn and Suites Lake Mary Florida here.
Thursday July 7th, 2016 - 2:51PM
WASHINGTON—The Hotel del Coronado in Coronado, CA, has achieved the Silver level of Green Seal's Standard for Hotels and Lodging Properties (GS-33).
Thursday July 7th, 2016 - 10:02AM
IRVINE, CA—Hotel Hermosa reopened after 11 months of extensive renovations. The refurbished hotel is located in Los Angeles' South Bay at 2515 Pacific Coast Hwy. The property has 81 guestrooms and loft suites.
Thursday June 30th, 2016 - 9:49AM
SINGAPORE—M Social Singapore, a 293-room hotel situated along the Singapore River, has opened.
Monday June 27th, 2016 - 9:37AM
BOSTON—Boston Park Plaza has completed a comprehensive, multi-year, $100-million renovation and restoration project. The extensive transformation includes the complete overhaul of the hotel's 1,060 guestrooms and corridors, main entrances and lobby, meeting spaces, mezzanine levels, the addition of a library room and the replacement of all furniture and fixtures.
Wednesday June 22nd, 2016 - 10:33AM
MCLEAN, VA—Hilton Garden Inn has opened its second property in Hawaii, the Hilton Garden Inn Waikiki Beach. It is the brand's first property in the Waikiki area of Honolulu, its 700th worldwide and its largest property of all time. The newly redeveloped Hilton Garden Inn Waikiki Beach, managed by Evolution Hospitality, brings 623 new rooms to the brand.
Tuesday June 21st, 2016 - 9:07AM
PARIS—AccorHotels has expanded the MGallery by Sofitel brand with the opening of Hotel Paris Bastille Boutet here in the capital's 11th district near Le Marais, as well as Hotel Santa Teresa in Rio de Janeiro.
Monday June 20th, 2016 - 11:46AM
DANA POINT, CA—Monarch Beach Resort has completed a comprehensive, yearlong $40-million renovation. Following the recently refreshed 400 guestrooms, the redesign features a new Miraval Life in Balance Spa by Miraval Group; a transformed culinary program and five new dining concepts; an expansive new pool area complete with private cabanas and a new arrival experience.
Friday June 17th, 2016 - 9:14AM
MCLEAN, VA—Hilton Worldwide has signed the new Resort at Mahogany Bay Village to Curio – A Collection by Hilton in San Pedro, Belize.
Thursday June 16th, 2016 - 9:45AM
SAO PAULO—Hilton São Paulo Morumbi revealed a multimillion-dollar dollar renovation featuring a new restaurant, Armazem Morumbi, renewed coffee shop concept at Caffè Cino and an updated lobby.
Thursday June 16th, 2016 - 9:09AM
BOSTON—Royal Sonesta Boston unveiled 28,000 sq. ft. of newly renovated meeting and event space featuring contemporary furnishings and views of the Charles River and Boston skyline.
Wednesday June 15th, 2016 - 9:52AM
DALLAS—Rockbridge has revealed details of The Adolphus Hotel's final phase of a comprehensive renovation, which was conceived in collaboration with SWOON, a Dallas-based design firm. The renovation includes a transformed lobby, public spaces, new restaurant concepts and retail options.
Wednesday June 8th, 2016 - 9:18AM
OAKLAND, CA—For many guests, they don't realize what they had until it's gone—and that is peace and quiet. For hotels with noisy guestrooms, there is the inherent risk of bad online reviews and fewer bookings. Learn what you can do to cut the racket, restore your property's reputation and ensure a peaceful night's sleep.
Tuesday June 7th, 2016 - 9:28AM
ORLANDO, FL—Margaritaville Resort Orlando has unveiled the first rendering of the new property here, which is being developed by Encore Capital Management.
Tuesday June 7th, 2016 - 8:21AM
NEWPORT BEACH, CA—Last week, sister publication Hotel Business Design and Gensler hosted a Hospitality Talk focused on mega-trends in the industry: technology, Millennials, the sharing economy and community building. Sponsored by Evolve, Global Allies, Littman Brands Contract, Grohe and Daltile, the session was moderated by Nicole Carlino, senior associate editor.
Wednesday June 1st, 2016 - 11:48AM
LIMA, PERU—The Westin Lima Hotel & Convention Center has received the Existing Buildings: Operations & Maintenance (EBOM) LEED Certificate awarded by the U.S. Green Building Council to existing buildings, which have eco-friendly operation and maintenance practices.
Tuesday May 31st, 2016 - 2:08PM
CANTON, OH—Hall of Fame Village LLC, led by Industrial Realty Group (IRG), has revealed the design, created by Dallas-based HKS Architects, for a four-star upscale hotel that will be a key component of Hall of Fame Village, a nearly $500-million development project of the Pro Football Hall of Fame's campus here.
Friday May 27th, 2016 - 10:04AM
BONN, GERMANY—The Marriott Bonn World Conference Hotel is scheduled to open here at the end of the month. With 17 floors, 336 rooms including 30 suites, two presidential suites and an array of modern facilities, the hotel is one of the largest in Germany, according to the company.
Thursday May 19th, 2016 - 9:41AM
NEW YORK—The InterContinental New York Barclay has reopened following an extensive $180 million, 20-month top-to-bottom renovation with more than 420,000-sq.-ft. of redesigned interiors.
Tuesday May 17th, 2016 - 10:52AM
LONG ISLAND CITY, NY—The Courtyard by Marriott Long Island City/New York Manhattan View has opened here, featuring a new lobby space as well as the brand's latest contemporary room design.
Tuesday May 17th, 2016 - 9:03AM
LITTLETON, MA—The Courtyard by Marriott Boston Littleton has opened here, featuring a new lobby space as well as Courtyard's latest contemporary room design.
Monday May 16th, 2016 - 9:03AM
PARSIPPANY, NJ—While notable, high-end art fairs roll into New York City this weekend, Super 8 Hotels hosted the first-ever exhibit enticing gallery-goers to snatch a piece of art—for free, according to the company.
Thursday May 5th, 2016 - 10:11AM
KUALA LUMPUR, MALAYSIA—St. Regis Hotels & Resorts, part of Starwood Hotels & Resorts Worldwide Inc., has opened its 38th hotel here. Owned and developed by ONE IFC Sdn Bhd, The St. Regis Kuala Lumpur is part of a mixed-use development in the Kuala Lumpur Sentral Precinct (KL Sentral).
Tuesday May 3rd, 2016 - 9:47AM
ATLANTA—Winter Construction will begin mobilizing this month to build a new seven-story boutique hotel in Savannah, GA. The hotel is being developed by RBHD and owned by Rockbridge, and will span an entire city block in the historic River Street district.
Friday April 29th, 2016 - 10:11AM
NEW YORK—The Bernic, a 96-room, 21-story new-build hotel, is slated to open in July at 145 E. 47th St. here, with a new restaurant and sky lounge in tow.
Tuesday April 26th, 2016 - 10:54AM
VERNON HILLS, IL—American Hotel Register Company has opened a new showroom inside its world headquarters here.
Monday April 25th, 2016 - 9:50AM
MIAMI—The former U.S. Immigration Naturalization Service (INS) building here will be redesigned by architecture and design firm Stantec and converted to a 139-key Hilton Garden Inn.
Wednesday April 20th, 2016 - 9:42AM
EAST ELMHURST, NY—LaGuardia Plaza Hotel (LPH), an independent hotel located in the New York City borough of Queens, has joined the NYC Carbon Challenge for Hotels.
Friday April 8th, 2016 - 9:48AM
LONDON—Generator Hostels has opened its newest property in Amsterdam.
Thursday April 7th, 2016 - 9:31AM
LOS ANGELES—L.E. Hotels has added four boutique properties to its portfolio: Hotel Los Gatos in Los Gatos, CA; Grand Hotel Imperiale Resort in Moltrasio, Lake Como, Italy; Hotel Beta Porto in Porto, Portugal; and Hotel Principe Real in Lisbon, Portugal.
Friday March 25th, 2016 - 10:13AM
QUEBEC CITY, CANADA—Tribute Portfolio, part of Starwood Hotels & Resorts Worldwide, Inc., has debuted in Canada with the signing of Hotel PUR Quebec, a Tribute Portfolio Hotel, owned by Hotel PUR Quebec LP and managed by Crescent Hotels and Resorts Canada Company.
Thursday March 24th, 2016 - 9:39AM
MCLEAN, VA—Following a multimillion-dollar renovation, Redmont Hotel Birmingham has joined Curio - A Collection by Hilton as the brand's second hotel in Alabama after the recent opening of The Admiral Hotel Mobile.
Tuesday March 8th, 2016 - 10:14AM
MCLEAN, VA—Hilton Worldwide has unveiled plans to bring Canopy by Hilton to the U.K. with Canopy by Hilton London City. Following a franchise agreement with 4C Hotel Group, the new hotel, located in Aldgate, will open in early 2018.
Monday March 7th, 2016 - 10:22AM
SAN FRANCISCO—Viceroy Hotel Group's newest property Hotel Zeppelin San Francisco has opened here. The 196-room hotel is located two blocks from Union Square and within walking distance to FiDi.
Monday March 7th, 2016 - 9:26AM
MEMPHIS—The Guest House at Graceland, located near Elvis Presley's Graceland mansion here, is nearly eight months away from its grand opening. Dignitaries, designers and construction teams associated with the hotel held a topping off ceremony at the hotel construction site to put the final beam in place.
Friday March 4th, 2016 - 9:35AM
It's Business as Usual: Occupancy, ADR, RevPAR Hold Steady
Well, it's halfway through 2016, and not much has changed since the beginning of the year. Despite the terrorism acts in Orlando, FL and Turkey, and the Brexit referendum, little has happened to affect the travel industry. Overall, things might have slowed a bit, but a majority of the fundamentals are holding steady, including occupancy, ADR and RevPAR. I've been asked why this cycle has lasted longer than those preceding it. As far as I am concerned, it's due to a single phrase: Never Again.
Well, it's halfway through 2016, and not much has changed since the beginning of the year. Despite the terrorism acts in Orlando, FL and Turkey, and the Brexit referendum, little has happened to affect the travel industry. Overall, things might have slowed a bit, but a majority of the fundamentals are holding steady, including occupancy, ADR and RevPAR. I've been asked why this cycle has lasted longer than those preceding it. As far as I am concerned, it's due to a single phrase: Never Again.
Click Here to read the full article.
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Of Course Dzhokhar Tsarnaev Is Not an Enemy CombatantJohn McCain and Lindsey Graham's ludicrous, harmful campaign to get him declared one.
The Spectator
"The Insane Root That Takes the Reason Prisoner"Macbeth, Boston, and the two paradoxes of evil.
We Are All Russians NowEven American neocons were for Chechens before they were against them.
Hang Up And Listen
Hang Up and Listen: The Cross Between Akili Smith and Aaron Brooks EditionSlate's sports podcast on the aftermath of the Boston Marathon bombing, the NBA playoffs, and stereotyping black quarterback prospects.
What If Dzhokhar Tsarnaev Decides Not to Talk?The police should be able to use all civilized means to make him—as the Fifth Amendment should allow.