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Theranostics 2014; 4(8):798-807. doi:10.7150/thno.8934
Research Paper
Enhanced Plasmonic Resonance Energy Transfer in Mesoporous Silica-Encased Gold Nanorod for Two-Photon-Activated Photodynamic Therapy
1. Institute of Biomedical Engineering and Nanomedicine (I-BEN), National Health Research Institutes, Zhunan, Miaoli 350, Taiwan.
2. Department of Chemistry, National Taiwan University, Taipei 106, Taiwan.
3. Department of Radiology, The University of Chicago, Chicago IL, 60637, USA.
Chen NT, Tang KC, Chung MF, Cheng SH, Huang CM, Chu CH, Chou PT, Souris JS, Chen CT, Mou CY, Lo LW. Enhanced Plasmonic Resonance Energy Transfer in Mesoporous Silica-Encased Gold Nanorod for Two-Photon-Activated Photodynamic Therapy. Theranostics 2014; 4(8):798-807. doi:10.7150/thno.8934. Available from http://www.thno.org/v04p0798.htm
The unique optical properties of gold nanorods (GNRs) have recently drawn considerable interest from those working in in vivo biomolecular sensing and bioimaging. Especially appealing in these applications is the plasmon-enhanced photoluminescence of GNRs induced by two-photon excitation at infrared wavelengths, owing to the significant penetration depth of infrared light in tissue. Unfortunately, many studies have also shown that often the intensity of pulsed coherent irradiation of GNRs needed results in irreversible deformation of GNRs, greatly reducing their two-photon luminescence (TPL) emission intensity. In this work we report the design, synthesis, and evaluation of mesoporous silica-encased gold nanorods (MS-GNRs) that incorporate photosensitizers (PSs) for two-photon-activated photodynamic therapy (TPA-PDT). The PSs, doped into the nano-channels of the mesoporous silica shell, can be efficiently excited via intra-particle plasmonic resonance energy transfer from the encased two-photon excited gold nanorod and further generates cytotoxic singlet oxygen for cancer eradication. In addition, due to the mechanical support provided by encapsulating mesoporous silica matrix against thermal deformation, the two-photon luminescence stability of GNRs was significantly improved; after 100 seconds of 800 nm repetitive laser pulse with the 30 times higher than average power for imaging acquisition, MS-GNR luminescence intensity exhibited ~260% better resistance to deformation than that of the uncoated gold nanorods. These results strongly suggest that MS-GNRs with embedded PSs might provide a promising photodynamic therapy for the treatment of deeply situated cancers via plasmonic resonance energy transfer.
Keywords: Gold nanorods, photodynamic therapy, plasmonic resonance energy transfer, surface plasmon resonance, two-photon luminescence.
Gold nanomaterials, and gold nanorods (GNRs) in particular, have recently received significant attention due to their photophysical and photochemical properties [1-3]. GNRs possess transverse and longitudinal surface plasmon resonances (SPRs) that correspond to electron oscillations perpendicular and parallel to the direction of the nanorod's major axis, respectively. The SPR band can be easily tuned from visible to infrared wavelengths through adjustments of the nanorod's aspect ratio. In addition, this unique SPR property contributes a local field enhancement of the radiative rate of electron-hole recombination processes, resulting in a considerable enhancement of the photoluminescence. The photoluminescence from GNRs is of particular interest in the fields of nanoscience and nanotechnology for various potential applications, including biomolecular sensing and imaging [4-8]. Photoluminescence from bulk gold was first reported by Mooradian in 1969 [9]. Subsequently, the mechanism and phenomenon was correlated with the electronic structure of gold nanoclusters, elucidated by Apell and Boyd [10,11]. Excitation takes place via transition of electrons from the d- to the sp-band, to generate electron-hole pairs. The observed emission results from the radiative electron-hole recombination of the excited electrons in the sp-band with the holes in the d-band, with SPR enhancement [11-13]. The luminescence of GNR is resonantly enhanced by a factor of approximately 106 compared with that of bulk gold, a phenomenon known as the lightning-rod effect [14-16]. Heat is also generated as a consequence of electron-phonon collisions, garnered as the source for photothermal therapy.
Two-photon luminescence (TPL) from GNRs has considerable potential in biomedical imaging because of its high resolution, low photo-damage, good biocompatibility and low attenuation by water and biomaterials, enhancing photon penetration depth in tissue [17]. Although the quantum yield of emission of GNR is quite small, the fact that GNRs possess very high two-photon absorption cross-sections, about two order of magnitude higher than that of normal organic fluorophores [18], compensates enough to make GNR attractive as a two-photon luminescence imaging agent. For example, Chen et al. investigated a polystyrenesulfonate-coated GNR for two-photon photoluminescence imaging and in situ real-time photothermolysis in cancer cells [19]. Wang et al. also studied the in vitro and in vivo luminescence of GNRs in mouse ear blood vessels by two-photon activation and determined that the TPL intensity from a single GNR was 58 times brighter than that of a single rhodamine molecule [18]. For the application of photodynamic therapy (PDT), Yeh et al. coated poly(styrene-altmaleic acid) and indocyanine green onto the surface of GNRs, to form a nanoplatform for NIR optical imaging and photo-thermal/photodynamic therapy [20]. GNRs have also been used as a carrier and fluorescence quencher for PSs [21-23]. PSs have been conjugated onto the surface of GNRs via a protease-cleavable peptide linkage. Tumor-selective PDT and photothermolysis therapy (PTT) were performed after tumor targeting [22]. Photo-thermal effects generated by GNRs have also been exploited for externally controlled drug release via NIR lasers. Zhang et al. coated mesoporous silica on the outside of GNRs that served as multifunctional theranostic platforms: (a) the mesoporous SiO2 shell could provide a high surface area for the drug payload, while (b) the inner gold nanorod functioned as a two-photon imaging agent, hyperthermia agent, and a light-mediated trigger for drug release [24]. Same material with different formation, gold nanocages (AuNCs), was also employed for combination of two-photon photo-thermal/photodynamic cancer therapy. The assembly of photosensitizer, hypocrellin B and photo-thermal transducer, AuNC with utilization of two-photon techniques results in one-off administration and irradiation for antitumor treatment. With NIR two-photon irradiation, AuNCs can convert light into heat and excite PS to generate singlet oxygen species [25].
However, studies have shown that repeated irradiation of GNRs and gold nanocrystals with intense laser pulses causes the GNRs to deform into gold nanospheres. Typically, pulse energies on the order of only 10 μJ are sufficient to completely convert gold nanorods into gold nanospheres [26-29]. The deformation of GNRs into spheroids inflicts a collateral spectral shift of peak surface plasmon resonance absorption, as well as a decrease in TPL intensity. To combat such losses, several groups have employed silica shell encasements of their nanorods as stabilizers to preclude thermal deformation of GNRs [30-32]. In previous work, we enveloped GNRs in a silica-matrix to enhance their stability during photoacoustic wave induction [30]. Our silica-coated GNRs demonstrated significantly greater resistance to degradation of signal intensity than that of the uncoated GNRs, significantly improving their use as high-contrast agents for photoacoustic imaging.
In this work, we report on the development of mesoporous silica-coated gold nanorods (MS-GNRs) bearing PSs, forming a stable and functionalizable probe for TPA-PDT. The mesoporous SiO2 shell deposited around our GNRs not only provided a large surface area for conjugating therapeutic moieties, but also served as a platform for intra-particle/plasmonic energy transfer between the GNRs and the payload materials. Providing confinement by mesoporous silica, the deformation of GNRs was significantly suppressed, with their TPL stability being greatly improved. For therapeutic application, not like widely used photothermal therapy, we incorporated a PS (Pd-meso-tetra(4-carboxyphenyl) porphyrin, PdTPP) into the nano-channels of the GNR's mesoporous silica shell. PdTPP is a metallo-porphyrin that not only could be used to measure oxygen distributions in tissues via oxygen-dependent quenching of phosphorescence but also used as an efficient PS [33]. By conjugating PdTPP with MS-GNR, this nanoplateform can be made to switch between being a phosphorescence probe for oxygen sensing/imaging (diagnostics) and a PS for PDT (therapeutics) by simply changing the energy of photoirradiation. It opened the possibility of on-site diagnostic and therapeutic application in the future. Highly efficient excitation of the PS was achieved by proximal energy transfer from the GNRs surface plasmons to the PSs and then to neighboring oxygen molecules via collision. Following two-photon excitation of GNRs, cytotoxic singlet oxygen was generated and verified both in vitro and in vivo in a breast-cancer mouse models. As TPA-PDT employs near-infrared light, our PSs functionalized MS-GNRs achieve much greater penetration depths than PDT methods, making them well suited especially for the PDT treatment of deeper internal organs.
Experimental Section
Materials. Hexadecyltrimethylammoniumbromid (CTAB, 99%, Acrôs organics), gold(III) chloride trihydrate (HAuCl4 ∙ 3H2O, sigma-Aldrich), tetraethyl orthosilicate (TEOS, 99.0%, Aldrich), N,N-dimethylformamide (DMF, 99.8%, Sigma-Aldrich), L-ascorbic acid (AA, 99.7%, sigma-Aldrich), Sodium borohydride (NaBH4, 99.9%, Fluka), Silver nitrate solution (AgNO3, 0.01 mol/l, Fluka), N-octane (98%, Sigma-Aldrich), di(N-succinimidyl) carbonate (DSC, 95% Fluka), N,N-diisopropylethylamine (99.5%, Sigma-Aldrich), Deionized water (18 MΩ) was used in all the experiments.
Synthesis of MS-GNR. Gold nanorods were synthesized by seed-mediated growth according to previously described procedures [34]. First, 5 mL of CTAB solution (0.20 M) was mixed with 5 mL of HAuCl4 solution (0.5 mM). Subsequently, 0.60 mL of ice-cold NaBH4 solution (0.01 M) was added to the mixture and vigorously stirred for 2 minutes at 28°C, which resulted in the formation of a brownish-yellow seed solution. The growth solution was prepared by adding 0.2 mL of AgNO3 (4 mM) and 5 mL of HAuCl4 (1 mM) solutions to 5 mL of CTAB (0.20 M) solution under gentle mixing followed by the addition of 70 µL of L-ascorbic acid (0.0788 M) solution. To grow the nanorods, 12 μL of the seed solution was added to the growth solution at 27-30 °C under gentle stirring for 30 seconds. The color of the solution changed to burgundy-red within 10-20 minutes. The solution was then aged for an additional 24 hours at 28 °C before being centrifuged at 12,000 rpm for 30 minutes. For large-pore mesoporous silica coatings, 333 μL n-octane and 0.10 mL of 0.1 M NaOH solution were added to 10 mL of the prepared GNR solution under stirring. Subsequently, 60 μL of 20% TEOS in methanol was injected four times into the mixture at 30-minute intervals. The mixture was then allowed to react for 16 hours and was continued for an additional 4 hours after 35 μL of APTMS (95%) was added [35]. The resulting MS-GNR particles were then centrifuged and washed with DI water and ethanol at least three times to remove the CTAB. The surfactant templates were removed via the reaction with 0.1 M HCl for 20 hours followed by three washes with ethanol.
Synthesis of MS-GNR-PdTPP. We covalently linked the sensitizer group PdTPP to the surface of mesoporous silica by our previously reported method [36]. 40 mg of Pd-meso-tetra(4-carboxyphenyl) porphyrin (PdTPP) and 150 mg of DSC were mixed with 97 μL of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide in DMF (20 mL) solution for 2 hours. The activated PdTPP was then reacted with 100 μL of APMTS for 1 hour and subsequently purified via passage through a PD-10 column (Amersham Biosciences). MS-GNR particles (20 mg) were mixed with 400 μL of APTMS-modified PdTPP in ethanol solution, and the mixture was stirred continuously at room temperature for 20 hours. Solid samples were collected via centrifugation at 12,000 rpm for 20 minutes; the samples were subsequently washed, and the pellet was redispersed with deionized water and ethanol several times. The amount of PdTPP loading in the MS-GNRs was determined by measuring the absorbance of the reaction solution at 400 nm (i.e., the Soret band of PdTPP).
Two-photon luminescence and photostability measurements. The two-photon luminescence and photostability measurements were performed using an inverted scanning confocal microscope (Zeiss LSM 710). A femtosecond Ti:Sapphire laser (MaiTai, Spectra-Physics) was used as the excitation source to generate the TPL from MS-GNR. The output laser pulse was centered at 800 nm, and the pulse duration was ~200 femtoseconds with a repetition rate of 80 MHz. The laser beam was directed into the microscope using a water-immersion objective lens (N.A=1.4) to focus the laser beam onto the MS-GNRs, which were dispersed onto a clean glass coverslip with water. The TPL from MS-GNR was collected through the same objective, separated from the excitation laser by a dichroic beamsplitter, and then detected with a photomultiplier tube (PMT) placed in the back port of the microscope. A bandpass filter (400 to 690 nm) was placed before the PMT to minimize the scattering of photons from the excitation source. For the photostability measurements, bare GNR or MS-GNR particles were irradiated under repetitive laser pulses with average power of 120 mW and the TPL was monitored as a function of time. For two-photon luminescence imaging, cells were monitored with excitation power of 4 mW after treatment of the MS-GNRs or MS-GNR-PdTPPs in a serum-free medium for 1 hour at 37 °C.
Singlet oxygen measurements. The chemical oxidation of 9,10-anthracendipropionic acid (ADPA) in an aqueous suspension of the nanoparticles was used to determine the singlet oxygen generation [37]. Aqueous solutions of ADPA (20 μL, 12.2 mM) were mixed with MS-GNR-PdTPPs solution in D2O (0.5 ml), whereas GNR solution with ADPA or PdTPP solution with ADPA was used as the control. All solutions were then irradiated under femtosecond laser with same power of 350 mW at 800 nm (Spitfire Pro, Spectra-Physics). The change in the absorbance of the ADPA at 378 nm was monitored as a function of time. All experiments were carried out in the dark room during the measurement.
In vitro two-photon luminescence images and PDT effects. MDA-MB-231 breast cancer cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS) at 37 °C under 5% CO2. Cells were seeded at a density of 3×104 on a 35 mm dish and incubated for 24 hours. To obtain two-photon luminescence images, the cells were treated with 50 μg/mL MS-GNR-PdTPPs in a serum-free medium for 1 hour at 37 ºC in darkness and then washed twice with phosphate buffered saline (PBS). The cell nuclei were stained with 1 μM Hoechst 33342 for 10 minutes and washed three times with PBS. To determine the in vitro PDT effects, 50 μg/mL MS-GNRs or MS-GNR-PdTPPs were added to the cell dish with serum-free medium for 1 hour. Cells were washed with PBS to remove the excess nanoparticles and placed in fresh medium that contained YOPRO-1 and propidium iodide (PI) dyes (Molecular Probes, USA). GNRs were excited using a Ti:sapphire laser (Mai Tai, Spectra-Physics) at 800 nm with an output power of 3 mW. For cell morphology and fluorescence imaging, YOPRO-1 was excited at 488 nm and monitored using a 525±5 nm bandpass filter while PI was excited at 535 nm and monitored using a 620±5 nm bandpass filter. Images were collected using a confocal microscope (Zeiss LSM 710).
In vivo two-photon excited PDT effect. All experiments that involved animals were performed in accordance with the guidelines of the Management Group of Animal Experiments (MGAE) in Taiwan (NHRI-IACUC-101079-A). Male nude mice (nu/nu; 20-25 g; 6-8 weeks of age; BioLasco Taiwan) were anesthetized by isoflurane inhalation and subcutaneously inoculated with MDA-MB-231 cells (2 × 106 cells in 0.1 mL PBS). In vivo two-photon excited PDT experiments were performed when the tumor volume reached approximately 200 mm3. The nude mice were anaesthetized with 80 mg/kg ketamine and 13 mg/kg xylazine i.p. and received intra-tumor injections of sonicated MS-GNR-PdTPPs at a concentration of 16 mg/kg in 100 μL of saline solution. Control groups of mice received intra-tumor injections of saline, MS-GNRs or PdTPP alone. At 1 hour post-injection, the tumors in mice were irradiated using a Ti:Sapphire femtosecond amplifier (Spitfire Pro, Spectra-Physics). The femtosecond laser amplifier output was 1.2 W with a repetition rate of 1 kHz, pulse duration of 100 fs, central wavelength of 800 nm, collimated beam diameter of ~7 mm and Gaussian beam profile. An optical chopper (SR541, Stanford Research Systems) was used to block the laser and reduce the number of laser pulses onto the mice to 1/12 to prevent direct photothermal heating damage to mice skin tissue. An average power of 100 mW was measured and irradiated onto the mice with exposure time of 16 minutes. The total exposure energy is 96 J. For the histological evaluation, tumors were sectioned for hematoxylin and eosin, DAPI, TUNEL and caspase-3 staining. The slices were examined using an Olympus BX51 microscope.
Transmission electron microscopy (TEM). TEM imaging of GNR and MS-GNR particles was performed using a Libra 120 TEM transmission electron microscope (Carl Zeiss). A carbon-coated 200 mesh copper grid was pre-rinsed with 5 μl of ethanol and then deposited with 5 μL of the suspension of GNR or MS-GNR particles in ethanol. After 3 minutes, the sample was dried with filter paper and further dried with a flow of N2 gas. The sample was then viewed on the electron microscope, and images were obtained using a digital camera attached to the microscope. For TEM imaging of tumor slides, mice were sacrificed at 24 hours after MS-GNR-PdTPP treatment, and their tumor specimens were fixed overnight in glutaraldehyde buffered (2.5%) with phosphate buffered saline (PBS; 0.1 M, pH 7.4). Tissues were then washed three times in PBS and post-fixed for 1 hour in a solution that contained OsO4 buffered (2%) with PBS. Tissues were subsequently washed 3 times in H2O and dehydrated stepwise in ethanol. Tissues were then polymerized using Spurr resin at 68 °C for 15 hours and embedded specimens subsequently sectioned into 70 nm slices and viewed on a Hitachi H-7650 TEM operated at 80 kV.
Results and Discussion
1. Enhancement of the photostability of GNRs by the mesoporous silica confinement
A description of the two-photon-activated photodynamic therapy using mesoporous-silica-encased gold nanorods (MS-GNR-PdTPP) is shown in Scheme 1.
Schematic illustration of two-photon-activated photodynamic therapy (TPA-PDT) using mesoporous silica-encased gold nanorods. Pd-porphyrins (Green sphere), conjugated onto the walls of nanochannels within the mesoporous silica shell, are activated via intra-particle plasmonic energy transfer (ET) from two-photon-excited gold nanorods, to generate cytotoxic singlet oxygen.(Click on the image to enlarge.)
In this approach, gold nanorods were synthesized via a seed-mediated, surfactant-assisted growth procedure [34], followed by its coating with mesoporous silica in a single step [35]. Pd-porphyrins were conjugated to the walls of nano-channels within the mesoporous silica shell via covalent bonds according to our previous method [36]. Following two-photon excitation, two sequential energy transfers were used to generate cytotoxic singlet oxygen: the first energy transfer (1st ET), from the gold nanorod's surface plasmon to a proximal Pd-porphyrin within the silica shell, and the second energy transfer (2nd ET) from the Pd-porphyrin to proximal oxygen molecules. Figure 1a shows TEM images of the MS-GNR with an average particle size of approximately 80 nm. The average aspect ratio (length/diameter) of the gold nanorods was 3.4 ± 0.80. The inset of Figure 1a shows an enlarged image in which the mesoporous structure of the shell is clearly observed. The thickness of the mesoporous silica layer was less than 20 nm. With a femtosecond pulsed laser beam directed into the microscope and onto a clean glass coverslip with water-dispersed MS-GNRs, photoluminescence measurements from the fixed scan area (175 µm × 175 µm) were made. As shown in Figure 1b, a typical photoluminescence spectrum of an aqueous suspension of MS-GNRs excited at 800 nm via a femtosecond laser reveals a broad emission band in the visible region of 450-650 nm. The oscillation structure in this range has been previously observed by Wang et al. [18] Boyd et al. had assigned them as transitions between the Fermi level and the various d bands at various symmetry points [11]. Two-photon luminescence of MS-GNRs was confirmed by the non-linear dependence of the photoluminescence intensity on the excitation power. TPL measurements demonstrated a quadratic dependence of 1.98 ± 0.30 on the incident power, which confirmed that the excitation occurred via a two-photon absorption process (Figure 1c). Figure 1d illustrates the relationship between the excitation spectrum of the two-photon luminescence (open triangles) and the longitudinal plasmon band (solid line) of the MS-GNRs. The significant overlap between the two spectra indicates that the observed two-photon luminescence intensity is governed by the local field enhancement originating from the gold nanorod's surface plasmon resonance. Evidently, MS-GNRs exhibit optical properties that are quite similar to those of bare GNRs.
TEM image and photophysical properties of MS-GNRs. (a) TEM image of MS-GNRs, with insert showing an enlarged view of its mesoporous structure. (b) Photoluminescence spectrum of MS-GNRs in an aqueous solution irradiated with femtosecond laser at 800 nm. (c) Dependence of the integrated photoluminescence intensity of (b), versus the excitation power; slope, obtained from the regression fitting, is ~2 indicating that the measured photoluminescence is indeed TPL. (d) Steady-state absorption of MS-GNRs (solid line) and the TPL excitation spectrum (open triangles).(Click on the image to enlarge.)
In Figure 2, we illustrate the robust stability of the two-photon luminescence intensity of MS-GNRs under repeated laser excitation, as compared with that of un-coated GNRs. Notably, although a small amount of residual CTAB remained on the surfaces of un-coated GNRs during the seed-mediated GNR synthesis, CTAB did not provide nearly as much protection of the nanorods from thermal deformation as did the mesoporous silica shell. Aqueous suspensions of GNR or MS-GNR particles were dispersed on clean glass coverslips, sealed by a rubber cassette to prevent dehydration and any consequent change in nanoparticle concentration. The average of two-photon luminescence intensities in a fixed regions-of-interest (ROIs) was measured and plotted as a function of time. The two-photon luminescence of uncoated GNRs (Figure 2a, black line) decayed rapidly to 39.4% of its initial value during the first 20 seconds of exposure to 120 mW laser irradiation, and decreased further to 32.8% of its initial value after 40 seconds. In contrast, under identical laser irradiation conditions, the TPL of MS-GNRs (Figure 2a, red line) decayed to 94% of its initial maximal intensity during the first 20 seconds and remained relatively unchanged thereafter; with the MS-GNRs' TPL intensity being 260% greater than that of uncoated GNRs. After 100 seconds, the integrated TPL intensity of MS-GNRs was 220% greater than that of the uncoated GNRs.
TEM images of Figures 2b and 2c show the morphology of MS-GNRs, and GNRs respectively, after 100 seconds of laser irradiation. GNRs with a mean aspect ratio of 3.4± 0.8 before laser irradiation absorbed strongly at the 800-nm excitation wavelength and underwent sufficient heating to undergo a shape transformation of many of the nanoparticles. The gentle surface melting of the GNRs leads to shorter, wider rods that possessed an average aspect ratio of 2.7± 1.0. Some of them have even melted into spherical shape (Figure 2c). In contrast, the shape of the nanorod cores within MS-GNRs possessed an average aspect ratio of 3.3± 0.7 after laser irradiation (Figure 2b). Thus, post-irradiated MS-GNRs retained most of the structural features of pre-irradiated MS-GNRs, consistent with the results of the stability of two-photon luminescence intensity measurements.
Enhancement of photostability of GNRs via mesoporous envelopment. (a) The TPL intensities of GNRs (■) and MS-GNRs (red ●) monitored as a function of time, under the exposure of 120 mW repetitive laser pulses. TEM images of MS-GNRs (b) and GNRs (c) following 100 seconds of laser irradiation.(Click on the image to enlarge.)
2. Photodynamic therapy by intra-particle plasmonic energy transfer
Since the two-photon luminescence stability of gold nanorods is greatly enhanced by encapsulating the nanorods within a mesoporous silica matrix, we postulated that MS-GNRs could serve as sustained energy sources for PDT via a plasmonic energy transfer to the Pd-porphyrins adsorbed on the nearby silica shell under two-photon absorption. To substantiate this mechanism, we first examined the effects of conjugating Pd-porphyrins to MS-GNRs. In Figure 3a we present TEM images of MS-GNRs in which Pd-porphyrins had been conjugated onto the nano-channel walls of the mesoporous silica nanoparticles. As might be expected from the doping, the mesoporous structure of MS-GNR-PdTPPs in Figure 3a is darker than the structure of MS-GNR shown in Figure 1a. The surface charge of the MS-GNR-PdTPPs showed a positive ξ-potential value of 4.63 mV, indicating that the nanoparticles could be endocytosed well by MDA-MB-231 breast cancer cells (Figure 3c) [38]. Intra-particle plasmonic energy transfer was characterized by monitoring the emission spectrum within the visible region (450-650 nm) while exposed to 800-nm two-photon excitation. Luminescence of MS-GNRs was clearly noted within the visible spectrum (Figure 3b, black line) - advantageous for resonant energy transfer to the Pd-porphyrins. With the conjugation of Pd-porphyrins onto the nano-channel walls of MS-GNRs, the emission spectrum of MS-GNR-PdTPPs in Figure 3b (red line) showed a significant decrease at wavelengths 500-600 nm, suggesting that highly efficient energy transfer had taken place between the gold nanorods and Pd-porphyrins following two-photon excitation. To evaluate the energy transfer of Pd-porphyrins to neighboring oxygen molecules, we monitored the generation of singlet oxygen via anthracene-9,10-dipropionic acid (ADPA), a singlet oxygen quencher which reacts with singlet oxygen and leads to reduction of its optical absorption at 378 nm [37]. Figure 3d shows the decrease in optical absorption at 378 nm of an ADPA solution bearing MS-GNR-PdTPPs as a function of irradiation time reflecting efficient singlet oxygen production from the MS-GNR-PdTPP via 2-step serial energy transfers following two-photon excitation, with ADPA suspensions of PdTPP and GNRs serving as controls.
To characterize the cytotoxicity of two-photon-induced MS-GNR-PdTPP photodynamic therapy, two indicators cell damage were employed: YOPRO-1 and propidium iodide (PI). Because neither of these two dyes can penetrate cell membranes, counter-staining irradiated cells for YOPRO-1/PI signal reflected cell/nuclear membrane compromised. Repeated irradiation on MS-GNR-PdTPP-treated cells with a 800 nm femtosecond laser (3 mW) revealed increasing YOPRO-1 (green) and PI (red) penetration into cells as a function irradiation time, as shown in Figures 4a-c. Fluorescence intensity measurements of YOPRO-1 and PI, integrated and plotted as a function of time (Figure 4g), demonstrated that the fluorescence intensity of YOPRO-1 (green) and PI (red) increased by 18- and 94-fold respectively, following 194 seconds of laser irradiation. By contrast, no detectable change in the low levels of YOPRO-1 and PI fluorescence was observed for cells treated with MS-GNR (Figures 4d-f and 4h).
(a) TEM imaging of MS-GNR-PdTPPs; scale bar: 0.10 µm (b) Energy transfer in MS-GNR-PdTPPs, showing the TPLs of MS-GNRs (black) and MS-GNR-PdTPPs (red). (c) Confocal microscopy imaging of the endocytosis of MS-GNR-PdTPPs, with the nuclei stained by Hochest 33342 (blue signal); white foci are the TPLs of MS-GNR-PdTPPs under 800 nm laser excitation; scale bar: 10 µm. (d) Decay of optical absorption of ADPA at 378 nm, caused by the generation of singlet oxygen from GNRs (○), PDTPPs (▲) and MS-GNR-PdTPPs (■) as a function of laser irradiation exposure time.(Click on the image to enlarge.)
Confocal fluorescence microscopy images showing the TPA-PDT performance (1O2 cytotoxicity) of MS-GNR-PdTPPs (a)-(c) and MS-GNRs (d)-(f) in treated breast cancer cells under 3 mW repetitive laser irradiation of 3 durations: 0, 108, and 194 seconds. YOPRO-1 (green) and propidium iodide signals (red) denote leakage of the cell and nuclear membranes, respectively. Scale bar: 10 µm. Fluorescence intensity of YOPRO-1 and propidium iodide as a function of irradiation period for cells treated with MS-GNR-PdTPPs (g) and MS-GNRs (h).(Click on the image to enlarge.)
TEM images of a 70 nm-thick section of tumor harvested from an anesthetized mouse 24 hours after administration of MS-GNR-PdTPPs and TPA-PDT; scale bar: 500 nm. Insert shows an enlarged image of localized MS-GNR-PdTPPs within the tumor; scale bar: 100 nm.(Click on the image to enlarge.)
To evaluate the in vivo efficacy of two-photon-induced MS-GNR-PdTPP photodynamic therapy, nude mouse models were implanted with 2 × 106 MDA-MB-231 breast cancer cells and treated with 16 mg/kg of MS-GNR-PdTPPs by intra-tumor injection. In vivo localization of MS-GNR-PdTPPs was accomplished by TEM imaging of the tumor tissue from an anesthetized mouse 24 hours after injection of nanoparticles and subsequent irradiation. As Figure 5 shows, MS-GNRs-PdTPPs were endocytosed by cells without suffering any structural defects (see inset of Figure 5).
To assess in vivo TPA-PDT efficacy, a breast tumor xenograft mouse model was irradiated with 800 nm femtosecond laser pulses for a total exposure energy of 96 J (~250 J/cm2), 1 hour after injection of MS-GNR-PdTPP (Figure 6g-i) nanoparticles. An optical chopper was used in this system to reduce the delivered number of laser pulses to its 1/12. It prevented the tissue damages caused by direct laser irradiation and the following induced GNR photothermal effect. By such configuration, the photothermal effects, not like in other studies [24-25], could be minimized to enable analysis of only the photodymanic phenomena of PS induced by the enhanced plasmonic energy transfer of GNR. The mouse was sacrificed 24 hours post-irradiation and its tumor harvested/sectioned into 5 mm thick slices. Tissue sections were then stained with one of two apoptosis indicators - Caspase-3 and TUNEL - to elucidate cytotoxicity. Hematoxylin and eosin (H&E) and DAPI stains were also applied to characterize each specimen's morphology. The results of the staining are shown in Figure 6 for tumors subject no treatment (i.e., the blank controls; Figures 6a-c), to irradiation alone (Figures 6e-g), treatment with MS-GNRs followed by irradiation (Figures 6m-o), and treatment with MS-GNR-PdTPPs but without irradiation (Figures 6q-s); for comparison to the results of TPA-PDT experiments (Figures 6i-k). Caspase-3 activity was assessed by caspase-3 immunohistochemistry with caspase-3 antibodies and displayed by DyLight (ex. 654 nm, em. 673 nm)-conjugated goat-anti-mouse secondary antibodies (Figure 6, third row, red). Apoptosis-induced DNA fragmentation was revealed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (Figure 6, second row, green). As Figure 6 shows, none of the tumor tissues were stained by casepase-3 and TUNEL except for those subjected to combined MS-GNR-PdTPP treatment and laser irradiation. Represented tumor images (n=3 for each group) acquired 14 days after treatments were shown for quantitative analysis of tumor growth (Figure 6, bottom row). Tumors grew 5.12 times (control, figure 6d), 4.62 times (laser, figure 6h), 4.2 times (MS-GNR-PdTPP, figure 6t) and 3.41 times (MS-GNR, figure 6p) compare to the original size on the first day. By contrast, inhibition of tumor growth was observed with combined MS-GNR-PdTPP treatment and laser irradiation (1.2 times, figure 6i). Despite relatively large pulse energies (1.2 mJ/pulse) employed, the optical chopping reduced exposure levels sufficiently as to permit heat dissipation - No apparent direct (Figures 6e-h) and limited indirect (Figures 6m-p) photothermal heating effects were observed under the laser irradiations used in this experiment. Thus simple, optically chopped femtosecond laser can be used to perform TPA-PDT at the relatively safe average power of ~100 mW irradiated on a 7 mm circular area (i.e., 250 mW/cm2), with minimal collateral damage to neighboring tissue.
In vivo studies illustrating TPA-PDT therapeutic benefits derived from intra-tumor injection of MS-GNR-PdTPPs. Histologic analyses of harvest tumor sections 24 hours post-irradiation were made with hematoxylin and eosin (first row, scale car: 100 µm), TUNEL (second row, green coloring), caspase-3 immuno-histogram (third row, red coloring) and DAPI (blue coloring) staining. Scale bar: 25 µm. Represented tumor images (n=3 for each group) acquired 14 days after treatments were shown for quantitative analysis of tumor growth (fourth row). Scale bar: 5 mm.(Click on the image to enlarge.)
In this report, we describe the development of a novel intra-particle plasmonic energy transfer nanocomposite for two-photon photo-dynamic therapy comprised of mesoporous silica-encased gold nanorods. Enveloping the gold nanorods with mesoporous silica confers a number of advantages to the use of bare or non-mesoporous silica coated GNRs; these include:
- Stabilization of the gold nanorods' shape against thermal deformation following repeated laser irradiation.
- Enhanced two-photon excited luminescence intensity and imaging lifetime of the nano-platform, which extended the possibility for deeper and longer imaging in vitro and in vivo.
- Large surface area for conjugation of photosensitizers.
- Scaffold for intra-particle plasmonic energy transfer between the GNRs and the photosensitizers preventing photosensitizers from GNR quenching by directing conjugation. It improves the subsequent energy transfer efficiency of photosensitizers to oxygen molecules for generation of cytotoxic singlet oxygen.
Using a 2-step sequential intra-particle energy transfer upon two-photon excitation (from gold nanorod → Pd-porphyrin → oxygen molecule,), we observed substantial cytotoxic sequelae in both in vitro cell studies and in vivo murine models of breast cancer.
Taken together, the intense two photon luminescence coupled with good stability of mesoporous silica encased gold nanorods, integrated with intra-particle energy transfer activated singlet oxygen generation, make mesoporous silica-encased gold nanorods a promising nanosystem for the efficient in vivo tracking and photodynamic therapy of malignancies in deep tissues.
GNR: gold nanorod; MS-GNR: mesoporous silica-coated gold nanorod; GM: Units of Goeppert-Mayer; PDT: photodynamic therapy; TPA-PDT: two-photon activated-photodynamic therapy; TPL: two-photon luminescence; SPR: surface plasmon resonances.
The study was supported by the National Health Research Institutes of Taiwan (MED-099-PP-04 and NM-099-PP-01) and by the National Science Council of Taiwan (NSC 98-2221-E-400-001 and NSC 99-2113-M-400-001-MY3). We thank Chun-Wei Shih for performing the confocal microscopic image acquisition and Ms. Yu-Ching Chen for assistance with the TEM measurements.
Competing Interests
The authors have declared that no competing interest exists.
1. Xia Y, Li W, Cobley CM, Chen J, Xia X, Zhang Q, Yang M, Cho EC, Brown PK. Gold nanocages: from synthesis to theranostic applications. Acc Chem Res. 2011;44:914-924
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7. Tong L, He W, Zhang Y, Zheng W, Cheng JX. Visualizing systemic clearance and cellular level biodistribution of gold nanorods by intrinsic two-photon luminescence. Langmuir. 2009;25(21):12454-12459
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14. Mohamed MB, Volkov V, Link S, El-Sayed MA. The 'lightning' gold nanorods: fluorescence enhancement of over a million compared to the gold metal. Chem Phys Lett. 2000;317:517-523
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16. Eustis S, El-Sayed MA. Why gold nanoparticles are more precious than pretty gold: Noble metal surface plasmon resonance and its enhancement of the radiative and nonradiative properties of nanocrystals of different shapes. Chem Soc Rev. 2006;35:209-17
17. Durr NJ, Larson T, Smith DK, Korgel BA, Sokolov K, Ben-Yakar A. Two-photon luminescence imaging of cancer cells using molecularly targeted gold nanorods. Nano Lett. 2007;7:941-945
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Author contact
Corresponding author: Leu-Wei Lo, Institute of Biomedical Engineering and Nanomedicine (I-BEN), National Health Research Institutes, Zhunan, Miaoli 350, Taiwan, Phone: +886-37-246-166 ext. 37115, Fax: +886-37-586-440. E-mail: lwloorg.tw; or Chung-Yuan Mou, Department of Chemistry, National Taiwan University, Taipei 106, Taiwan, Phone: +886-2-3366-8205, Fax: +886-2-3366-8670. E-mail: cymouedu.tw.
Received 2014-2-25
Accepted 2014-4-17
Published 2014-5-30


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Prime Minister Binyamin Netanyahu sent a condolence letter Monday to the Sassoon family, which tragically lost seven children in a deadly house fire over Shabbat.
Elian (16), David (12), Rivka (11), Yehoshua (10), Moshe (8), Sara (6), and Yaakov (5) Sassoon died after hot plate in their house in Flatbush, New York apparently malfunctioned.
Following a funeral procession that began Sunday in Brooklyn, the seven children were laid to rest Monday afternoon in Jerusalem, where the family had lived before moving to the US.
In the letter, addressed to Gabriel Sassoon, Netanyahu expressed his sympathy to the grief-stricken father.
"Dear Gabriel, with deep shock I heard about the tragic death of your beloved children: Elian, David, Rivka, Yehoshua, Moshe, Sara, and Yaakov. From what I've heard about them, I learned they were valued children who were a source of pride and joy for you."
"Every one of your children was a world, unique and special. There is no greater grief than the loss of sons and daughters, and it's an even greater agony when it comes to young children," Netanyahu empathized.
"The people of Israel feel your pain," the Prime Minister stressed.
Netanyahu concluded the letter by wishing Gavriel the inner strength to cope with the disaster that struck the entire family.
"I wish a speedy and full recovery to your wife Gail and daughter Tzipora," Netanyahu wrote. Both escaped by jumping out of a window are currently being hospitalized in critical condition.
"Your people shares in the deep loss of your children, seven pure souls."

subset: commoncrawl


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Nigeria imported 4.79 billion litres of premium motor spirits (PMS), also called Petrol in the second quarter of 2018, according to the latest report by the National Bureau of Statistics (NBS).
A further breakdown of the petroleum products importation revealed that 1.11 bln litres of automotive gas oil (AGO), 43.79 mln litres of household kerosene (HHK). The report said 200.39 mln litres of aviation turbine kerosene (ATK) were imported into the country in Q2 2018.
It indicated that the months of April 2018 recorded the highest volumes of premium motor spirits (PMS) imported into the country at 1.78 bln litres while the highest volume of automotive gas oil (AGO) and household kerosene (HHK) were imported in June 2018.
The report showed that statewide distribution of truck-out volume for Q2 2018 showed that 4.89bn of premium motor spirits (PMS), 1.17 bln litres of automotive gas oil (AGO), 168.83 mln litres of household kerosene (HHK) and 176.98 mln litres of aviation turbine kerosene (ATK) were distributed nationwide during the period under review.


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The electric power sector consists of facilities which generate electricity primarily for use by the public. Energy is used for the generation, distribution, and transmission of electric power.
Between 2014 and 2015, electric power sector energy use increased 0.2 percent to 403.8 trillion British thermal units (Btu). Natural gas use increased 4 percent although consumption of natural gas comprised 1 percent of the sector's total consumption. Petroleum use decreased 84 percent although consumption of petroleum products comprised less than 1 percent of the sector's total consumption. Nuclear electric power use increased 2 percent and renewable energy use increased 22 percent. Coal use decreased 4 percent.
The principal fuels used at electric utilities are coal (61 percent) and nuclear electric power (27 percent). Renewable energy (11.2 percent), natural gas (1.1 percent), and petroleum (0.02 percent) make up the other 12 percent of fuels consumed. Petroleum was comprised totally of distillate fuel oil.
In 2015, consumption of solar thermal and photovoltaic energy and geothermal energy in the electric power sector was comparatively small and rounded to zero. More information on solar energy, geothermal energy, and wind energy in Nebraska is available.
Hydroelectric power data was included in renewable fuels, but is shown separately in the graphs and data tables below.


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- I am no longer doing designs. My portfolio will be either taken down or updated to reflect this;
- I might be launching an Etsy store, but no promises;
- I'll be adding a page for my schedules (Twitch streams and YouTube uploads, if I start doing YouTube videos again);
- I'm going to make an increased effort to update some things with my stream setup, starting now; and
- I'm finally updating this site! Yay! (this post counts, but more to come, probably)
Have any thoughts or comments? Post them below, and I'll make sure to get back to you.


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Prism Bariatric Bed Classic
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Pay justice fights
Luton airport cleaners are on a week-long strike as part of a campaign against their "Scrooge" bosses' pay policies.
The workers who are responsible for maintaining a safe and clean environment for staff and passengers at Luton Airport are currently struggling to make ends meet on the minimum wage of £7.83.
Cleaning firm Sasse, which was awarded the contract by Luton Airport in April this year and increased its turnover by 21 per cent to £9m in 2017, have offered a three year pay deal to increase workers' pay.
By 2021, however, Sasse workers will still be earning below the real Living Wage of £9.
Unite regional officer Jeff Hodge said, "Bosses at Luton Airport are behaving like Scrooge. By 2021 cleaners at Luton Airport still won't be earning a real Living Wage.
"With cleaners out on strike for a full seven days in the run up to Christmas the stoppage will bring cleaning services at the UK's fifth busiest airport to a virtual standstill."
The strike, which 100 per cent of members balloted voted for, began earlier this week (December 4) and will finish on Tuesday (December 11).
Meanwhile, Unite members employed by Clece Care Services at Luton Airport have suspended their planned strike action while the workers vote on a revised offer from the company.
Toxic combination
Clece's Luton Airport workforce, who are responsible for helping passengers with mobility restrictions, including disabled passengers, are on a toxic combination of zero hours and the minimum wage.
Hodge called on Luton London Airport management to get their "house in order".
The airport announced profits of nearly £40m and carried almost 16m passengers in 2017 but has refused to intervene to insist its contractors pay at least the Real Living Wage and treat workers with dignity.
Hodge said, "This year alone there have been five separate disputes over pay involving workers (at Luton). The airport is suffering reputational damage.
"With healthy profits and growing passenger numbers, Luton and its contractors have no excuse for paying workers below the Real Living Wage. Workers at Luton Airport are intensifying their campaign for pay justice."


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This is so absolutely beautiful! I don't know the name of the choir but as soon as I find out, I will post it! It's Blessed!
This rendition of 'Lord I Thirst For You' brought tears to my eyes immediately as I thought of how much I love Him and how much He has blessed me!!


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The Opening Ceremony of Bayburt University 2018-2019 Academic Year was held in the Conference Hall of the Faculty of Education and was attended by the President of the Strategy and Budget Directorate Naci Ağbal, Governor Ali Hamza Pehlivan, Mayor Mete Memiş, Garrison Commander David Balibaşa, Rector Prof. Dr. Selçuk Coşkun, Police Chief Sezayi Er, academicians, press members and students.
Befor the speech of Rector Prof. Dr. Selçuk Coşkun, Governor Pehlivan and Mayor Memiş put emphasize on the importance of Bayburt University for the city and added that they intend to increas the the number of jointly produced projects.
Rector Prof. Dr. Selçuk Coşkun started his speech by underlining the duties and responsibilities of universities, academicans and students during the days of the economic war. Speaking about the activities of the Bayburt University, he talked about the newly opened departments, the current status of the Bâbertî Complex, the projects being implemented, new faculties, number of students and other developments. Addressing the academics and students, Coşkun stated that the new generation of universities is obliged to train the students who produce and that information should be transcended beyond stocking. Sharing the plans and targets of Bayburt University in the new academic year with the guests, Coşkun said that they are working on a training model that is focused on innovations, digitalization processes and information technologies. Coşkun mentioned the importance of working together, making effort and producing. He ended his speech by transferring the advices of the elders to the students.
After the speech of Rector Prof. Dr. Selçuk Coşkun, President of Strategy and Budget Directorate Mr. Ağbal was called to the podium to give the first lecture of the new academic year. He started his speech by expressing his gratitude to those who contributed to the development of the Bayburt University, especailly Türkish President Mr. Recep Tayyip Erdoğan.
At the beginning of his thematic lesson 'Education, Technology, Innovation: Key Trends for the Future?', which he called symbolic speech, he showed the first speech of the founder of the Alibaba e-commerce site during honorary doctorate. Indicating that the world education system has changed, Ağbal said that the educational expenditures of the countries are increasing day by day and added that todays education concept is not storing information, but to produce, reproduce and enrich through imagination. Ağbal also stated that the production has become more optional in our age, where the transition from detachable products to personalized and non-digital based products has been experienced and mentioned many details such as Big Data, Data Mining, Internet of Things, Block Chain, Robotic Production. Ağbal concluded that West-based technology production started to shift to the Far East as a result of R & D investments and we had to produce if we want to have a say in this sector.
At the end of the ceremony, academicians who received Associate Professor and Professor degrees, and academicians who achieved success with various projects were presented.


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The latest THR long-form roundtable has arrived! We've already shared the in-depth discussions with the directors, actors, and actresses roundtables, and now we've got the hour-long chat between some of 2017's most celebrated screenwriters. The latest roundtable Q&A includes Jordan Peele (Get Out), Darren Aronofsky (mother!), Aaron Sorkin (Molly's Game), Emily V. Gordon (The Big Sick), Fatih Akin (In the Fade), and Andrew McCarten (Darkest Hour), and dives into all kinds of subjects related to writing movies.
Aronofsky talks knowing that mother! was going to be divisive, the different interpretations of mother! and if any of them changed his perspective on the film. Gordon talks about writing as a means of making people feel less alone and the nuances of getting the autobiographical story of The Big Sick right. Peele talks about why he changed Get Out's ending and how the theatrical version allows him to have his cake and eat it too. Akin talked about making controversial, political films like In the Fade and how he handles the heated backlash they provoke. They also discuss the demands and compromises of writing stories based on true people, working in genre filmmaking, which historical figures they would invite to a dinner, their best piece of writing advice, and more. Watch the full roundtable discussion below.
For more of the latest on how awards season is shaping up, check out the links below:
- 'Three Billboards' Wins Big at the BAFTAs as the Oscar Race Gets Clearer
- First Round of Oscar Presenters Includes Tiffany Haddish, Chadwick Boseman, and Daniela Vega
- Oscar Beat: Best Picture Predictions – Do We Have a Frontrunner Yet?


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Register now for Storytime Programs at Roselle Public Library
Registration for Preschool Storytime (ages 3-5 years), Toddler Tales (ages 20-35 months with parent or caregiver), and Tots & Tykes (ages 6-19 months with parent or caregiver) is open. The First Fall Session of Storytime Programs begins September 9 and continues through October 16. Preschool Storytime meets on Wednesdays, with a choice of either a 10:00-10:30 a.m. class or a 1:00-1:30 p.m. class. Toddler Tales meets on Tuesday mornings from 10:00 a.m.-10:30 a.m. Tots & Tykes meets Tuesday mornings from 11:30 a.m. - 12 noon.
Child must be the required minimum age by the date the program session begins. Parents are required to stay in the Youth Services Department during Preschool Storytimes. Please, no siblings in the Toddler Tales or Tots & Tykes classes - only the registered child with parent or caregiver. Roselle Public Library cardholders have priority - spaces are limited.
To register or ask about the first fall session of storytime programs contact, or visit, the Youth Services "Ask Me" desk (630)529-1641 x351, or register online through our website.


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Like a kid in the days leading up to Christmas, I had been anxiously anticipating this club hike since the first time I saw it on the Smoky Mountain Hiking Club calendar. And, like the kid jumping out of bed before sunrise to check under the tree, not even an absurdly early start time could dampen my enthusiasm. I could barely go to sleep the night before with thoughts of thrilling adventures following Harrell and Hoover through one of the Smokies' wildest areas racing through my mind.
A group of fourteen hardy adventurers, a mixture of old friends and new faces soon to become friends, met at the Alum trail head. After brief introductions and exchanges of pleasantries and insults, we were quickly on our way.
A short stroll on the trail served as an appetizer for the main course, which began with a hop across the crystal clear and cold Styx Branch.
Greg Hoover seemed right at home walking up an exquisite little cascade on Alum Creek that he had hiked past innumerable times.
We tried, some more successfully than others, to keep our feet dry as we leaped from rock to rock and scrambled over, under, and past various obstacles.
Unfortunately, however, the obstacles sometimes win. While holding on to a rhodo bush, Chad slipped on a rock. His shoulder was ripped out of socket. It's a quite common injury when scrambling through the mountains, but it was an injury that necessarily ended his day. In severe pain, he headed back down the creek, escorted by Hoover, toward a doctor to have his dislocated shoulder reset. I felt horrible for Chad that he had suffered such misfortune on his first outing with us and would be missing an incredible hike. I hope he will be back with us soon.
Now with just twelve, the diminished group proceeded on up the mountain.
We came upon a nice waterfall, lacking an abundance of water, but embellished with a stately old log.
We continued to follow the small branch higher and higher through rough, but enchanting, terrain.
After a bit of work, we encountered the first scar in the enormous bowl of Anakeesta Canyon.
I had assumed Harrell was leading the group up this scar and along the tops of the other scars in the bowl, but he had other plans. I don't believe in letting a good scar go to waste, so I ran ahead of the group and took a short detour up to the top.
From the top, I could see little specks of my comrades trudging by along the bottom.
So as not to get too far behind, I skied down the loose rubble at breakneck speeds and intercepted the tail end of the gang at the bottom.
We all regrouped and recharged at a flat open spot, steeling ourselves for the intense climbing that lay ahead.
Further upstream, a fallen log served as a giant ladder providing access to an enormous scar. I was delighted to begin the real ascent.
It's always comforting to be out with a selfless health professional like Cindy. I never know when I might gash my head open or something.
It didn't take long for Tim to find himself in a tricky situation.
Everyone was working for the payoff of spectacular views.
We all like to identify distant peaks and tell stories of our explorations on each of them.
Climbing a scar is an alternation between focus on the details of the rock in front of your face and the overwhelming expanse of miles of unbroken mountain scenery stretching to the horizon.
It's soothing to be alone in the mountains, but there is nothing like the camaraderie of being a part of a large group of like-minded adventurers.
Harrell displayed his athletic prowess.
Past more scars down the valley, we could look out across Parton's Peaks and Peregrine Peak toward the looming Mt. LeConte.
Tim and I separated from the group to scamper up some interesting rocks.
Extremely steep and scary rock walls towered over us.
We could look along the side of Anakeesta Ridge to the scoured Chimneys far down the valley.
The bluffs pushed Tim and me further apart. He found himself in a place he didn't want to be in. He was a mere speck lost in the immensity of the Anakeesta Canyon.
The higher we climbed, the more spectacular became the scenery. The famous Y Scar on the side of No-Name Ridge came into view.
We pushed through thick brush, up steep slopes, and along precipitous ledges.
Harrell expertly led the group through treacherous terrain.
With sweat dripping, hearts thumping, and muscles burning, we pulled ourselves ever higher.
Just when we began to believe there was no top, we suddenly stood upon it, swathed in the ecstasy of conquering the mountain.
The views off the other side toward Newfound Gap were exhilarating. I was ecstatic that we still had much adventure left in order to get down there.
I doubt anybody in the group would have rather been anywhere else.
I was happy to get the chance to hike with Ed again and be a part of one of the countless adventures he's enjoyed in the Smokies through the decades.
The south-facing ridge gathered the warm sunshine and filled my soul with mountain music.
I took a little different route off the ridge crest for a ways and took a look back up at Harrell and the crew relaxing at the top.
I climbed back up to rejoin them, where I corralled everyone for a group photo.
Then the game was on as we began the descent of the famous Anakeesta Scar.
Pants and flesh were shredded by the sharp anakeesta rock as we spent a large part of the time on our hands and butts.
Ken Wise, Gentleman of the Forest, added a touch of class to the rather crass gang traversing the mountains.
Between the times when you are trying to not tumble down the hillside, it is essential to stand still, look around, and attempt to absorb the stunning beauty of the place.
It was about here that I accidentally sent a basketball-sized rock down on Ed. I yelled out a warning, and he instantly spun around to face the tumbling boulder, assuming a ninja-like athletic stance. He watched the rock speeding toward him, bouncing around randomly like an unpredictable bull. At the last second, like a grizzled matador, he gracefully sidestepped the charging beast as if he had done it a hundred times before. He probably had.
We gathered at a rare flat spot to make sure nobody had rolled away.
We picked our way down the last bit of open rock, pushed through some thick forest, crawled through a giant log maze, and hopped down a dry stream bed. Then, just like that, it was all over.
And thus concluded one of the grandest SMHC adventures ever. I thank the thirteen other folks that contributed to a day that won't be forgotten.
Views: 577
Well, that must be the mother of all off-trail ventures. Great to be a able to tag along in spirit following the play-by-play photos. Early shot of Ken, I thought, "Does that dude have on a necktie?" Never, ever in my life have I encountered a hiker in a tie, let alone off-trail. Among my favorite shots are the cliff face of the waterfall and the wide open views out across the valley. Thanks for taking us along is spirit.
Yes, I am hoping this image of Ken replaces the one imprinted in my brain at Three Forks.
Thanks, guys. Karl, I think Ken was going for the vintage hiker fashion from the club's early days. Or maybe he's just a nut. And, the temperature was very warm, so shorts and a t-shirt were quite comfortable. In fact, after the hike, I stopped by the Sinks and took my first couple dives of the new season. And, Hoover, we hate you missed it, but it was a noble thing you did helping a fallen hiker out. I will be happy to go back up there with you anytime (as long as we leave Harrell at home so we don't have to start walking in the dark).
Thanks David for sharing the hike/climb. What a great adventure ! The photos show how rugged the area is and also how beautiful.
That Ken Wise is a real dandy.
Nice trip and pics. I may give it another shot later this spring, or maybe in the fall. It's actually been several years since I've been on the Canyon side. Both sides are quite a playground, for sure. Glad you enjoyed it. Thanks for all your blogs and pics.
I was hoping someone was going to do a blog on that hike. Thanks for all the photos, I was trying to imagine what that terrain was going to look like and your extensive picture documentation almost made me feel like I was there.
Incredible pictures and adventure. I saw that hike coming up when the SMHC schedule was published. I was training and looking forward to joining you guys until an unexpected family issue prevented it. I liked Ken's attire for such a rugged hike. Was he trying to emulate in appearance former famous members of SMHC? Tim's attire always makes you think the temperature must have been 30 degrees warmer. And David, I'm glad you managed to avoid additional injury.


text: C. V. Raman Polytechnic, a sister concern of C. V. Raman College of Engineering is built on a sprawling beautifully landscaped area just on the outskirts on beautiful city of Bhubaneswar. CVRP is committed to impart "Quality Education & Training" and aims to be a global institution through continual improvement. The institute facilitates faculty and support staff to work as a team and update their knowledge and skills to match the global industrial and technological development through defined objectives.


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Okay, so, I can't find any official 'blurb' for Doctor Who Magazine #400 (other than the one from their facebook page, which I've already posted)so I'm going to have to type the details myself. The cover, because it's the 400th issue (congradulations to DWM) there's a special cover featuring David Tennant reading issue #400 with the cover showing the cover and the cover and... you get the idea (see the cover at the bottom of this article).
This issue comes free with a poster of the 10th Doctor reading DWM in the TARDIS (see below for cropped version) and a fantastic competition to go on the set of Doctor Who and see one of the specials being filmed! Here's part of the poster:DWM #400 Includes:
- A competition to go and see a Doctor Who special being filmed
- Double-sided poster
- Sarah Jane Adventures Series Two preview
- Colin Baker joins the Time Team!
- "The sheer briliance of Doctor Who!"
- Russell T Davies interview and more
- 'Who on Earth is...' with David Tennant
- Comic Strip: Think Twice - Part one
- You Are Not Alone
- Special article on 'Four Hundred DWMs!'
- Latest books', episodes' and audio books' reviews
- WIN! Loads of fantastic Doctor Who goodies!
- and the regular: Production Notes (from RTD himself), Gallifrey Guardian and Galaxy Forum
All that and more, only in the fantastic 400th issue of Doctor Who Magazine!


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Albea Psychology Center
At the Albea Psychology Center we have more than 30 years of experience helping people with their mental and sexual health. We have a team of professionals of the highest category, who do everything to understand the problems of our patients and help them find a solution.
URL: http://www.centroalbea.com
Category: Services , Health
Date added: 2019-04-03


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My Town GR: Proos Manufacturing expanding and hiring | Business
Title (Max 100 Characters)
GRAND RAPIDS, Mich.—An auto parts supplier in Grand Rapids is expanding, adding 50 new jobs.
Proos Manufacturing will invest $5.7 million into renovating its Oak Industrial Drive facility to ramp up its fabrication and stamping operations. When the expansion is finished, the manufacturer will have 113 employees.
The plans were announced Wednesday, after Proos Manufacturing secured a $200,000 Michigan Business Development Program grant. The funding is part of a $715,000 incentive package by The Right Place, Inc., the City of Grand Rapids and the Michigan Economic Development Corporation.
"Managing growth is just as challenging as managing decline. However, with the assistance of The Right Place, MEDC, and the City of Grand Rapids, Proos Manufacturing is now able to start another exciting chapter of our 94-year history," said Amy Proos, CEO. "The Right Place and MMTC-West have been valuable business partners in our company's success. With their assistance, we continue to grow, create jobs, and invest in West Michigan."
Proos Manufacturing was recently named Automotive Supplier of the Year at the 2014 Michigan International Auto Show.
The company's headquarters will remain at 1037 Michigan Street NE in Grand Rapids.


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The scientist Harrison Wells has has had a lot of conflicting names and labels on The Flash. He's a mentor and a murderer, a mastermind and a man out of time. Even Harrison Well's evil endgame of The Flash doesn't add up. On one hand, Eobard Thawne himself has told the audience that he just wants to get home to his time. However, the operating system Gideon said pretty plainly that Thawne was there to kill Barry. Something is up.
We know that killing The Flash is why Eobard Thawne traveled back to the past in the first place. He was chasing Barry Allen and ended up in Allen's family home. What if — and this is probably the case so get your ibuprofen ready for the following headache scenario — what if past Thawne was there to kill future Barry and future Barry was there to save his mother's life, which inevitably caused her death? Because why else would Barry lead someone so dangerous to his homestead? It has to be some kind of loop like that, which would render her demise unavoidable.
At the end of last Tuesday's episode "Grodd Lives," Harrison Wells (I know he's Eobard Thawne, but I came to love this shady character under a different name first and I'm having a hard time letting go.) finished work on a a kind of thumb drive inside a vial of some sinister substance that he explained was his key to going home. What is it? Has he been harvesting this from all of the metahumans that crossed his path? Is he regaining a power, or creating a portal?
This turn of events in Harrison Wells' storyline is so mysterious, we don't even now where he is. Entertainment Weekly's Chancellor Agard speculated that Wells and Eddie are inside the particle accelerator, which at the very least will provide us with more fun references from Cisco. However, even Harrison Wells needs fresh air, and he would surely have been detected leaving and entering S.T.A.R. Labs by Cisco and Caitlin.
Maybe I've been watching too much Once Upon A Time, but I'm starting to wonder if Harrison Wells' ticket home will somehow give him an advantage in this epic battle against his rival, Barry. Is Harrison Wells trying to create an alternate universe? He's seemed pretty strictly adherent to keeping the future on track, but he could be waiting for the opportune moment to change the time line.
We also know that, while Eobard was born in a time that's centuries to come, a lot is riding on an event less than 10 years in the future, in which Barry Allen will go missing. This makes me think that Eobard Thawne needs to time travel again so that he can retrieve Barry Allen from 2024. Some of us need to get groceries on the way home and some of us have nemeses to kidnap.
Will Harrison Wells ever cease to be frustrating? Wanting to get home is an honorable goal. Eobard Thawne is basically Dorothy from The Wizard of Oz with super powers. However, it doesn't change the fact that the Reverse-Flash has it out for Barry Allen. There's no way that The Flash's Big Bad is just going to leave and be out of our hair forever.
Image: Cate Cameron/The CW


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Nicholas Carroll returns with a EuroFantasyLeague preview ahead of the upcoming round of fixtures. London and Madrid derbies headline the weekend matches however a number of stars are in doubt following the international break. Arsenal hosts Tottenham in Saturday's early Premier League match, with Harry Kane keen to return to to the playing field having missed out on England's two friendlies.
Liverpool and Manchester United are firm favourites to take maximum points at home in their respective matches while over in Serie A, Napoli will be looking to keep a hold of top spot when they host AC Milan.
Meanwhile, many fantasy owners will be looking for some improvement from Real Madrid's biggest stars when they take on city rivals Atletico Madrid in one of La Liga's biggest clashes.
Below is our weekly preview:


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Snow Tonight
School Forecast for:
Monday through Friday
Last Updated: 4PM Thursday February 17, 2018
Delay No School Early Dismissal
The Forecast
4PM Saturday
Snow is currently overspreading Pennsylvania, and should arrive in New York within the next four hours. Tonight's storm will be a quick-hitter, and will likely be over by the time most of us wake up tomorrow. Make sure you get outside and enjoy the snow tomorrow because whatever falls probably won't last long — rain showers enter the picture by Monday evening with temperatures in the 60s by Tuesday and Wednesday. Stay safe if you must travel tonight.
Click image for larger and clearer version.
8AM Saturday
Don't be fooled by this morning's beautiful sky, a storm system will spread snow over the region tonight. Trends over the past 24 hours have been for slightly more snow than previously forecast. It now appears that's most of Ulster County will likely fall within the 3 to 6 inch range. The "extreme range" for this event is one to seven inches. Expected start time: around 7pm today. Expected end time: around 5am Sunday. Driving conditions are expected to deteriorate late this evening, and remain poor through Sunday morning.
12PM Thursday
I don't know about you, but I can't help but feel that Old Man Winter has been sleeping on the job for the past week or so. This morning's temperatures in the mid-40s along with tonight's likely rain showers aren't what I personally like to see in mid February. In either case, it appears that Winter may be waking up for at least a few hours this weekend. A nor'easter is likely to develop off the Mid-Atlantic coast this weekend. Over the past 36 hours there has been an increasing probability that this storm will impact our region with snow heading into Sunday …especially south of Albany… and there is the potential for measurable snowfall Saturday night. The amount of snow that falls, if any, will be dependent on the track of the storm. It is too early to speculate at exact snowfall totals as there is still the potential for anywhere from 0 to 7 inches with the more likely range currently being toward the middle of those extremes. We'll see how the outlook unfolds heading into tonight
12PM Sunday
The Kingston area has picked up about half an inch of pure rain since yesterday evening. Rain will continue through about 4PM today. Temperatures will peak around 40 degrees around 12AM Monday, and then gradually fall to the lower 30s by dawn. There could be slick spots from today's wet weather, but a widespread black ice event is not currently anticipated for the morning. As we progress through the week, winds flowing from the southwest will allow temperatures to build. By Thursday we could reach the low 50s. Since 1949, local temperatures on February 15th (Thursday) have only reached the 50s on eight occasions. A frontal boundary will pass through the region Thursday night into Friday, likely triggering rain and then possibly some snow showers. Currently, neither Thursday nor Friday look to be complete washouts.
Click image for larger and clearer version.
The pattern that has emerged entering mid-February is one that is very unfriendly for snow. The pattern favors conditions such as this week with winds from the south and southwest prevailing, resulting in warmer than average temperatures and mainly rain events with some snow possibly tacked on toward the beginning or end. This does not bode well for us snow lovers. It is entirely possible that this February will not see any significant snowstorms, and as mentioned in the post below from February 1st, seasons that record no weeks with at least 10 inches of snowfall in February have an 80% chance of ending below average. The season snow total currently stands at 28.1 inches, which is about three inches below the 13-year average. The average season total by the end of March is about 43 inches.
February is Here
Updated: February 1, 2018
~ January Recap ~
January was quite a wild ride. The month kicked of with record breaking cold temperatures. New Year's Day recorded a record low of -10 degrees followed by a week of frigid temperatures. The cold reached its most extreme point on January 7th when the low bottomed out at -14 degrees. But the cold was far from permanent; within less than a week we were setting record high temperatures. January 12th recorded a record breaking 62 degrees for the afternoon high, while the following day reached 59 degrees. The warmth was accompanied by heavy rains of over an inch that resulted in flooding and ice jams in various parts of Ulster County; the Wallkill River in New Paltz was particularly affected. By the 16th snow was the headliner with an 8 inch snow storm accounting for more than half of the month's snowfall. A second major rain storm dropped half an inch on the 23rd before the month coasted to a calm finish.
~ The Month Ahead ~
So far this season, Kingston's total snowfall has continued to hover right around the 13-year average. The season snow total as of February 1st is 22.5 inches which is exactly on par with last year's 22.1 inches that had fallen by this date. As we head into February we can expect an uptick in the snowiness. February is historically our most impactful month by far, ad it is when we see a higher frequency of more impactful storms. Of all the weeks between December and March that have ever recorded at least 10 inches of snow, about half of them have occurred in February alone. Seasons that record no weeks with at least 10 inches of snowfall in February have an 80% chance of ending below average, while seasons that have at least one week with 10 inches of snow in February have less than a 40% chance of ending below average. The outlook over through the first half of this month favors a return to wintery conditions. Below average temperatures are favored over above average temperatures, and the emerging pattern favors active weather with multiple opportunities for accumulating snow.
Image caption: So far this winter's snowfall (pink line) has been near average.
Click image for larger and clearer version.
Image caption: Kingston remains on track to see about 48 inches of snow.
Click image for larger and clearer version.
The Extended Outlook offers a general sense of upcoming conditions, but is not a specific forecast. Conditions depicted may change as the they get closer in time.
Click image for larger and clearer version.All 2017 Photos of The Month have been updated.
Check them out via the link on the side and feel free to share any of your photos on Facebook!
Kingstonsnows ♦ Kingston, NY
86852 Visiting Snowflakes
Since January 2009
Archive of Updates


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Waterfall Inspired Archway of Flowers for Laura & Michael by Bradgate Flowers
Bradgate Flowers of Leicestershire have been creating tranquility for a Stapleford Park wedding
I met Laura and Michael back in 2016, such a lovely couple with a very different outlook on the theme for their wedding day of June 2017 at Stapleford Park, Leicestershire! My brief was in Laura's words, "natural, lots and lots of greenery, with white flowers and lots of scent, must have scent!" But Laura's biggest ask was…
"A waterfall inspired archway"
The arch was to be covered from top to bottom in soft Italian ruscus, scented white stocks, and placed between two large pillars in the Orangery. This was going to be the main feature of the day, where the couple would take their vows, be followed out by their guests through the arch and onto the beautiful manicured gardens of Stapleford Park where they would be showered with real white rose petal confetti.
Wanting to give Laura the ultimate archway to celebrate their marriage meant a lot of lost sleep the night before! You would think after more than 30 years of being a florist I would get used to the pro's and con's – the truth is you never get used to them! With Friday already a scorcher and Saturday's forecast set to be a fabulous 29c, (you can't ask more from the weather for your wedding day!) panic started to set in and "what if….." were the only words I kept hearing myself say! But I have worked with stocks thousands of times – why am I worrying? Maybe it's because I'm to create this beautiful statement feature, the feature that the bride wanted most on her florals list, even above the bouquet she was to hold. The feature that will stand right there in front of all those guests possibly melting in the heat, trying to survive and last the entire day in the unforgiving heat. The worry of the Orangery with its glass roof and an outside temperature set to be min 29c. I was awake all night – this was a florists nightmare!
On arrival Saturday morning, it was already 19.5 at 7.30 am. I walked through the Main Hall into the Orangery and a weight was instantly lifted. The room was air conditioned! My face beamed, I was so happy and couldn't help but say to myself "what was all the worry for, everything is going to be just fine!" and it was. With my face beaming I set to work, using layers and layers of soft Italian ruscus to create the main structure, then individually wiring and hanging 200 stems of white scented stocks to create the waterfall illusion and here it is – my waterfall inspired arch in all it's glory having survived the day, bringing Laura and Michael's floral dreams to reality.
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Quizzes and Trivia
These are the qualities that characterize us as human beings, after that propel us, on occasion, en route for greatness. Three Wonder Stories Admiration 1. Mar 19, In the spring ofConner traveled to Montreal to see her once all over again, this time to interview her for ABC.
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We've already announced that one half of the Axe Murder Boyz, Bonez Dubb would be hosting his own podcast titled "The Bonecast" very soon. Check out our original post HERE. We now have a date, sponsor info, and more flavor!
Episode 1 is premiering live exclusively on YouTube, Tuesday, August 27th at 8:00pm EST! Subscribe at YouTube.com/BonezAMB and click the lil' bell to get notifications.
Don't think this is just a music-related podcast though because Bonez will be covering all forms of entertainment including movies, music, underground music, gaming and more! Basically, it's a mix of underground culture and pop culture.
Special guests will also be hitting up the Bonecave (I made that up) every now and again to chat as well, so tune in to see who'll be on the show, ninjas!
If you're interested in being a sponsor for this podcast you can hit up TheBonecast.bigcartel.com.
Check out the different tiers available:
Less than a week away, homies! Don't miss it!
from Faygoluvers


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- Level of difficulty: 4****
- Finished size: 66" x 66"
- Foundation papers included
- Technique: paper piecing
Brilliant spiked auroras radiate out from bright stars in this fiery quilt.
Embellish with crystals or metallic thread to make this quilt sparkle and glow.
Material requirements include: 1-1/4" star backgrounds; 3/4 yd lt yellow; 1-1/4 yds center square background; 1-5/8 yds middle square background; 2-1/3 yds outer square background; 2-3/4 yds sashing; 3/4 yd black for spikes.
Pattern Details
66" x 66"
- Jacqueline De Jong Be Colourful Quilts


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Cinos the Anti-Sonic is a character that appears in Sonic the Hedgehog in the Fourth Dimension. He was an alternate version of Sonic that was turned into his opposite by the use of the ROCC at the same time Kintobor became Robotnik.[1]
Cinos had white fur. His expressions appeared to be those of complete evil, as shown when he and Sonic merged.[1]
When Sonic and Tails went back in time to undo their undoing of Kintobor's transformation, the version of Sonic from that point in time got caught in the ROCC's blast, turning him into an evil being filled with Chaos Energy. Cinos proved to be more than a match for Sonic and the two Tailses, whirling at high speeds, until his sneaker got caught on the floor and leaving him vulnerable. Using this opportunity, Tails found three gold rings to use on Cinos, believing they could "neutralize his energy" as Sonic put it. It did not work so well, so Sonic touched Cinos instead and slowly absorbed him, as he was his temporal duplicate.[1]
Powers and abilities
Cinos was every bit as speedy as the real Sonic. His body was also apparently filled with Chaos Energy.[1]
Cinos' Chaos Energy-infused body proved to be his downfall, as rings can be used to neutralize his energy.[1]
- Cinos's name is "Sonic" spelled backwards, reflecting how Cinos is a evil counterpart of Sonic.


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Custom Design - Wormy Chestnut Side Table
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