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Editorial Academy
Zoledronic acid suppresses transforming growth factor-β-induced fibrogenesis by human gingival fibroblasts
Yuko Komatsu
Miho Ibi
Naoyuki Chosa
Seiko Kyakumoto
Masaharu Kamo
Toshiyuki Shibata
Yoshiki Sugiyama
Akira Ishisaki
Affiliations: Division of Cellular Biosignal Sciences, Department of Biochemistry, Iwate Medical University, Iwate 028‑3694, Japan, Department of Oral and Maxillofacial Surgery, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan, Division of Oral and Maxillofacial Surgery, Department of Reconstructive Oral and Maxillofacial Surgery, Iwate Medical University, Iwate 020-8505, Japan
Published online on: May 10, 2016 https://doi.org/10.3892/ijmm.2016.2582
Copyright: © Komatsu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Bisphosphonates (BPs) are analogues of pyrophosphate that are known to prevent bone resorption by inhibiting osteoclast activity. Nitrogen-containing BPs, such as zoledronic acid (ZA), are widely used in the treatment of osteoporosis and bone metastasis. However, despite having benefits, ZA has been reported to induce BP-related osteonecrosis of the jaw (BRONJ) in cancer patients. The molecular pathological mechanisms responsible for the development of BRONJ, including necrotic bone exposure after tooth extraction, remain to be elucidated. In this study, we examined the effects of ZA on the transforming growth factor-β (TGF‑β)-induced myofibroblast (MF) differentiation of human gingival fibroblasts (hGFs) and the migratory activity of hGFs, which are important for wound closure by fibrous tissue formation. The ZA maximum concentration in serum (Cmax) was found to be approximately 1.47 µM, which clinically, is found after the intravenous administration of 4 mg ZA, and ZA at this dose is considered appropriate for the treatment of cancer bone metastasis or bone diseases, such as Erdheim-Chester disease. At Cmax, ZA significantly suppressed i) the TGF‑β-induced promotion of cell viability, ii) the TGF‑β-induced expression of MF markers such as α-smooth muscle actin (α-SMA) and type I collagen, iii) the TGF‑β-induced migratory activity of hGFs and iv) the expression level of TGF‑β type I receptor on the surfaces of hGFs, as well as the TGF‑β-induced phosphorylation of Smad2/3. Thus, ZA suppresses TGF‑β-induced fibrous tissue formation by hGFs, possibly through the inhibition of Smad‑dependent signal transduction. Our findings partly elucidate the molecular mechanisms underlying BRONJ and may prove to be beneficial to the identification of drug targets for the treatment of this symptom at the molecular level.
Bisphosphonates (BPs) are analogues of pyrophosphate that are known to prevent bone resorption by inhibiting osteoclast activity (reviewed in ref. 1). BPs are used for the treatment of various bone diseases, including osteoporosis and bone metastasis in cancer with or without hypercalcemia (2). Nitrogen-containing BPs, such as zoledronic acid (ZA), are used in the treatment of osteoporosis and bone metastasis (reviewed in ref. 3), as well as in the treatment of other bone diseases, such as Erdheim-Chester disease (4). In addition, they are more potent inhibitors of bone resorption than non-nitrogen-containing BPs (reviewed in ref. 5). However, despite their several benefits, ZA induces BP-related osteonecrosis of the jaw (BRONJ) in cancer patients (6,7). BRONJ is defined as necrotic bone exposure in the oral cavity continuing for >8 weeks in BP-treated patients who have not had received head and neck radiation therapy (8). The molecular mechanisms responsible for the development of BRONJ remain to be elucidated; however, some risk factors, including periodontitis with bacterial plaque in the oral cavity have been associated with this symptom (reviewed in ref. 7). Previous studies have suggested that infectious agents, including Actinomyces, play important roles in the etiology and progression of BRONJ (9–11). Interestingly, Kobayashi et al reported that ZA promoted the adherence of Streptococcus mutans to hydroxyapatite and the proliferation of oral bacteria obtained from healthy individuals, suggesting that ZA increases bacterial infection (12). Wound closure by oral epithelial cells (ECs) and gingival fibroblasts (GFs) is important, not only for successful wound healing, but also for the protection of the socket from oral bacterial infection following tooth extraction. Thus, it is possible that an oral bacterial infection may induce BRONJ in cooperation with other risk factors, such as diabetes mellitus with steroid intake (13) or with microvascular disease (14), smoking, prosthetic trauma, and implant treatment (15). In addition, the ability of GFs to migrate and synthesize type I collagen is essential for the formation of rigid gingival connective tissue to cover the intraoral bone exposure, which protects the alveolar bone of the maxilla and the jaw from infection with oral bacteria. However, the mechanisms through which ZA affects the ability of human GFs (hGFs) to migrate and synthesize the connective tissue at the molecular level remain to be elucidated.
When tissue injury occurs, the blood coagulation cascade is initiated, resulting in the formation of fibrous connective tissue, which functions as a scaffold for the influx of inflammatory cells. Platelets also aggregate at the site of injury, and become activated by binding to the negatively charged extravascular fibrous tissue, and release various growth factors, including transforming growth factor-β (TGF-β) (reviewed in ref. 16). TGF-β1 is known to be expressed in serveral types of cells, including hGFs, and is involved in the proliferation and differentiation of these cells (17,18). Thus, the functions of TGF-β seem to be autocrine or paracrine as regards the regulation of hGFs during oral inflammation and the wound healing processes at the site of injury. The TGF-β superfamily consists of two families: the TGF-β/activin/Nodal family and the bone morphogenetic protein (BMP)/growth and differentiation factor (GDF)/Mullerian inhibiting substance (MIS) family (reviewed in ref. 19).
The TGF-β superfamily ligands initiate a cascade of signaling events by binding to their respective type I and type II receptors in the extracellular space. Following this, two type I and two type II receptors form a tetrameric complex. In this ligand-bound complex of type I and type II receptors, the type II receptor kinase activates the type I receptor kinase. The type I receptor induces intracellular signal transduction by phosphorylating the receptor-regulated Smads (R-Smads) (reviewed in refs. 20–23). Smads are central signal transducers of TGF-β superfamily and are composed of 3 groups. The first group comprises the R-Smads; Smad1, Smad5 and Smad8 are primarily activated by the BMP-specific type I receptors, whereas Smad2 and Smad3 are activated by TGF-β-specific type I receptors. The second group contains the common mediator Smad (Co-Smad; e.g., Smad4). The third group comprises the inhibitory Smads (I-Smads; e.g., Smad6 and Smad7). Activated R-Smads form complexes with the Co-Smad, which enter the nucleus, and, together with other cooperative proteins, positively or negatively control the transcription of specific target genes. I-Smads suppress the activation of R-Smads by competing with R-Smads for type I receptor interaction and by recruiting specific ubiquitin ligases, resulting in their proteasomal degradation (reviewed in ref. 24).
TGF-β has the ability to induce the differentiation of various types of cells into myofibroblasts (MFs), which typically exhibit the formation of F-actin stress fibers (reviewed in ref. 25). We have previously demonstrated that TGF-β induces the expression of the MF markers, α-smooth muscle actin (α-SMA) and type I collagen, in fibroblastic cells derived from periodontal ligament (26). Sobral et al reported that TGF-β induced the differentiation of hGFs into MFs in a Smad-dependent manner (27). Thus, TGF-β is now known to induce MF differentiation from fibroblasts, which preferentially form a fibrous tissue. TGF-β is also known to induce migratory activity in various types of cells, including fibroblastic cells in a Smad-dependent manner (28–30). In addition, Bakin et al reported that TGF-β regulates the migratory activity of ECs in a p38 mitogen-activated protein kinase (MAPK)-dependent manner (31), suggesting that TGF-β induces cell migratory activity through MAPKs as opposed to Smads. Moreover, TGF-β plays important roles in physiological wound closure by promoting cell migration and type I collagen synthesis. On the other hand, the abnormal and persistent appearance of MFs causes scar formation or fibrosis (32,33). The abnormal potentiation of TGF-β signaling in MFs possibly causes fibrogenic diseases. Therefore, MFs represent key players in the physiological reconstruction of connective tissue following injury and in generating the pathological tissue deformations that characterize fibrosis (34).
In this study, we investigated the mechanisms through which ZA, at its maximum concentration in serum (Cmax), which clinically, is usually found after the intravenous administration of 4 mg ZA, which is the appropriate amount for the usual treatment of bone metastasis (35,36) or bone diseases such as Erdheim-Chester disease (4), affects TGF-β-induced intracellular signal transduction and MF differentiation of hGFs, which are important processes for the progression of fibrogenesis during inflammation.
Recombinant TGF-β was purchased from Peprotech, Inc. (Rocky Hill, NJ, USA). The TGF-β type I receptor inhibitor, SB-431542, which preferentially suppresses the activation of the intracellular signal transduction of Smad2 by this receptor (37,38), and sometimes broadly suppresses the TGF-β1-induced activation of p38 MAPK, and extracellular signal-regulated kinase (ERK) as opposed to Smad2 (30) was purchased from Calbiochem (La Jolla, CA, USA). Zometa® obtained from Novartis Pharmaceuticals (Tokyo, Japan) was used as ZA in all our experiments. The Cmax of ZA in an adult human body is approximately 1.47 µM within 15 min after its intravenous administration (4 mg/5 ml) according to the user instructions provided with Zometa®. This is the appropriate amount of ZA for the treatment of cancer bone metastasis (32,33), and is referred in the internal document ZOMU00007 belonging to Novartis Pharmaceuticals.
hGFs were isolated and cultured as described in our previous study (39). Briefly, the cells were isolated from the fresh gingival tissue biopsy samples of 3 volunteers, and were maintained in a Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (both from Invitrogen, Gaithersburg, MD, USA). Informed consent was obtained from all the volunteers prior to obtaining the samples, and the Ethics Committee of Iwate Medical University approved the research protocol (approval no. 1126). Subsequently, one hGF culture retaining a high proliferative potential among the 3 cultures was used. NIH3T3 mouse embryonic fibroblasts (used as a standard control for the fibroblasts), obtained from RIKEN Cell Bank (Tsukuba, Japan), were cultured with DMEM supplemented with 10% FBS and penicillin-streptomycin (both from Invitrogen).
The status of cell viability was evaluated using an alamarBlue assay (AbD Serotec, Oxon, UK) according to the manufacturer's instructions. This assay reagent includes an indicator that fluoresces and undergoes colorimetric changes when reduced by mitochondrial respiration, which is proportional to the number of living cells. For the viability assay, the cells were seeded in 96-well plates at a density of 2.48×103 cells/well and cultured for 48 h in medium containing 10% FBS, with or without ZA at the indicated concentrations (0.0147-147 µM). Some of the cells were subsequently treated with TGF-β1 (1–5 ng/ml) for 24 h following treatment with ZA. The medium was replaced with DMEM containing 10% alamarBlue solution to evaluate the viability of the cells, and the cells were cultured for an additional 4.5 h. The absorbance in each well was measured using an ELISA plate reader (Tosoh Corp., Tokyo, Japan). The data were presented as values of Abs570 − Abs600. Each experiment was repeated 3 times, with 5-wells dedicated for each time point.
RNA isolation and RT-qPCR
Total RNA from the hGFs and NIH3T3 cells was isolated using ISOGEN II reagent (Nippon Gene, Toyama, Japan) according to the manufacturer's instructions. First-strand cDNA was synthesized from total RNA using the PrimeScript RT reagent kit (Takara Bio, Shiga, Japan). PCR was subsequently performed on a Thermal Cycler Dice Real-Time system using SYBR Premix Ex Taq II (both from Takara Bio) with specific oligonucleotide primers (human α-SMA forward, 5′-ATACAACATGGCATCATCACCAA-3′ and reverse, 5′-GGGCAACACGAAGCTCATTGTA-3′; mouse α-SMA forward, 5′-CAGATGTGGATACAGCAAACAGGA-3′ and reverse, 5′-GACTTAGAAGCATTTGCGGTGGA-3′; human TGF-β type I receptor forward, 5′-GCTGCTCCTCCTCGTGCT-3′ and reverse, 5′-TTGTCTTTTGTACAGAGGTGGC-3′; human TGF-β type II receptor forward, 5′-CTGCACATCGTCCTGTGG-3′ and reverse, 5′-GGAAACTTGACTGCACCGTT-3′; and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-ATGGTGGTGAAGACCCCACT-3′; and mouse GAPDH forward, 5′-TGTGTCCGTCGTGGATCTG-3′ and reverse, 5′-TTGCTGTTGAAGTCGCAGGAG-3′). The mRNA expression levels of α-SMA, TGF-β type I receptor and TGF-β type II receptor were normalized to those of GAPDH, and the relative expression levels were presented as the fold increase or decrease relative to the control.
The cells were lysed in RIPA buffer [50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS] or lysis buffer [20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA and 1% Triton® X-100] containing protease and phosphatase inhibitor cocktails (both from Sigma, St. Louis, MO, USA). The protein content of the samples was measured using BCA reagent (Pierce, Rockford, IL, USA). Samples containing equal amounts of protein were separated on 10% SDS-polyacrylamide gels and transferred onto a polyvinylidenedifluoride membrane (Millipore Corp., Bedford, MA, USA). After being blocked with 1% BSA or 1% skim milk in T-TBS (50 mM Tris-HCl, pH 7.2, 150 mM NaCl and 0.05% Tween-20), the membrane was incubated with primary antibodies including anti-α-SMA rabbit polyclonal antibody (1:1,000; ab5694; Abcam, Cambridge, UK), anti-Smad2/3 purified mouse monoclonal antibody (1:1,000; 610842; BD Transduction Laboratories™, Franklin Lakes, NJ, USA), anti-phospho-Smad2/3 (#8828), anti-p38 MAPK (#9212), anti-phospho-p38 MAPK (#9211), anti-c-Jun N-terminal kinase (JNK; #9251) and anti-phospho-JNK polyclonal antibodies (#9252) (1:1,000; all from Cell Signaling Technology, Inc., Beverly, MA, USA) and anti-α-tubulin mouse monoclonal antibody (1:25,000; #3873, Cell Signaling Technology, Inc.) as a loading control for normalization. The proteins of interest were then detected using appropriate horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Inc.) and an Amersham ECL™ Prime Western Blotting Detection reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).
Immunofluorescence analysis of cultured cells
For immunofluorescence analysis of the cultured cells, the hGFs were subcultured on non-coated cover glass slips (Matsunami Glass Ind., Ltd. Kishiwada, Japan) at a density of 2.5×104 cells/glass slip (BD Biosciences, Franklin Lakes, NJ, USA) and maintained in DMEM supplemented with 10% FBS, with or without ZA (1.47 µM) for 48 h. The culture medium was replaced with DMEM without FBS, supplemented with or without TGF-β1 (5 ng/ml). The cells were maintained in this medium for 4 or 5 days, fixed in 4% paraformaldehyde (Nacalai Tesque, Inc., Kyoto, Japan) for 15 min and permeabilized with Triton X-100 (Sigma). Following background reduction with normal goat serum, the cells were labeled with anti-α-SMA rabbit polyclonal antibody (1:100; ab5694; Abcam), or anti-collagen type I rabbit polyclonal antibody (1:100; 600-401-103-0.1; Rockland, Inc., Rockland, ME, USA) at room temperature for 1 h. After being washed with phosphate-buffered saline (PBS) to remove the excess primary antibody, the cells were incubated with Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1:1,000; A-11034; Molecular Probes, Leiden, The Netherlands) and DAPI (1:1,000; KPL, Gaithersburg, MD, USA) for 30 min at room temperature. After being washed with PBS to remove the excess secondary antibody, the fluorescent signal was detected using an Olympus IX70 fluorescence microscope with the LCPIanFI 20 objective lens (Olympus Co., Tokyo, Japan).
Evaluation of the migratory activities of hGFs and NIH3T3 cells
Cell migration assays were performed using Transwell® membrane cell culture inserts (8 µm pore size; Corning Inc., Corning, NY, USA) according to the manufacturer's instructions. The hGFs were cultured with or without ZA (1.47 µM) in DMEM supplemented with 10% FBS for 48 h at a cell density of 6.0×105 cells in 10 cm culture dish. The cells were then placed at a density of 1.0×105 cells in the cell culture inserts in the each well of a 24-well cell culture plate in DMEM with 0.1% BSA. The cells were allowed to migrate through the porous membrane that bordered the upper cell culture insert and the lower well of 24-well culture plate which contained DMEM in the presence or absence of TGF-β (5 ng/ml) for 6 h at 37°C. TGF-β was added to the culture medium in the lower well of 24-well culture plate. In some cases, SB-431542 was added to both the upper and lower culture media. The cells on the upper side of the membrane were wiped, and the membrane was fixed in 4% paraformaldehyde in PBS. After being washed with PBS, the cells that had migrated onto the underside of the membrane were labeled with DAPI (1:1,000; KPL) and counted. The values were shown as average of those from 3 wells. The evaluation of the migratory activity of the NIH3T3 cells was performed as described above without pre-treatment of the cells with ZA.
The hGFs were cultured with or without ZA (1.47 µM) in DMEM supplemented with 10% FBS for 48 h at a density of 6.0×105 cells in a 10-cm culture dish. The cells (1.0×105 cells) were suspended in PBS containing 0.5% FBS and 2 mM EDTA and incubated with anti-TGF-β receptor type I (1:100; ab30103) or type II (1:50; ab78419) (both from Abcam) primary antibodies for 1 h at 4°C. For the negative control experiments, the cells were incubated with the same protein amount of normal control IgG (sc-2028; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) as each specific antibody. The cells were then incubated with PE-conjugated secondary antibodies (#732988 for ab31013; #732970 for ab78419) for 30 min in the dark. Acquisition was performed using an EPICS XL EXPO 32 ADC system (Beckman Coulter, Fullerton, CA, USA).
The data are presented as the mean ± SD (n=3 or 5 experiments). The data were statistically analyzed using the Student's t-test, and P<0.01 (indicated by an asterisk) was considered significant. The results shown in all experiments are representative of at least 3 separate experiments.
ZA suppresses the TGFβ1-induced increase in the viability of hGFs
Treatment with ZA for 48 h in medium containing 10% FBS at the concentrations of 0.0147 µM (1/100 of Cmax), 0.147 µM (1/10 of Cmax) and 1.47 µM (Cmax) did not affect the viability of the hGFs (Fig. 1A) and the NIH3T3 cells which were used as a standard control for the fibroblasts (Fig. 1B). However, at 10-100-fold higher concentrations (14.7–147 µM) than the Cmax, ZA significantly suppressed the viability of the hGFs (35–58% suppression of the control) and that of the NIH3T3 cells (29–80% suppression of the control) in a dose-dependent manner. Thus, ZA (Cmax) does not affect the viability of hGFs and normal standard fibroblasts at 48 h following administration.
Zoledronic acid (ZA) suppresses the transforming growth factor-β1 (TGF-β1)-induced increase in the viability of human gingival fibroblasts (hGFs) at the maximum concentration in serum (Cmax). (A) hGFs and (B) NHI3T3 cells were seeded in 96-well plates at a density of 2.48×103 cells/well and cultured for 48 h in medium containing 10% fetal bovine serum (FBS) with or without ZA at the indicated concentrations (0.0147–147 µM). The medium was replaced with DMEM containing 10% alamarBlue solution, and the cells were cultured for an additional 4.5 h. To evaluate the viability of the cells, the absorbance (Abs570−Abs600) in each well was measured using a plate reader. (C) Firstly, hGFs were seeded in 96-well plates at a density of 2.48×103 cells/well and cultured for 48 h in medium containing 10% FBS with or without ZA (Cmax) (lower, and upper graphs, respectively). The cells were subsequently stimulated with TGF-β1 (1–5 ng/ml) for 24 h. The viability of the hGFs was then measured as described above. The data are presented as the means ± SD (n=5). *P<0.01 was considered significant.
In order to elucidate the mechanisms through which ZA affects TGF-β-induced fibrogenesis by hGFs, the hGFs were pre-treated with ZA (Cmax) for 48 h and subsequently treated with TGF-β1 at the indicated concentrations for the indicated periods of time. Finally, the effect of pre-treatment with ZA (Cmax) on TGF-β-induced functions in hGFs was investigated using the following experiments:
We examined whether ZA (Cmax) affects the viability of hGFs stimulated with TGF-β1 (1–5 ng/ml). As shown in Fig. 1C (upper graph), TGF-β1 (1–5 ng/ml) increased the viability of the hGFs (23–47% promotion of the control). However, ZA (Cmax) suppressed the TGFβ1-induced increase in the viability of the hGFs (Fig. 1C, lower graph; compare same TGF-β1 concentrations between graphs).
ZA (Cmax) suppresses the TGF-β-induced MF differentiation of hGFs
TGF-β1 (5 ng/ml) significantly upregulated the α-SMA mRNA expression level in the hGFs (Fig. 2A, bars 1 and 2 from left). ZA (Cmax) completely suppressed the TGF-β1-induced increase in the mRNA expression of α-SMA in the hGFs (Fig. 2A, bars 2 and 4 from left). In addition, western blot analysis revealed that ZA (Cmax) clearly suppressed the TGF-β1-induced upregulation of α-SMA expression at the protein level in the hGFs (Fig. 2B). Immunofluorescence staining also revealed that ZA (Cmax) clearly suppressed the TGF-β1-induced upregulation of α-SMA expression (Fig. 2C, left panels) and type I collagen expression (Fig. 2C, right panels) at the protein level in the hGFs. We confirmed that the TGF-β1-induced increase in the mRNA expression of α-SMA in the hGFs was significantly inhibited by SB-431542, an inhibitor of TGF-β type I receptor (Fig. 2D), indicating that TGF-β1 specifically induced the MF differentiation of hGFs through ligand-receptor interaction. Thus, these results indicate that ZA (Cmax) suppresses the TGF-β-induced MF differentiation of hGFs.
Zoledronic acid (ZA) at the maximum concentration in serum (Cmax) inhibits the transforming growth factor-β (TGF-β)-induced myofibroblast (MF) differentiation of human gingival fibroblasts (hGFs). (A) hGFs were cultured for 48 h in growth medium supplemented with 10% fetal bovine serum (FBS) in the presence or absence of ZA (1.47 µM or Cmax value of an adult human; ZA administered intravenously at 4 mg/5 ml). The culture medium was replaced with DMEM without FBS supplemented with or without TGF-β1 (5 ng/ml) and maintained for 24 h. The relative expression level of MF marker α-smooth muscle actin (α-SMA) was analyzed by RT-qPCR. Data represent the means ± SD (n=3). *P<0.01 was considered significant. (B) The status of MF differentiation of hGFs at the protein level was evaluated by western blot analysis with anti-α-SMA antibody. The cells were cultured with or without ZA (Cmax) as described in (A), and were subsequently cultured in DMEM without FBS supplemented with or without TGF-β1 (5 ng/ml) for 4 days [α-SMA, upper panel; α-tubulin (internal control), lower panel]. (C) The cells were first cultured with or without ZA (Cmax) as described in (A), cultured in DMEM without FBS supplemented with or without TGF-β1 (5 ng/ml) for 4 (α-SMA; left panels) or 5 (type I collagen; right panels) days. The cells were then immunostained with anti-α-SMA (left panels) (green) and anti-type I collagen (right panels) (green) antibodies. Nuclei were fluorescently counterstained with DAPI (blue). Scale bar, 50 µm. (D) hGFs were cultured for 48 h in growth medium supplemented with 10% FBS, and the medium was replaced with DMEM without FBS supplemented with or without TGF-β1 (5 ng/ml). The cells were maintained for 24 h. Some of the cells were treated with the TGF-β receptor inhibitor, SB-431542 (10 µM), which was added to the culture medium 30 min prior to stimulation with TGF-β1. The relative mRNA expression level of MF marker α-SMA was analyzed by RT-qPCR. Data represent the means ± SD (n=3). *P<0.01 was considered significant.
ZA (Cmax) inhibits the TGF-β-induced migratory activity of hGFs
As shown in Fig. 3A and C, the migration of the hGFs through the porous membrane that bordered the upper and lower chambers was significantly enhanced by stimulation with TGF-β1 (5 ng/ml). However, ZA (Cmax) completely suppressed the TGF-β1-induced migratory activity (Fig. 3A and B). In addition, we confirmed that the TGF-β-induced migratory activity was markedly inhibited by SB-431542 (Fig. 3C and D), indicating that TGF-β1 specifically induced the migration of hGFs through ligand-receptor interaction. These results indicate that ZA (Cmax) inhibits the TGF-β-induced migratory activity of hGFs.
Zoledronic acid (ZA) at the maximum concentration in serum (Cmax) inhibits the transforming growth factor-β (TGF-β)-induced migratory activity of human gingival fibroblasts (hGFs). Cell migration assays were performed using Transwell® membrane cell culture inserts as described in the Materials and methods. (A and B) hGFs were cultured with or without ZA (1.47 µM) for 48 h. The cells were placed in the cell culture inserts in the each well of 24-well cell culture plate. The cells were then allowed to migrate through the porous membrane that bordered the upper cell culture insert and the lower well of 24-well culture plate in the presence or absence of TGF-β1 (5 ng/ml). (C and D) hGFs were placed in the cell culture inserts as described above. The cells were allowed to migrate through the membrane in the presence or absence of TGF-β1 (5 ng/ml). In some cases, SB-431542 was added to both the upper and lower culture media. The cells that had migrated onto the underside of the membrane were labeled with DAPI and visualized using a fluorescent microscope (A and C) and counted (B and D). Data represent the means ± SD (n=3). *P<0.01 was considered significant.
ZA (Cmax) suppresses the expression of TGF-β type I receptor on the surfaces of hGFs and the TGF-β-induced phosphorylation of Smad2/3 in hGFs
In order to gain insight into the molecular mechanisms underlying the suppressive effects of ZA (Cmax) on TGF-β-induced fibrogenesis by hGFs, we investigated whether ZA affects the expression of TGF-β type I and II receptors on the surface of hGFs. In flow cytometric analysis, hGFs had two peaks in the histogram of TGF-β type I receptor-positive cells, that is, 2 groups of hGFs weakly or strongly expressed TGF-β type I receptors, respectively (Fig. 4A, upper left panel). Intriguingly, ZA (Cmax) lowered the ratio of the number of cells strongly expressing TGF-β type I receptors on their surface against that of total hGFs by 19.6 to 6.4% (Fig. 4A, upper panels). By contrast, hGFs had only one peak in the histogram of TGF-β type II receptor-positive cells (Fig. 4A, lower left panel). ZA (Cmax) did not change the position of the peak in the histogram of TGF-β type II receptor-positive cells (Fig. 4A, lower panels). Moreover, RT-qPCR analysis revealed that ZA (Cmax) did not decrease the total amount of both TGF-β receptor type (types I and II) expression at the mRNA level (Fig. 4B, upper and lower graphs, respectively), suggesting that ZA (Cmax) suppressed the expression of TGF-β type I receptor on the surface of hGFs. In addition, ZA (Cmax) suppressed the TGF-β1 (5 ng/ml)-induced phosphorylation of Smad2/3 (Fig. 4C). We confirmed these results by reproducing them in independent experiments.
Zoledronic acid (ZA) at the maximum concentration in serum (Cmax) suppresses the expression of transforming growth factor-β (TGF-β) type I receptor on the surface of human gingival fibroblasts (hGFs) and the phosphorylation of Smad2/3 in hGFs. (A) hGFs were cultured with or without ZA (Cmax) for 48 h as described in Fig. 2 at a density of 6.0×105 cells in a 10-cm culture dish. The expression levels of TGF-β type I (upper panels), and II (lower panels)-receptors on the surface of hGFs were evaluated by flow cytometry using anti-TGF-β type I receptor (red) and anti-TGF-β type II receptor (red) antibodies or the same amount of normal control IgG (blue). (B) hGFs were cultured for 48 h in growth medium supplemented with 10% fetal bovine serum (FBS) in the presence or absence of ZA (Cmax). The relative mRNA expression levels of TGF-β type I receptor (upper graph) and TGF-β type II receptor (lower graph) were analyzed by RT-qPCR. Data represent the means ± SD (n=3). *P<0.01 was considered significant. (C) hGFs were cultured with or without ZA (Cmax) as described in (A). The cells were subsequently cultured with or without TGF-β1 (5 ng/ml) for 1 h. The phosphorylation of Smad2/3 was evaluated by western blot analyses with anti-Smad2/3 and anti-phospho-Smad2/3 antibodies.
Wang et al reported that TGF-β1 increased the viability of human dermal fibroblasts (40). We found that ZA (Cmax) reduced the TGF-β1-induced increase in the viability of hGFs (Fig. 1C). These results suggest that ZA (Cmax) possibly suppresses the TGF-β1-induced fibrogenesis in oral gingival tissue. Lu et al reported that TGF-β1 promoted the viability of pulmonary artery endothelial cells through Smad2-mediated signal transduction (41). We found that ZA (Cmax) suppressed the TGF-β1 (5 ng/ml)-induced phosphorylation of Smad2/3 (Fig. 4C), implicating that ZA (Cmax) may possibly suppress the TGF-β1-induced promotion of the viability of hGFs through suppression of Smad2/3 activities.
Pan et al or Koch et al reported that 5 or 50 µM ZA induced the osteoblastic differentiation of osteoblast precursor cells (42,43). In addition, Chen et al reported that 10 µM ZA induced the dendritic cell differentiation of monocytes (44). However, the mechanisms through which ZA (Cmax) affected the differentiation ability of cells derived from the oral cavity remained to be elucidated. In this study, we demonstrated that 1.47 µM ZA (Cmax) significantly suppressed the TGF-β1-induced MF differentiation of hGFs (Fig. 2A, B, and C). These results strongly suggest that ZA (Cmax) inhibits wound healing in the oral cavity by the attenuation of type I collagen synthesis in MFs differentiated from hGFs following TGF-β1 stimulation. In addition, it is generally known that MFs play an important role in generating a contractile force, facilitating wound closure (reviewed in ref. 16), suggesting that ZA (Cmax) also interferes with this process through the suppression of the TGF-β1-induced MF differentiation of hGFs. Of note, we found that ZA (Cmax) clearly suppressed the TGF-β1-induced phosphorylation of Smad2/3 in hGFs (Fig. 4C). Smad2/3 are known to be key signaling molecules inducing the MF differentiation of fibroblasts (27). These results suggest that ZA (Cmax) suppressed TGF-β1-induced MF differentiation of hGFs through inhibition of Smad2/3 phosphorylation by TGF-β1.
Pabst et al reported that ZA at 50 µM, a much higher concentration than Cmax, significantly decreased the migratory activity of human oral keratinocytes at 72 h following treatment with ZA (45). However, it remained to be clarified whether ZA (Cmax) affected the migratory activity of various cells derived from the oral cavity, including hGFs. On the other hand, TGF-β is known to induce migratory activity in various types of cells, including fibroblastic cells, in a Smad-dependent manner (28–30). In this study, we demonstrated that ZA (Cmax) significantly suppressed the TGF-β1-induced migratory activity of hGFs (Fig. 3A and B). Wound healing or mucous re-epithelialization is a complex process involving the proliferation and migration of mesenchymal cells, such as fibroblasts and MFs, which construct granulation tissue for wound closure and then induce mucous re-epithelialization on it (reviewed in ref. 46). As described above, ZA (Cmax) suppressed TGF-β1-induced phosphorylation of Smad2/3 (Fig. 4C). These results suggest that ZA (Cmax) suppresses wound closure with granulation tissue in the oral cavity by the inhibition of the TGF-β1-induced migration of mesenchymal cells, such as hGFs by suppressing Smad2/3 phosphorylation by TGF-β1. On the other hand, Ozdamar et al (47) reported that the phosphorylation of polarity protein Par6 is required for TGF-β1-dependent epithelial-to-mesenchymal transition (EMT) in mammary gland ECs and controls the interaction of Par6 with the E3 ubiquitin ligase Smurf1. Smurf1, in turn, targets the guanosine triphosphatase RhoA for degradation, thereby leading to a loss of tight junctions (47). In addition, RhoA is generally known to be a regulator of cell migratory activity in various types of cells by promoting stress fiber assembly and the resultant cell adhesion (48). Intriguingly, serine-to-alanine point mutation [Par6(S345A)] of the TGF-β1-induced phosphorylation site in Par6 suppressed the TGF-β1-induced EMT in a Smad-independent manner. It remains to be clarified whether ZA (Cmax) suppresses the TGF-β1-induced migratory activity of hGFs through affecting Par6/Smurf1/RohA signal transduction pathway in a Smad-independent manner.
Saito et al demonstrated the negative effects of ZA on the re-epithelialization of oral mucosa in a three-dimensional in vitro oral mucosa wound healing model (49). They demonstrated histologically that ZA downregulated the expression of TGF-β type I and II receptors and Smad3 phosphorylation in oral keratinocytes. However, they used ZA at 10 µM, which was a much higher concentration than Cmax, for evaluating the effects on TGF-β1-induced signal transduction in human oral keratinocytes. Moreover, they did not elucidate how TGF-β-induced intracellular signals affect the function of human oral keratinocytes at the cellular and molecular levels. In this study, we demonstrated that ZA (Cmax) lowered the ratio of the number of cells strongly expressing TGF-β type I receptors on their surface against that of total hGFs, but did not affect the expression status of TGF-β type II receptors on hGFs (Fig. 4A). This finding suggests that ZA (Cmax) preferentially suppresses the expression of TGF-β type I receptor over that of TGF-β type II receptor on the surfaces of hGFs. By contrast, as described above, a high concentration of ZA (10 µM) seemed to downregulate the expression of both TGF-β receptor types (types I and II). In addition, we found that ZA (Cmax) suppressed the TGF-β1-induced phosphorylation of Smad2/3 in hGFs (Fig. 4C), implicating that the suppression of the TGF-β1-induced phosphorylation of Smad2/3 in hGFs by ZA (Cmax) may be caused by the inhibition of TGF-β type I receptor expression on the surfaces of hGFs. However, it remains to be clarified whether ZA (Cmax) directly suppresses the TGF-β1-induced phosphorylation of Smad2/3, or indirectly suppresses that through the inhibition of the TGF-β type I receptor expression on the surface of hGFs. On the other hand, Okamoto et al demonstrated that ZA (30-50 µM) induced apoptosis and S-phase arrest in mesothelioma by inhibiting the functions of Rab family proteins (50). The Rab family is generally known to control the endosomal trafficking of membrane molecules, such as growth factor receptors (reviewed in ref. 51). It is also generally known that Rab5, activated by a guanine nucleotide exchange factor (GEF), directs TGF-β-activated receptors into the endocytic pathway that promotes TGF-β-induced Smad2/3-dependent signals (52). In addition, Kardassis et al suggested that TGF-β receptors may be recycled on the cell membrane in a Rab-11-dependent manner following clathrin-dependent internalization of TGF-β receptors, which may positively affect the activities of Smad2/3-dependent TGF-β-induced intracellular signals mediated by these receptors (53). Our laboratory is currently investigating whether ZA (Cmax) suppresses the function of Rab family members that may upregulate the activity of TGF-β1-induced Smad2/3-dependent intracellular signals in hGFs at the receptor level, thereby resulting in downregulating TGF-β1-induced fibrogenic activity of the cells.
As described above, it has been reported that the TGF-β-induced activation of p38 MAPK positively regulates migratory activity (31). In fact, TGF-β1 upregulated the phosphorylation of p38 MAPK in hGFs; however, ZA (Cmax) did not suppress the TGF-β1 (5 ng/ml)-induced phosphorylation of p38 MAPK in hGFs (data not shown), suggesting that p38 MAPK activity is not related to the suppressive effects of ZA (Cmax) on TGF-β1-induced fibrogenic activity of hGFs. It is plausible that the suppression of type I receptor expression on the surface of hGFs by ZA (Cmax), as shown in Fig. 4A, may not be sufficient to suppress the TGF-β1-induced p38 MAPK phosphorylation. On the other hand, the activation of MAPKs such as JNK and p38 MAPK, but not ERK, is necessary for the progression of the hypoxia-induced MF differentiation of fibroblasts (54). Although, in western blot analysis, the phosphorylation of JNK was not detectable in hGFs before/after TGF-β1 (5 ng/ml) stimulation (data not shown), it remains to be clarified whether JNK affects the TGF-β1-induced fibrogenic and migratory activities of hGFs. In addition, the mechanisms through which p38 MAPK affects the TGF-β1-induced fibrogenic and migratory activities of hGFs also remains to be clarified.
In conclusion, in the present study, it was suggested that ZA (Cmax) attenuates TGF-β1-induced wound closure by inhibiting the formation of granulation tissue by hGFs stimulated with TGF-β1 that was derived from inflammatory tissue, possibly through the suppression of Smad2/3 signaling. Our findings partly clarify the molecular mechanisms underlying BRONJ and would benefit research into drug targets at the molecular level for the treatment of this symptom.
This study was supported in part by Grants-in-Aid for Scientific Research (KAKENHI) (grant nos. 24791981 awarded to M.I., 25463053 awarded to N.C., 26462823 awarded to S.K., 22592076 awarded to M.K., 26293426 awarded to T.S., 24593002 awarded to Y.S., and 26670852 awarded to A.I.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Grant-in-Aid from the Dental Society of Iwate Medical University; and Grant-in-Aid for Strategic Medical Science Research Centre from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, 2010–2014.
zoledronic acid
BRONJ
BPs-related osteonecrosis of the jaw
gingival fibroblasts
hGFs
human GFs
TGF-β
transforming growth factor-β
growth and differentiation factor
Mullerian inhibiting substance
R-Smads
receptor-regulated Smads
Co-Smad
common mediator Smad
I-Smads
inhibitory Smads
myofibroblasts
α-SMA
α-smooth muscle actin
MAPKs
mitogen-activated protein kinases
extra-cellular signal-regulated kinase
maximum concentration in serum
phosphate-buffered saline
GAPDH
glyceraldehyde 3-phosphate dehydrogenase
SDS-polyacrylamide gel electrophoresis
c-Jun N-terminal kinase
epithelial-to-mesenchymal transition
guanine nucleotide exchange factor
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Komatsu, Y., Ibi, M., Chosa, N., Kyakumoto, S., Kamo, M., Shibata, T. ... Ishisaki, A. (2016). Zoledronic acid suppresses transforming growth factor-β-induced fibrogenesis by human gingival fibroblasts. International Journal of Molecular Medicine, 38, 139-147. https://doi.org/10.3892/ijmm.2016.2582
Komatsu, Y., Ibi, M., Chosa, N., Kyakumoto, S., Kamo, M., Shibata, T., Sugiyama, Y., Ishisaki, A."Zoledronic acid suppresses transforming growth factor-β-induced fibrogenesis by human gingival fibroblasts". International Journal of Molecular Medicine 38.1 (2016): 139-147.
Komatsu, Y., Ibi, M., Chosa, N., Kyakumoto, S., Kamo, M., Shibata, T., Sugiyama, Y., Ishisaki, A."Zoledronic acid suppresses transforming growth factor-β-induced fibrogenesis by human gingival fibroblasts". International Journal of Molecular Medicine 38, no. 1 (2016): 139-147. https://doi.org/10.3892/ijmm.2016.2582


��Its been so long since my last blog, I kinda forgot how to do this haha. �Anyways hows everyone doing, Im still just plugging away here in Syracuse. �It has been an up and down season so far and I mean I started off pretty slow and really had to work hard to get out of that funk I was in. �Personally I have been working on my mechanics so that I could stay taller as I deliver the pitch and make the ball have more of a down ward angle on it, so that is the thing I have been working on and the last 5 or 6 starts it has been working out and I feel great doing it now. �As for this team man I really cant say that I have had more fun with any other team, this group of guys here are a lot of fun and we have been winning so thats always a plus. �other than that so far I am just focusing on baseball and the wedding this off season and having a blast doing both : ). �Well I know that this isn't much since I haven't blogged in over 2 months but I will try to do a better job, i cant keep making the excuse that I'm super busy haha. �Plus Its my birthday so Happy Birthday to me haha..
This blog goes out to the Adenhart's in this tough time, I know that this is late but I just wanted to write a blog and tell you how this tragic time has touched ball players everywhere. I can only talk for our team, we were all watching Sportscenter and getting ready to watch the Masters and to start our season, not even knowing what happened in Anaheim the night before. When the news passed threw the bottom line that morning our whole team couldn't believe the horrible news, the shocked look on everyones face with that gloss in all our eyes. I don't think one person on our team new Nick on a personal level but it felt like we all did that day, he was a part of our baseball family and it felt like that we lost one of our family members. I mean as a human being you don't ever like to hear about anyone die or anything like that but when it happens like this it really hits home. So our prayers go out to the Adenhart's and to all the other 3 family's involved.
Help the Nationals' "First Ladies" fight Alzheimer's!
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�� � I was thinking, and thats not always a good thing. (haha!) �Since I have become a "Blogger" I have been half and half talking about baseball, and fun times off the field that have nothing to do with baseball or anyone involved, well today I want to talk about and try and get some feedback from everyone here on what they think because everyone calls me an idiot when I bring this up. �Ok so one day I woke up and looked outside and there was no clouds in the sky, looked like it was going to be just a beautiful hot and sunny day right, wrong I went outside and it was freezing cold and I'm talking sun was shining like no other. �Then on other days you could go out and its overcast and its warmer outside. �So this brings me to my conclusion, I said that the clouds on an overcast day serve as like a blanket and the sun heats them up, therefore making us underneath the clouds feel warmer, but everyone just laughs and says I'm wrong. �I might have to bring in Bill Nye the science guy and thats fine. �But why does it seem to be colder on days when the sun is shining and there is not a cloud in the sky and warmer on the days where its more overcast? �Don't get me wrong, I am no scientist, and I have had my fare share of times when I argue about things I have no clue about and think they are right and I end up being wrong, I just want to see some paper work on this topic. �Is that so much to ask, so thats what's going on at Spring Training 2009. �Have a good one.
�� So I was talking to Mike Hinckley the other day and he comes up to me and says "can you grow a beard Bally," I said yes I can and yes I have before, he then goes on to tell me that he had the "Sickest" beard of all time this offseason. �I have been known to grow a pretty sweet beard myself, so you guys be the judge.


Raja Yoga meditation requires the individual to study spiritual knowledge. Understanding of the knowledge is essential to its practice and application. The aim of Raja Yoga is self-sovereignty and self-mastery and so it is important for the individual to be discerning on his or her spiritual journey. The process of learning is a simple one: listening to and understanding the teachings;contemplating and making sense of how to apply the teachings in life; inculcating the teachings and emerging the innate qualities into the awareness of the self; experiencing the meaning of insightful wisdom and deciding the quality of one's actions.


text:
North East travel disruption as rain causes chaos
THE persistent heavy downpours that have swamped much of the country will continue today and cause some areas to experience a whole month's rainfall in just 24 hours, weather experts say.
Newcastle floods
Road users in the North East are being advised to consider whether their journey is necessary before attempting to travel tonight and tomorrow (26 September) as strong winds and heavy rain continue.
The Met Office's amber alert for severe wet and windy weather remains in place with very difficult driving conditions expected to continue over the next 24 hours.
North Northumberland and the Borders have been particularly badly affected by the storms with rain and gale force winds lashing the area.
Commuters hoping to travel by rail from Berwick, Alnmouth and Morpeth were left stranded after all trains north and south on the East Coast mainline were cancelled with no replacement bus services operating at the moment. There was no word on when services may resume with passengers travelling between Edinburgh and Newcastle diverted via Carlisle.
North east floods - School closures on September 25
Communities across England and Wales are bracing themselves for the risk of floods from rivers and surface water as up to 100mm, almost four inches, of rain could fall by the end of the day, the Met Office said.
In England the Environment Agency has issued 55 flood warnings for the South West, the Midlands, Wales, the North East and North West, and 164 less serious flood alerts in the South East and East Anglia.
In Morpeth, Northumberland, some people were evacuated from their homes as floodwater rose.
A Northumbria Police spokeswoman said: "Residents in an area of Morpeth are being evacuated as a precaution.
"We are working with the local authority to take them to an alternative location."
A map of North East warnings is below:
The Environment Agency urged people to stay safe in the face of flooding, keeping away from swollen rivers and not to attempt to drive through floodwater. The agency said it expected to issue a significant number of flood warnings and alerts for rivers in the coming days and had sent out teams across the country to check on flood defences, clear blockages and monitor river levels.
Alison Baptiste, flood risk manager at the Environment Agency, said: "We are already seeing travel disruption due to the wet weather and could well see flooding across the country this week.
"We strongly urge people to sign up to flood warnings, keep a close eye on local weather forecasts and be prepared for the possibility of flooding.
"We also ask that people stay safe by staying away from swollen rivers and not attempting to drive through floodwater."
Newcastle flooding causes commuter misery - VIDEO & GALLERY
:: Have you been affected? Leave your comments below, and email pics of the wet weather to pics@ncjmedia.co.uk

redpajama_set_name: RedPajamaCommonCrawl


Avalon Resort is listed among the most popular and reputed four star properties in Mussoorie. The resort is located in close proximity to the bus stand and therefore offers easy connectivity to major commercial centers and tourist hotspots of the hill station. The Avalon resort lies in scenic location that offers unrivalled view of the mighty mountains on one side and the gracious Doon valley on the other, making it ideal place for nature lovers and vacationers. Patrons are offered choice of Studio Rooms, Club Suites and Royal Club Suites as accommodation options, depending upon their requirements and budget range. All the rooms offered by Avalon resort are designed by experts and therefore sport contemporary décor and charming interiors. They are also fitted with best of amenities and facilities to keep guests pampered and satisfied.
Avalon Resorts has also developed an onsite multi cuisine restaurant on its property that offers Indian, continental and Chinese culinary specialties to guests. Apart from this, to make guests stay more comfortable and enjoyable the resort offers other facilities and services like travel desk, sightseeing arrangements, laundry facility, doctor on call service, parking space, conference center with advanced audio visual facilities, round the clock room service and many more. Scenic location and lavish accommodation options offered by Avalon Resorts makes it one of the finest stay options in Mussoorie.
Studio Rooms are the simplest kind of accommodation choice offered by Avalon Resorts in Mussoorie. These Studio rooms are ideally suited to solo guests and business travelers who are looking for a short term stay with all necessary comforts and amenities. The Studio Rooms are designed by expert decorators and reflect charming amalgamation of contemporary décor theme and local cultural elements. The interiors are furnished, premium Italian furniture, with superior materials and decorative artistic pictures that give it imperial feel and charm. The Studios consist of a large bedroom that is equipped with plush double bed, comfortable two seater sofa unit, wardrobes, luggage rack, a small kitchenette with mini bar or fridge, microwave, toaster, kettle and a lavish bathroom with shower. Other amenities provided in Studio Rooms include LCD Television with satellite connectivity and subscription to premium channels, high speed internet access through Wi-Fi connectivity, electric blankets, hot and cold water supply, telephone with direct dial facility and many others. Premium facilities and charming décor of the Studio Rooms make them ideal stay choice for couples.
If space is your prime requirement while picking out a vacation stay choice in Mussoorie, then the One Bedroom Club suites will surely serve your purpose. These suites are designed for small families and groups traveling to Mussoorie and therefore are designed in such a way that they cater to different needs of all the members of the group. As far as décor of the suites is concerned, the One Bedroom Club Suites are crafted carefully to meet tastes and preferences of guests. Patrons will find contemporary décor, Italian furniture and premium furnishing in the Club Suites. The Club Suite consists of plush bedroom, spacious living room and dining area along with a separate kitchenette. The living room has a specially designed pull out bed for two that sleeps two people. The Club Suite also has spacious balcony that allows guests to enjoy the scenic natural beauty of Mussoorie right from the comfort of their rooms. As far as other amenities are concerned, the Club Suites boast of LCD Television with satellite connectivity and subscription to premium channels, high speed internet access through Wi-Fi connectivity, electric blankets, hot and cold water supply, telephone with direct dial facility and many others. Lavish luxuries and scenic view of the valley make Club Suites stand out and apart from other accommodation options available in Mussoorie.
Planning a family trip to Mussoorie with your family of group of friends, then you might need an stay choice that offers ample space and all the required amenities to cater to different needs of all the members of the group. The Two Bedroom Royal Club Suites offered by Avalon Resorts does exactly this and much more. The Royal Club Suites are the best accommodation category offered by the resort and justifies this tag by offering the best in terms of space, facilities and features. The Royal Club Suite consists of two separate bedrooms with attached bathrooms, living room, dining area and a lavish balcony that overlooks the gracious Doon valley. The Royal Club Suites are independent cottages and therefore offer complete privacy to guests, who can enjoy their vacation with their loved ones in complete peace of mind. To keep guests pampered and satisfied the Royal Club Suites are equipped with modern amenities and facilities like LCD Television with satellite connectivity and subscription to premium channels, high speed internet access through Wi-Fi connectivity, electric blankets, hot and cold water supply, telephone with direct dial facility, TV in master bedroom and many more. The lavish nature and classy setup of Royal Club Suites make them stand out and apart from the regular accommodation choices available in Mussoorie.
Avalon Resorts is an experiential property and therefore offers all the necessary facilities and features on site to the guests. In line with this, the resort has developed a multi cuisine restaurant that caters to culinary requirements of patrons staying here. The restaurant is one of the finest diners in the region and offers an array of north Indian, south Indian, Chinese and continental preparations. Patrons can also pick some of the delectable local dishes that are served here. All the food items are prepared by experienced chefs using organic ingredients and rare spices that impart unique and distinct flavor the food. Another features of the multi cuisine restaurant is the comfortable seating arrangement and neutral décor of the dining hall, which makes it ideal for casual lunches or dinners on special occasions. Guests have also been highly appreciative of the strict cleanliness and hygiene standards followed by the restaurant, which inspires confidence in the kind of food served here.


With the most extensive profile identification guide in the world, we can manufacture a bespoke filler to your requirements. Each foam filler is carefully produced by Brett Martin to the highest standards of accuracy, design and quality possible. Products such as Polyethylene, Supaseal and Very High Temperature Material (VHTM) are ideal solutions for all types of cladding and rooflights, airsealing, and fire or acoustic sealing.
So, if your after some product advice or would like to discuss your project requirements contact our Sealant Shop on 024 7660 2022 or email roofsealants@brettmartin.com.
Produced to the highest standards of accuracy, design and quality.
Ideal solutions for sealing rooflights, airsealing and fire/acoustic sealing.
We can manufacture a filler to your bespoke requirements.
Suitable for settings such as eaves, ridge and roof intersections.
Available in a wide range of colours and thicknesses.
Can be supplied with a wide range of options.
Polyethylene (PE foam) is a closed cell physically cross-linked polyethylene laminated with strengthening interfaces. PE foam fillers are widely used and cost effective for standard applications.
Supaseal foam is a physically cross-linked material, blending two flexible polymers into a closed cell foam. Unique to Brett Martin, Supaseal is an excellent foam filler which is high in durability and flexibility.
Firestop is a mineral wool which is essential when a fire rating is specified. This filler can provide up to 6 hours fire protection.
At Brett Martin, our precision cutting machinery produces foam fillers to exceptionally high levels of accuracy, optimising seals and minimising energy loss. If your requirements are more specific, we can manufacture bespoke fillers for an extensive range of settings (eaves, ridge and other roof intersections).
Virtually every roofing and cladding profile is held on our database and others can be quickly added. At Brett Martin, we ensure that fillers are always precision matched to sheeting panels. Sheet manufacturer name or reference can identify the correct profile or profile drawings can accurately match dimensions.
The choice of flute depends on which side of the profiled sheet is to be filled.
Typically used at the ridge, filling the inter-profile between ridge cap and corrugated roof sheet, this filler fits on the outside of the sheet. For a reverse profile the large flute filler goes outside the sheet.
Typically used under the roof sheet at the eaves, the small flute (when integrated) fills the inter-profile cavity between sheet and structure.
Bonded butyl sealants can be applied to the profile, the base or both, offering easier location and improved weather seal.
Large and small flute fillers are manufactured with a standard 6mm base. Extended base depth available on request.
Fillers to be fitted along roof hips and valleys must be cut to the angle formed by the flashing or valley and short dimension of the profiled sheet *(true angle). It must also be determined if the filler is left or right handed.
*This depends on both the plan angle and pitch of the roof; we can calculate this if required.
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Made to order FIRE STOP C144 is designed to be fitted above compartment walls and into corrugations where complete resistance to fire is required.
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Can be supplied as strips/ angle cut on request.
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Essential when fire rating is specified.
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Provide firestopping for vertical and horizontal construction joints.
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Finally, there has been some sanity in Uganda law because the law that sentences gay with HIV to death and life in prison for being gay has been scraped.
Entry filed under: News, Religion, spirituality. Tags: Christian, gay, News, Religion, Uganda.


text:
Q: Consulta MySQL where 'al menos' contenga uno de los valores Tengo un buscador en mi pag web en la que se puede buscar usuarios o #etiquetas. Este esta formado por un form alojado en el menubar, este buscador. Este buscador esta formado simplemente por un input
 y por ub button. Para buscar una etiqueta simplemente se coloca un # antes de la palabra a buscar y para buscar un usuario pues sin #. Ejemplo:
Ejemplo buscar usuario : pepito
Ejemplo buscar etiqueta : #programacion
Aqui dejo mi barmenu el cual contiene el form buscador

  Inicio
  <button class="navbar-toggler" type="button" data-toggle="collapse" data-target="#navbarSupportedContent" aria-controls="navbarSupportedContent" aria-expanded="false" aria-label="Toggle navigation">

  </button>

        Mi perfil (current)

          Mensajes

          Crear mensaje
          Ver mis mensajes

    <form class="form-inline my-2 my-lg-0" method="POST" action="buscador.php">
      <input class="form-control mr-sm-2" type="search" name="buscador" aria-label="Search" placeholder="#etiqueta1 usuario">
      <button class="btn btn-outline-success my-2 my-sm-0" type="submit">Buscar</button>
    </form>
    Salir

En concreto este es el buscador :
<form class="form-inline my-2 my-lg-0" method="POST" action="buscador.php">
      <input class="form-control mr-sm-2" type="search" name="buscador" aria-label="Search" placeholder="#etiqueta1 usuario">
      <button class="btn btn-outline-success my-2 my-sm-0" type="submit">Buscar</button>
    </form>

El fichero al que llama el action del form es el siguiente:
<?php

    include_once 'conexion.php';
    include_once "controllers/mensaje.php";

    $mensaje_controller = new Mensaje();

    $buscador = $_POST['buscador'];
    $esUsuario;

        if (substr($buscador, 0, 1) === '#') {
            //etiqueta

            $buscador = str_replace("#","",$buscador);
            $result = $mensaje_controller->verMensajesContengaEtiqueta($buscador);
            $esUsuario = false;
        }
        else{
            //usuario
            $mensaje_controller= new Mensaje();
            $result = $mensaje_controller->verMensajesUsuario($buscador);
            $esUsuario = true;
        }

         if ($result) {

            while ($row = mysqli_fetch_array($result)) {

                  $mensajes[] = [
                        'id' => $row['mensaje_id'],
                        'titulo' => $row['mensaje_titulo'],
                        'contenido' => $row['mensaje_contenido'],
                        'createAt' => $row['mensaje_createAt'],
                        'creador' => $row['mensaje_creador'],
                        'url_img' =>     $row['mensaje_url_img'],
                        'etiquetas'=> [
                            'ids'=> explode('|', $row['etiquetas_ids']),
                            'nombres'=> explode('|', $row['etiquetas_nombres'])
                        ]
                    ];
            }

         }

         //var_dump($mensajes);

     ?>

En este fichero buscador.php hace un poco como de organizador previo a la busqueda, es decir, mira si tiene #, si es que tiene llama a la funcion que busca etiquetas de mi controlador.Si no tiene # llama a la funcion que busca usuarios.
Bueno, pues para conseguir el funcionamiento de mi buscador estoy haciendo dos consultas:

La primera consulta (esta funcion seria si busca por usuario) debe obtener todos los mensajes de un usuario, esta funcion bien:
     SELECT
                          mensajes.id AS 'mensaje_id',
                          mensajes.titulo AS 'mensaje_titulo',
                          mensajes.contenido AS 'mensaje_contenido',
                          mensajes.createAt AS 'mensaje_createAt',
                          usuarios.usuario AS 'mensaje_creador',
                          mensajes.url_img AS 'mensaje_url_img',
                          GROUP_CONCAT(etiquetas.id SEPARATOR '|' ) AS 'etiquetas_ids',
                          GROUP_CONCAT(etiquetas.nombre SEPARATOR '|') AS 'etiquetas_nombres'

                     FROM etiquetas_en_mensajes

                    INNER JOIN mensajes ON mensajes.id = etiquetas_en_mensajes.id_mensaje
                    INNER JOIN etiquetas ON etiquetas.id = etiquetas_en_mensajes.id_etiqueta
                    INNER JOIN usuarios ON usuarios.id = mensajes.creador
                    WHERE usuarios.usuario = 'jose'

                    GROUP BY mensajes.id
                    ORDER BY mensajes.id DESC

La segunda consulta (esta funcion seria si busca por #etiqueta) la he hecho siguiendo el mismo patron de la primera consulta
        SELECT
               mensajes.id AS 'mensaje_id',
               mensajes.titulo AS 'mensaje_titulo',
               mensajes.contenido AS 'mensaje_contenido',
               mensajes.createAt AS 'mensaje_createAt',
               usuarios.usuario AS 'mensaje_creador',
               mensajes.url_img AS 'mensaje_url_img',
               GROUP_CONCAT(etiquetas.id SEPARATOR '|' ) AS 'etiquetas_ids',
               GROUP_CONCAT(etiquetas.nombre SEPARATOR '|') AS 'etiquetas_nombres'
            FROM
                etiquetas_en_mensajes
           INNER JOIN mensajes ON mensajes.id = etiquetas_en_mensajes.id_mensaje
           INNER JOIN etiquetas ON etiquetas.id = etiquetas_en_mensajes.id_etiqueta
           INNER JOIN usuarios ON usuarios.id = mensajes.creador
            WHERE
                    etiquetas_en_mensajes.id_etiqueta  IN  (
                      SELECT
                            etiquetas.id
                        FROM
                            etiquetas
                        WHERE
                            etiquetas.nombre = 'nombreEtiqueta'
                    )
            GROUP BY mensajes.id
            ORDER BY mensajes.id DESC

Esta ultima no lo hace mal del todo,  pero solo me tra la etiqueta que le digo que busque y deberia traer todas las demas etiquetas si es que las hay, asociadas al mismo mensaje

Por ejemplo:

Primera consulta: en las etiquetas(que es donde esta la problematica) tendria el valor etiqueta_1|etiqueta_2|etiqueta_3|etiqueta_4. Eso seria lo correcto.

Segunda consulta: Pero en mi segunda consulta que busco por nombreEtiqueta me sale lo siguiente: soloNombreEtiquetaPorlaQueBuscoHabiendoAParteDeEstaMasQueNoLasMuestra (En la subconsulta hago uso de IN porque esta me va a devolver una coleccion de datos)

La segunda consulta deberia decir: Buscame todos los mensajes donde al menos tengan la etiqueta X (pero traeme las demas etiquetas asociadas al mensaje tambien, la etiqueta X es simplemente el criterio de busqueda)
El diagrama de BD esta en esta otra pregunta:

A: Ya tienes vinculada la tabla etiquetas mediante INNER JOIN etiquetas... así que no te hace falta un where tan "complicado", quita:
          WHERE etiquetas_en_mensajes.id_etiqueta  IN  (
              SELECT
                    etiquetas.id
                FROM
                    etiquetas
                WHERE
                    etiquetas.nombre = 'nombreEtiqueta'
            )

y lo cambias por :
WHERE etiquetas.nombre LIKE '%nombreEtiqueta%'

El operador IN ,además de lento, tiene limitaciones respecto al
  tamaño de la lista donde buscar, evítalo en la medida de lo posible.
"The number of values in the IN list is only limited by the max_allowed_packet value"
Fuente: https://dev.mysql.com/doc/refman/8.0/en/comparison-operators.html#function_in

Revisa también búsqueda por patrón.

(edición)
Para no perder el resto de etiquetas, debido a la restricción del WHERE añadimos otra vinculación a la tabla etiquetas pero esta vez con un alias para poder referenciarla de forma unívoca en el SELECT:
(edición 2)
Verificado, funciona con INNER JOIN, se añade :
INNER JOIN etiquetas_en_mensajes AS em2 ON mensajes.id = em2.id_mensaje
INNER JOIN etiquetas AS e2 ON e2.id = em2.id_etiqueta

La consulta quedaría así:
   SELECT
          mensajes.*,
          GROUP_CONCAT(e2.id SEPARATOR '|' ) AS 'etiquetas_ids',
          GROUP_CONCAT(e2.nombre SEPARATOR '|') AS 'etiquetas_nombres'
     FROM mensajes
    INNER JOIN etiquetas_en_mensajes ON mensajes.id = etiquetas_en_mensajes.id_mensaje
    INNER JOIN etiquetas AS e  ON e.id = etiquetas_en_mensajes.id_etiqueta
    INNER JOIN etiquetas_en_mensajes AS em2 ON mensajes.id = em2.id_mensaje
    INNER JOIN etiquetas AS e2 ON e2.id = em2.id_etiqueta
    WHERE e.nombre LIKE '%nombreEtiqueta%'
    GROUP BY mensajes.id
    ORDER BY mensajes.id DES

Verificación: https://rextester.com/IDGT38023

redpajama_set_name: RedPajamaStackExchange


text:
NORTH MIAMI TO HOST SERIES OF CULTURAL ACTIVITIES DURING 2019 HAITIAN HERITAGE MONTH CELEBRATION
May is Haitian Heritage Month! The City of North Miami will continue its yearly tradition of celebrating the rich culture of the Republic of Haiti and the Haitian diaspora, with an enriching schedule of programs. Haitian culture will be celebrated through education, the arts, food, music, customs and tributes to Haitian pioneers.
Wednesday, May 1, join North Miami Mayor, Dr. Smith Joseph, at the City of North Miami's Haitian Heritage Month Kick-Off Celebration. At 5 p.m. the Mayor will host a Street Naming Ceremony in honor of Haitian folk singer, songwriter, acoustic guitarist and political activist, Joseph "Manno" Charlemagne. The ceremony will take place at the intersection of NW 7th Avenue and 125th Street. At 6 p.m. the Mayor's celebration will relocate to North Miami's MOCA Plaza for an evening of live Kompa performances by Bemol, Alamod and We Dem Zoes, plus a history corner, food from various regions of Haiti, and a showcase of MOCA's PÒTOPRENS art exhibition. The Mayor will also present a Key to the City to both Henri R. Ford, M.D., MHA, Dean of the University of Miami's Miller School of Medicine and Jean-Marie Denis aka, "Jan Mapou," a Haitian author, curator, community activist, and historian. MOCA Plaza is located at 770 NE 125th Street, North Miami.
Friday, May 3, at 6 p.m., the North Miami Public Library will host a Reception and Unveiling of the Haiti: An Island Luminous Kiosk. An Island Luminous combines rare books, manuscripts, and photos scanned by archives and libraries in Haiti and the United States, with commentary by over 100 authors from universities around the world. The NoMi Library is located at 835 NE 132nd Street, North Miami.
The Taste of Haiti Food Festival returns to North Miami on Saturday, May 11, at noon, in the MOCA Plaza. In conjunction with the Haitian Culinary Alliance, this free event promotes and introduces Haiti's cuisine and cultural richness to neighbors and friends throughout South Florida.
The 2019 NoMi Nights Concert and Haitian Flag Day Pre-Celebration is set for Thursday, May 16, at 6 p.m. in MOCA Plaza. Guests are encouraged to wear Haitian flags and colors. This event features a live concert performance by KAI, a local talent showcase, an appearance by Rara Lakay and returning hosts, Success Junior and Plus Daddy of Haitian American Productions.
Jazz At MOCA is Friday, May 24, at 8 p.m., in the MOCA Plaza. Jean P. Jam will be the featured performer at the free monthly concert event.
The 2nd Annual Haitian American Amateur Championship (HAAC) Golf Tournament and Luncheon will take place at 7:30 a.m., Saturday, May 25, at the Miami Shores County Club. This golf tournament and luncheon benefits the Golf Federation of Haiti Sister Cities Program and its goal of building a driving range and 3-hole golf course practice facility at Haiti's Olympic Sports Complex in Port-au-Prince. To register visit, www.HAAC.eventbrite.com. For more information, contact HAAC at 786-970-7403 or email TheHAAC@gmail.com. The Miami Shores Country Club is located at 10000 Biscayne Boulevard, Miami Shores.
For more information about the City of North Miami's 2019 Haitian Heritage Month Celebration, visit NorthMiamiFL.gov/Celebrate or call the Special Events Department at 305-895-9840


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The world's first fully automated metabolite extraction system.
In late 2013 Labman delivered the world's first fully automated metabolite extraction system to a large research institute in the USA. The system automatically processes samples from leaf through to phase separated extract in GC vials, ready for analysis. The system is used for the preparation of plant tissues and subsequent polar and non-polar metabolite extraction assays. Samples are produced ready for analysis on LC-MS or GC-MS instruments. The process is complex and laborious to undertake manually.


You've purchased your Merkur razor Vulfix shaving brush, now where are you going to put it? Give your razor and shaving brush a long life by keeping them clean and dry by investing in a stunning chrome shaving stand that will look great in any guy's bathroom. Take care of your shaving tools and they will take care of you!
The Universal Shaving Stand is the perfect way to safely store and display your double edge safety razor, Mach3 razor or Fusion razor.


text:
Chittoor () ist eine Stadt im indischen Bundesstaat Andhra Pradesh.

Chittoor liegt 120 km westlich von Chennai. Der Ponnai, ein linker Nebenfluss des Palar, fließt im Norden und Osten an der Stadt vorbei.

Im Juli 2012 wurde die Stadt zu einer Municipal Corporation erhoben.
In diesem Rahmen wurden 14 benachbarte Gram Panchayats eingemeindet.
Chittoor ist Verwaltungssitz des gleichnamigen Distrikts.
Die Stadt hatte beim Zensus 2011 160.722 Einwohner. Sie erstreckt sich über eine Fläche von 69,75 km².

Die nationale Fernstraße NH 4 (Bangalore–Chennai) führt an Chittoor vorbei. Der Bezirk Chittoor ist auch bekannt für feine Mangosorten und hat viele Mangomärkte.

Weblinks
 Chittoor Municipal Corporation (Stadtverwaltung)

Einzelnachweise

Ort in Andhra Pradesh
Municipal Corporation in Andhra Pradesh
Distrikt Chittoor

redpajama_set_name: RedPajamaWikipedia


travel guitars designed to meet Airlines' carry-on luggage size and weight requirements.
checking-in, carry-on, extra luggage costs, or lost and damaged checked instruments.
bevel, an armrest bevel, and a thin profile neck.
• Street: Black Fretted or Fretless $1199.99 w/travel bag.


text:
Amazon to Use Electrofuels in its Middle Mile Fleet
Published on: 22 Sep 2022
Amazon will begin using Infinium Electrofuels in the retailer's middle mile fleet as an ultra-low carbon alternative to traditional fossil fuels. The clean-burning electrofuels will be produced for Amazon by Infinium at one of the world's first electrofuels production facilities located in Texas.
Infinium Electrofuels, which are produced from carbon dioxide waste and renewable power, can be dropped into Amazon trucks with no engine modifications as an immediate replacement for petroleum-based fuel. These electrofuels dramatically reduce greenhouse gas emissions over traditional fossil fuels, making them among the strongest ultra-low carbon alternatives to traditional fuels without requiring costly engine modifications.
With the transportation sector accounting for approximately 25% of all CO2 emissions globally according to the UN Environment Programme, the ability to reduce emissions from trucks, aeroplanes, and ships without modifying existing infrastructure is critical to reaching a decarbonized world and a vision of net-zero CO2 emissions.
The Infinium Electrofuels production facility in Texas will use approximately 18,000 tons per year of CO2 waste that would have otherwise been released into the atmosphere, producing enough electrofuels to power vehicles in Amazon's middle mile fleet for approximately 5 million miles per year. The Infinium facility is due to begin production in 2023, and Amazon plans to initially begin using the electrofuels in the Southern California region.
Amazon previously supported Infinium's development of electrofuels technology through two rounds of investment through Amazon's Climate Pledge Fund, a $2 billion venture investment program that specifically invests in companies building technologies, products and services that will help Amazon and others accelerate the path toward net zero carbon future. While the new agreement is focused on providing Infinium Electrofuels for Amazon's middle mile fleet, Electrofuels can also be used to power aeroplanes and used as an ultra-low carbon fuel alternative in the production of plastics and other industrial materials.
Source: Environmental Leader
For more company-related news
More from Amazon.com, Inc.
AWS Aims to be Water Positive by 2030
Amazon will Invest $150 Million in Funds that Provide Capital to Underrepresented Entrepreneurs
Amazon Awards €100,000 Grant to SME to Make Environmentally Friendly Apparel for Children
Amazon Uses Green Hydrogen in Operations to Help Reduce Carbon Emissions
New Amazon Programme Provides Up to Five Small Businesses With $20,000


Oh my! DJ Gauffie strikes again with another gorgeous mix partly made up of Swedish components and partly of foreign ones but the result is all sorts of weekend brilliance!
First time we mentioned DJ Gauffie talking about his mashup of Lena Philipsson's "Idiot" and Taio Cruz's "Higher" and now he has mixed up Axwell's "I Found U", Martin Solveig "Hello" and Avicii's "Levels" - track that we surprisingly completely have missed but which is the main component of this mashup with its loud clubecstatic synths - the most appreciable dance-feature of this season.
What has come out of it this combination you can check right below and don't tell me it's not amazing!


text:
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Comics: A Global History, 1968 To The Present REVIEW
BOOK REVIEW Pretentious? Moi?
By sfx 02 May 2014
Comics: A Global History, 1968 To The Present book review .
The world is a big place, 45 years is a long time, and comics is a wide field. A survey of all this in just 300 pages would have to be pretty shallow and perfunctory. It may be lucky, then, that this book isn't quite what it claims to be. It's not "global", but concentrates on the US, Japan and France, with incidental reference to other places. It focuses mainly on Underground, alternative and avant-garde comix. It doesn't really start in 1968 either, or even justify why it should, except that Robert Crumb's Zap Comix appeared in that year
Sadly, it's still too sweeping, and not just in scope. It is a history in the sense that it tries to construct a narrative of movements and "development", but the connections it draws are often tenuous and evidence thin on the ground. It's packed with throwaway value judgements, repeatedly claiming that works are "considered to be" this or that without specifying by whom, or referring to works and artists as "major", "important" or even "successful" without defining these terms. It's thick with the jargon of pseudo-academic comics crit, but thin on intellectual rigour.
We're told, for example, that Jacques Tardi's "ornately patterned interiors recall Vuillard". Does this mean the comic artist quotes or riffs off the post-impressionist painter (which tells us about his aspirations) or just that this book's authors studied art history (which tells us about their pretensions)? They use "cross-hatching" to describe almost any rendering technique, including Virgil Finlay's stippling. There's a lot of stilted twaddle, and a few paragraphs of sheer gibberish that look like cut-and-paste blunders.
There are glaring omissions too: there's no mention of the UK's groundbreaking Action comic, Raymond Briggs, Joe Kubert's Yossel , the readership of albums in France, or Bryan Talbot's seminal work since Luther Arkwright - even the masterful Alice In Sunderland . There are silly errors, such as an offhand reference to "Marvelman (originally Miracle Man)" or the assertion that the X-Men had been "out of print for five years" before Giant-Size X-Men #1.
There are insights to be gleaned here, sure, and lesser-known gems to discover. So buy the book, scour it for these, and send the authors feedback about the rest. If it sells well enough, they might be allowed to write a second edition. Given another year of work, a more accurate title, plus about 100 pages more nuance and depth, that could become the illuminating overview this isn't.
Alex Summersby
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text:
Smiling Men with Bad Reputations je první sólové studiové album skotského hudebníka Mikea Herona, vydané v roce 1971 u vydavatelství Island Records (Spojené království) a Elektra Records (Spojené státy americké). Album produkoval Joe Boyd a vyšlo ještě v době, kdy byl Heron členem skupiny The Incredible String Band. Na albu se podílela řada dalších hudebníků, jako například Keith Moon a Pete Townshend (ze skupiny The Who), Simon Nicol, Dave Pegg a Richard Thompson (Fairport Convention) nebo John Cale (The Velvet Underground).

Seznam skladeb
Autorem všech skladeb je Mike Heron.

Obsazení
 Mike Heron – kytara, klávesy, zpěv
 John Cale – baskytara, kytara, klavír, harmonium, doprovodné vokály
 Gerry Conway – bicí
 Tony Cox – syntezátor
 Pat Donaldson – baskytara
 Dr. Strangely Strange – doprovodné vokály
 Ronnie Lane – baskytara
 Sue Glover – doprovodné vokály
 Mike Kowalski – bicí
 Malcolm Le Maistre – klarinet
 Sunny Leslie – doprovodné vokály
 Dave Mattacks – bicí
 Keith Moon – bicí
 Simon Nicol – kytara
 Dave Pegg – baskytara
 Dudu Pukwana – klavír, saxofon
 Rose Simpson – baskytara
 Liza Strike – doprovodné vokály
 Richard Thompson – kytara
 Pete Townshend – kytara
 Heather Wood – doprovodné vokály

Reference

Alba v angličtině
Debutová alba
Alba z roku 1971
Alba Elektra Records
Alba Island Records
Alba, která produkoval Joe Boyd


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