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Lomustine Nanoparticles Enable Both Bone Marrow Sparing and High Brain Drug Levels – A Strategy for Brain Cancer Treatments
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The blood brain barrier compromises glioblastoma chemotherapy. However high blood concentrations of lipophilic, alkylating drugs result in brain uptake, but cause myelosuppression. We hypothesised that nanoparticles could achieve therapeutic brain concentrations without dose-limiting myelosuppression.
Mice were dosed with either intravenous lomustine Molecular Envelope Technology (MET) nanoparticles (13 mg kg−1) or ethanolic lomustine (6.5 mg kg−1) and tissues analysed. Efficacy was assessed in an orthotopic U-87 MG glioblastoma model, following intravenous MET lomustine (daily 13 mg kg−1) or ethanolic lomustine (daily 1.2 mg kg−1 - the highest repeated dose possible). Myelosuppression and MET particle macrophage uptake were also investigated.
The MET formulation resulted in modest brain targeting (brain/ bone AUC0-4h ratios for MET and ethanolic lomustine = 0.90 and 0.53 respectively and brain/ liver AUC0-4h ratios for MET and ethanolic lomustine = 0.24 and 0.15 respectively). The MET formulation significantly increased mice (U-87 MG tumours) survival times; with MET lomustine, ethanolic lomustine and untreated mean survival times of 33.2, 22.5 and 21.3 days respectively and there were no material treatment-related differences in blood and femoral cell counts. Macrophage uptake is slower for MET nanoparticles than for liposomes.
Particulate drug formulations improved brain tumour therapy without major bone marrow toxicity.
KEY WORDSglioblastoma multiforme lomustine molecular envelope technology (MET) myelosuppression nanoparticles
Confocal laser scanning microscopy
Ethylene diamine tetraacetic acid
Glioblastoma multiforme
N-palmitoyl-N-monomethyl-N-N-dimethyl-N,N,N-trimethyl-6-O-glycol chitosan
High performance liquid chromatography
- MeCN
Molecular envelope technology
Magnetic resonance imaging
Trifluoroacetic acid
Glioblastoma multiforme (GBM) is the most frequently occurring primary brain tumour in adults (accounting for 65% of primary brain tumours) and is the most aggressive, incurable malignancy of the central nervous system (1). GBM continues to be associated with poor prognosis, in spite of more recent therapeutic advances, with less than a quarter of patients surviving for 2 years after diagnosis (1, 2, 3). The median survival time is 14 months and only a very small percentage (3–5%) of patients survive for more than 3 years (4). As for most cancers, there is no actual cure for GBM.
GBM is characterized by uncontrolled cellular proliferation, diffuse infiltration and significant angiogenesis (5) and it is initially diagnosed using magnetic resonance imaging (MRI), although image interpretation is sometimes problematic (6). Once diagnosed GBM is treated by surgical resection followed by radiotherapy and chemotherapy, however, for some tumours there is no acceptable treatment (7,8). Chemotherapy, when indicated, is very challenging due to the heterogeneous and infiltrating nature of tumours and therapeutic agents being unable to access the tumour site; the latter due to the blood brain barrier (5,9,10). Therefore, methods to increase the localisation of chemotherapeutics to the intracranial tumour site are needed. Nitrosoureas such as carmustine and lomustine and other alkylating agents, e.g. temozolomide have been employed in GBM chemotherapy, however dose limiting toxicities, such as myelosuppression, limit the effectiveness of these drugs (11, 12, 13, 14).
It is thus clear that increasing the drug levels at the tumour site while reducing drug levels in the bone marrow represent a significant challenge for the chemotherapy of GBM. One way of achieving high brain concentrations would be to increase the blood concentration of the drug via a dose intensification regimen. In the case of a lipophilic drug, we hypothesise that nanoparticles may allow high drug concentrations to be administered (dose intensification) such that high blood concentrations, and thus high brain tumour levels, of these drugs are achieved, without delivering high doses to the bone marrow. Dose intensification has been attempted in patients but requires autologous peripheral blood stem cell (PBSC) support via PBSC infusions (15).
We have chosen to test this bone marrow avoidance hypothesis with a class of nanoparticles which are known to evade liver capture on intravenous injection (16). These Molecular Envelope Technology (MET) nanoparticles are constructed from N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycol chitosan, a self-assembling polymer amphiphile (17). Drug loaded MET formulations were prepared and their biodistribution and pharmacodynamics/ toxic effects studied. We sought to provide a mechanistic explanation by also studying macrophage uptake of the MET particles.
Materials and Methods
All materials were obtained from Sigma Aldrich Corporation, MO, USA, unless otherwise stated. All solvents were obtained from Fisher Scientific, UK, Loughborough, United Kingdom.
Cell Culture
U-87 MG human glioblastoma cell line was purchased from American Type Culture Collection (ATCC® HTB-14™; ATCC, Manassas, VA, USA). U-87 MG cells were grown in Minimum Essential Medium (Life Technologies, Paisley, UK) supplemented with FBS (10% v/v; Labtech International Ltd, Uckfield, East Sussex, UK); sodium pyruvate (1 mM; Life Technologies) and L-Glutamine (2 mM; Life Technologies). Cells were maintained in culture (37°C in 5% CO2) for up to 14 days (splitting every 2–3 days when 75–80% confluence was reached in the 75 cm2 tissue culture flask) before they were used for tumour implantation.
Synthesis of the MET Polymer
MET Polymer Characteristics
Batch number
Mole% palmitoyl groups
Mole% quaternary ammonium groups
Molecular weight
Mw (kDa)
Mn (kDa)
Preparation of MET Lomustine Nanoparticles
The nanoparticle formulation was prepared by probe sonicating lomustine (2 mg ml−1), MET polymer (20 mg ml−1) soybean oil (10 mg ml−1) and polysorbate 80 (5 mg ml−1) in dextrose solution (5% w/v) on ice for 30 minutes (MSE Sonipreo 150, MSE UK, with the instrument set at 50% of its maximum output). Each time 26 ml of the MET formulation was prepared. The formulation was filtered (0.22 μm) and the amount of lomustine encapsulated was measured by HPLC. To achieve high drug concentrations, the formulation from above (26 ml) was lyophilised and reconstituted to 13 ml using double deionised water (Millipore Water Purification System, EMD Millipore Corporation, Merck KGaA, Darmstadt, Germany). Formulations were analysed by HPLC using an analytical C18 derivatised silica gel based (Onyx Monolithic: 5 μm; 100 x 4.6 mm; Phenomenex®, UK) column using an Agilent (Agilent Technologies 1200 Series) HPLC system.
Formulations were sized using a Malvern Nanosizer (Malvern Instruments, Malvern, UK) at a temperature of 25°C and data analysed using the Contin method of analysis.
Formulations were imaged using transmission electron microscopy (TEM). For TEM imaging, a drop of the formulation was placed on a formvar/carbon coated grid and excess sample was blotted off on a filter paper (Whatman No 1). The samples were then negatively stained (uranyl acetate 1% w/v) and left for 1–2 minutes to air dry. Subsequently, images were captured on the TE microscope using an AMT digital camera (5 mega pixels; AMT Deben, UK Ltd).
The stability of the dried lomustine nanoparticles was studied over a 7-day period, with formulations stored at room temperature and reconstituted periodically to produce a 1 mg ml−1 lomustine formulation, the drug content analysed and the particle size measured as described above.
Preparation of the Lomustine Ethanolic Formulation
The ethanolic lomustine formulation administered to male CD-1 mice as control in the pharmacokinetic studies was prepared as follows: 5 μl of polysorbate 80 was transferred into a glass vial containing 2 mg lomustine. The vial was vortexed for 1 minute and 895 μl of 5% w/v dextrose solution was added to it. The vial was vortexed for another 1 minute. It was then sonicated on ice for 30 minutes as described above. 100 μl of 10% v/v ethanol was added to the vial containing the lomustine, polysorbate 80 and 5% w/v dextrose solution and was vortexed for 1 minute. The content of the vial was filtered through a 0.22 μm syringe filter (Millipore).
The ethanolic lomustine formulation administered as control in the pharmacodynamics and toxicity studies was prepared by vortexing lomustine (2 mg) in absolute ethanol (100 μl) with polysorbate 80 (5 mg ml−1) in 5% w/v dextrose solution (final ethanol concentration of 10% v/v). The resulting colloidal mixture was then filtered (0.22 μm; Millipore syringe filter) to remove drug crystals and yield a non-particulate formulation.
The lomustine content of the formulations was determined by HPLC analysis of the filtrate.
Ethics Statement
All animals were housed at the UCL School of Pharmacy's Biological Services Unit (BSU) and were acclimatized in the BSU for 5–7 days before studies commenced. All animal studies were conducted in accordance with the policies and regulations of the Home Office as stipulated in the Animals and Scientific Acts 1986 UK, for the handling and care of laboratory animals used in scientific research, the recommendations of the BSU and with the approval of the ethics committee.
The MET lomustine formulation (1.04 mg ml−1) was intravenously administered to healthy male CD 1 mice (22–28 g) via the tail vein at a dose 13 mg kg−1 and in a dose volume of 289–357 μl. Control animals were administered an ethanolic lomustine formulation (0.37 mg ml−1) at a dose of 6.5 mg kg−1 and in a dose volume of 370–490 μl. Animals were killed at various time points and the blood, brain, liver and bone were sampled and the solid tissues stored at −80°C until analyses could be performed on them. Plasma was obtained by centrifugation of the blood samples (4000g, Hermle Z 323K centrifuge, HERMLE Labortechnik GmbH Siemensstr. 25 D-78564 Wehingen, Germany) at 4°C for 10 minutes and the plasma stored at −20°C until analyses could be performed.
Tissue Analysis
To thawed plasma samples (0.2 ml) was added chilled homogenising buffer [trizma HCl (50 mM), ethylene diamine tetraacetic acid [(EDTA); (0.1 mM), pH = 2, 0.2 ml], and carmustine (20 μg ml−1; 0.1 ml) as internal standard. This mixture was extracted with ethyl acetate (3 X 5 ml) and centrifuged (1000g) for 10 minutes at 4 oC. The combined organic layers were evaporated to dryness under a stream of nitrogen.
For analysis of brain, liver and bone samples, each tissue was weighed and homogenised in homogenising buffer (1 ml) and the homogeniser rinsings (2 X 1 ml) added to the homogenate. Carmustine (20 μg ml−1; 0.1 ml) was added to the aqueous layer as an internal standard and the homogenate extracted with ethyl acetate (3 X 10 ml). The mixture was centrifuged (1000g) for 10 minutes at 4 oC and the combined organic layers evaporated to dryness under a stream of nitrogen. The residues were subsequently reconstituted in mobile phase [acetonitrile (MeCN; HPLC grade): 0.02% v/v trifluoroacetic acid (TFA) in water (H2O): - 50: 50, 0.2 ml]. The reconstituted extracts were then subjected to HPLC analysis.
Analysis for lomustine content in the tissue samples was carried out using a gradient elution method with an initial condition of 15% MeCN in TFA (0.02% v/v> in water) and proceeding to 65% MeCN in TFA (0.02%v/v in water) over 10 minutes and at a flow rate of 2 mL min−1. The extracted samples were injected (20 μl) over an onyx monolithic C18 (5 μm, 100 x 4.6 mm) Phenomenex® column set at 40°C. Lomustine content was detected using a UV detector set at 230 nm. Calibration plot was done for plasma, brain, bone marrow and liver from which the actual concentration of lomustine in the samples was obtained. All results were expressed as mean ± standard deviation.
The pharmacokinetic parameters for the MET nanoparticle lomustine formulation was obtained using the non-compartmental method of the WinNonlin® software, version 4.1 (Pharsight Corporation, California 94040, USA).
Ex-Vivo Coherent Anti-Stokes Raman Spectroscopy (CARS) Imaging
Deuterated N-palmitoyl-N-monomethyl-N-N-dimethyl-N,N,N-trimethyl-6-O-glycol chitosan (MET) polymer was synthesised as previously described (19) and deuterated MET particles prepared as previously described (19). Male CD-1 mice (25–30 g) were intravenously dosed with deuterated MET particles (10.4 mg ml−1) at a dose of 75 mg kg−1 and in a dose volume of 200 μl. Animals were killed 1 hour after dosing. The subsequently harvested brains were stored in neutral buffered formalin (10% v/v). Fixed brains were cut into 0.5 mm thickness coronal slices with razor blades using a brain matrix (Zivic instruments, Pittsburgh, PA, USA). Brain slices were placed between two glass coverslips and sealed against dehydration prior to imaging using CARS microscopy, as previously described (20).
Bone Marrow Toxicity
Male CD-1 mice were randomly assigned to treatment groups and intravenously administered (via the tail vein) either MET lomustine (2.02 mg ml−1) at a dose of 13 mg kg−1 in a dose volume of ≈ 200 μl or ethanolic lomustine (0.16 mg ml−1) at a dose of 1.2 mg kg−1 in a dose volume of ≈ 200 μl daily for 10 consecutive days.
Subsequently, mice were killed on 1, 7, 14, 21 or 30 days after completion of the dosing and blood and femoral marrow cell counts were assessed to determine the effect of the treatments on the bone marrow.
Blood samples were obtained by cardiac puncture and transferred into EDTA coated tubes (BD Microtainer® tube with Dipotassium EDTA; Becton, Dickson and Company, New Jersey, USA). Full blood counts were carried out using an automatic haemocytometer (Sysmex Automated Haematology Analyzer KX- 21, Sysmex Corporation, Chuo-ku, Kobe 651-0073, Japan) to determine the levels of the various blood components (white blood cells, red blood cells and platelets). Femoral cells were obtained by flushing out the bone marrow with 1 ml PBS and the cell counts were determined by flow cytometry (MACSQuant Analyzer, Miltenyi Biotec, GmbH, Germany).
Macrophage Nanoparticle Uptake
Nile Red Loaded MET Nanoparticles
MET Nile Red formulations for flow cytometry studies were prepared by probe sonicating (QSonica sonicator, Connecticut, USA) Nile Red (50 μg ml−1) and the MET polymer (1 mg ml−1) in dextrose solution (5% w/v) on ice for 30 minutes, with the instrument set at 25% of its maximum output. This was followed by centrifugation (1000g) for 30 minutes at 4 oC to separate free Nile Red from encapsulated Nile Red. The supernatant was then carefully collected immediately after centrifugation. MET Nile Red formulations for confocal laser scanning microscopy (CLSM) experiments were prepared by adding a solution of Nile Red (100 μg ml−1, 100 μL) in ethanol to a dispersion of the MET polymer (1 mg ml−1) in dextrose solution (5% w/v), to a final volume of 10 ml and probe sonicated on ice for 30 minutes, with the instrument set to 25% of its maximum output. This was followed by centrifugation (1000 g) for 30 minutes at 4oC to separate free Nile Red from encapsulated Nile Red.
Nile Red Loaded Liposomes
Nile Red (50 μg ml−1), egg phosphatidyl choline (3 mg ml−1) and cholesterol (1.4 mg ml−1) were dissolved in chloroform (10 ml) and the resulting solution evaporated to dryness at 40°C using a rotary evaporator. The thin lipid film obtained was then hydrated with 5% w/v dextrose solution (5 ml) by shaking for 30 minutes at room temperature to yield a homogenous dispersion of egg phosphatidyl choline liposomes. This dispersion was subsequently probe sonicated on ice for 15 minutes, as described above. The liposome formulation was then centrifuged (1000g) for 30 minutes at 4 oC to eliminate free unencapsulated Nile Red and the supernatant was carefully collected immediately after centrifugation. These liposomes were used for the flow cytometry studies.
Liposomal Nile Red formulations for CLSM imaging were prepared by adding a Nile Red solution (100 μg ml−1, 100 μL) in chloroform to egg phosphatidyl choline (3 mg ml−1) and cholesterol (1.4 mg ml−1) dissolved in chloroform (10 ml) and following the methodology outlined above.
For both the Liposome and MET Nile Red formulations used for CLSM, the concentration of Nile Red [determined by fluorimetry, λexc = 488 nm and λem = 655 nm, (LS – 50 B, Perkin Elmer Inc., USA Spectrofluorimeter, with FL WinLab (Perkin Elmer Inc, USA) software] was adjusted so that both formulations had the same Nile Red concentrations (0.2 μg ml−1) prior to application to the cells.
Cell Uptake Experiments
J774A.1 cells were cultured in Dulbecco's Modified Eagle's Medium [(DMEM; ATCC® 30-2002™) modified to contain L-glutamine (4 mM), glucose (4.5 g L−1), sodium pyruvate (1 mM), and 1500 mg L−1 sodium bicarbonate] supplemented with 10% fetal bovine serum. The cells were maintained in culture for at least 14 days before they were used for uptake experiments. For flow cytometry and confocal imaging, cells were handled according to methods described by Fernando et al. 2010 (21) and Kim et al. 2012 (22) with some modifications, as outlined below.
Cells were seeded in 6 well plates at a density of 300,000 cells per well and incubated for 72 hours. Cells were then treated with either MET Nile Red (0.3 μg ml−1) or the liposome Nile Red (0.4 μg ml−1) formulations with some wells left untreated as control. Cells were treated for predetermined time periods of 5, 10, 30 minutes, 1 hour, 2 hours, or 4 hours. Cells were then washed 3 times with cold Dulbecco's phosphate buffered solution (DPBS, [calcium chloride anhydrous (CaCl2; 0.9 mM), magnesium chloride (MgCl2.6H2O; 0.5 mM), potassium chloride (KCl; 2.7 mM, potassium phosphate monobasic (KH2PO4; 1.5 mM), sodium chloride (NaCl 137.9 mM), sodium phosphate dibasic (Na2HPO4.7H2O; 8.1 mM), pH 7.0–7.2] (2 ml / well) and then incubated in cold PBS – EDTA (PBS- EDTA, [KH2PO4 (1.9 mM), NaCl (138.9 mM), Na2HPO4.2H2O (6.6 mM), Titriplex III (EDTA Na2; 1.4 mM), lithium chloride (LiCl; 10.1 mM); pH 7.5)] (1 ml / well) for 1–2 minutes. Cells were then harvested by gently scraping and pipetting the cell suspension which was then centrifuged and suspended in cold Ringers solution [Sodium chloride (38.5 mM), Potassium chloride (1.4 nM), Calcium chloride hexahydrate (0.5 mM), Sodium bicarbonate (0.6 mM); pH 7] and the nanoparticle uptake was quantified using the flow cytometer (MACSQuant Analyzer, Miltenyi Biotec, Germany) with uptake quantified with reference to the Nile Red fluorescence. A total of 20,000 cells were measured in each sample within the Nile Red positive channel. The experiment was carried out in quadruplet for each formulation and the untreated control cells.
For confocal microscopy imaging, cells were seeded in glass bottom 35 mm tissue culture dishes (MatTek, Corporation, Ashland, MA, USA) at a density of 75,000 cells per dish and incubated for 48 hours. Cell uptake of the formulations was then monitored in a time-lapse experiment set up on a Zeiss LSM 710 laser scanning microscopy imaging unit (LASOS Lasertechnik GmbH, Carl Zeiss, Franz-Loewen-Straße 2, 07745 Jena, Germany). Images were captured 3 minutes after treatment with the formulation and subsequently after every 5 minutes. The images were analysed using Zen 2009 software (Carl Zeiss Microscopy GmbH Carl Zeiss Promenade 10, 07745 Jena, Germany).
Brain Tumour Studies
Brain Tumour Model and Magnetic Resonance Imaging
Tumour bearing mice brains were embedded in a mixture (50: 50) of agarose gel (1% w/v) and formaldehyde solution (4% w/v) prior to imaging, to fix the tissue and minimize shifting of the samples during measurement. Images were collected using a 1.5 T Philips Intera Gyroscan magnet (Philips Healthcare, 5680 DA Best, The Netherlands). A high resolution T2-weighted Turbo Spin-Echo (TSE) sequence was used (Repetition time (TR) = 3000 ms, Echo time (TE) = 110 ms, flip angle = 90 degrees, number of slices = 54, voxel size = 0.14 X 0.14 X 0.14 mm3, 10 averages). High-resolution images were achieved by utilizing a microscopy coil, diameter 23 mm.
Image parameters were established in a preliminary experiment, so as to optimize tumour edge detection. OSIRIX software (Pixmeo SARL, Switzerland) was utilized to analyse the MRI images and to measure the tumour volume, after a manual determination of the edges in a slice-by-slice process.
Brain Tumour Treatment
Human glioblastoma (U-87 MG) cells were cultured in Minimum Essential Medium (MEM) supplemented with foetal bovine serum (FBS, 10% w/v); sodium pyruvate (1 mM; 5 ml) and L-Glutamine (1% w/v; 5 ml). Cells were maintained in culture for up to 14 days before they were used for tumour implantation.
Intracranial tumour models were established by orthotopic implantation of the glioblastoma cells (100,000 cells) in the left striatum (+0.5 mm anterior, – 2 mm lateral and to a depth of 3mm to the bregma, determined by a BENCHmark™ digital stereotaxic control panel), of stereotactically fixed anaesthetised (inhaled isoflurane) female CD-1 nude mice (20–30g in weight). Once tumours were established (7 days after implantation) mice were randomly assigned to treatment groups and injected daily (tail vein) with the lomustine formulations. Animals were either dosed with MET lomustine (2.6 mg ml−1) at a dose of 13 mg kg−1 and a dose volume of ≈ 200 μL or were dosed with ethanolic lomustine (0.18 mg ml−1) at a dose of 1.2 mg kg−1 and a dose volume of ≈ 200 μL. Animals were dosed on 10 consecutive days and the dose of the ethanolic lomustine formulation was the maximum dose volume that could be administered with repeat dosing. Animals were weighed daily and animals were killed once body weight had declined by 15% compared to the start of treatment. Examination of control animals once body weight had reached this threshold value revealed tumours which were 83.4 – 98.1 mm3 in volume, with a mean volume of 91.8 ± 7.57 mm3
Data were subjected to a one-way analysis of variance (ANOVA) with Tukey post hoc test using SPSS, version 17.0 software (SPSS Inc., Chicago, USA) for the pharmacokinetics experiment and Minitab® 16 software (Minitab, Inc., Pennsylvania, USA) for the brain tumour and bone marrow toxicity experiments. The treatment groups were compared two groups at a time for each time point and the significance level was set at 0.05.
Synthesis of the MET Polymer
Nanomerics' MET is based on the self-assembling polymer N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycol chitosan, which assembles into nanoparticles in aqueous media (17,18). Various batches of the MET polymer were synthesised and characterised (Table I).
Preparation of MET Lomustine Nanoparticles
The nanoparticle formulations presented as translucent liquids with a z-average mean
Ex-Vivo CARS Imaging
The deuterium labelled MET nanoparticles were visualised in ex-vivo brain samples after intravenous injection and were found to adhere to the brain endothelium, i.e. to the luminal side of the blood brain barrier (BBB). The MET polymer CARS signal was not seen in the brain parenchyma and as previously reported particles appear to adhere to the BBB (20) and are not delivered to the brain (16). However, MET nanoparticles are delivered to the BBB, adhere to the brain endothelial cells (Fig. 3), from where they presumably release loaded drug for transport across the BBB.
Lomustine Tissue AUC0-120 Following Intravenous Dosing
Formulation and dose
Brain AUC0-120min (μg g−1 min)
Bone AUC0-120min (μg g−1 min)
Liver AUC0-120min (μg g−1 min)
\( \frac{BrainAU{C}_{o-120 \min }}{BoneAU{C}_{o-120 \min }} \)
\( \frac{BrainAU{C}_{o-120 \min }}{LiverAU{C}_{o-120 \min }} \)
MET - Lomustine
13 mg kg−1
Ethanolic lomustine
6.5 mg kg−1
MET - Lomustine
6.5 mg kg−1
Lomustine Plasma AUC0-120 Following Intravenous Dosing
MET - Lomustine
Ethanolic lomustine
MET - Lomustine
13 mg kg−1
6.5 mg kg−1
6.5 mg kg−1
Plasma AUC0-120 (μg ml−1 min)
\( \frac{BrainAU{C}_{o-120 \min }}{PlasmaAU{C}_{o-120 \min }} \)
\( \frac{LiverAU{C}_{o-120 \min }}{PlasmaAU{C}_{o-120 \min }} \)
\( \frac{BoneAU{C}_{o-120 \min }}{PlasmaAU{C}_{o-120 \min }} \)
Lomustine Cmax Values
Dose (mg kg−1)
Plasma (μg ml−1) mean ± s.d
Brain (μg g−1) mean ± s.d
Liver (μg g−1) mean ± s.d
Bone (μg g−1) mean ± s.d
MET lomustine
1.84 ± 0.43*
2.74 ± 0.67*
9.85 ± 1.32*
1.82 ± 0.31
Ethanolic lomustine
0.84 ± 0.32
1.24 ± 0.40
3.00 ± 1.33
1.46 ± 0.15
MET lomustine
0.70 ± 0.15
0.76 ± 0.27
2.12 ± 0.98
0.78 ± 0.34*
lomustine is extensively distributed and metabolised in the plasma (25), but does lead to a proportional increase in brain AUC0-120 values (2.2 fold) and a limited increase in both bone (1.2) and liver (1.3) AUC0-120 values (Tables II and III).
At the highest dose, MET lomustine delivers 0.33% (0.77% per gram of brain) of the dose to the brain. It is clear that the MET formulation produces higher brain levels, while sparing the bone marrow and liver and this altered distribution is not underpinned by an increase in plasma exposure (as is seen with other pharmaceutical particulates such as liposomes (26,27)) but is due to differential tissue uptake mechanisms.
Animals administered 6.5 mg kg−1 lomustine either as the MET or ethanolic formulation showed no difference in the drug's plasma and brain Cmax, whereas the ethanolic formulation produced a significantly higher Cmax in the bone (including the bone marrow), when compared to the MET formulation (Fig. 4, Table IV), we thus conclude that the MET particles avoid the bone and bone marrow. Furthermore, with the 6.5 mg kg−1 dose, the MET particles are cleared faster from the liver when compared to the drug injected as an ethanolic solution (Fig. 4c) and we thus conclude that the MET particles are not retained by the liver. Lomustine chemotherapy is associated with dose limiting myelosuppression (15,28, 29, 30) and hepatotoxicity in dogs (31). Myelosuppression and hepatotoxicity are thus dose limiting adverse effects for the drug and any formulation that minimises bone marrow and liver exposure should have a positive impact on treatment.
Bone Marrow Toxicity
Full femoral cell counts (Fig. 5a) and white blood cell counts (Fig. 5d) remained unchanged for both formulations.
There were minor changes in red cell count (83.0 ± 8.0% of control values) and haemoglobin levels (84.5 ± 3.5% of control values) with the high dose (13 mg kg−1) MET formulation (Fig. 5b and e) on Day 1 with recovery after 7 days for the red cell count and recovery after 30 days for the haemoglobin levels. The changes in haemoglobin levels are unlikely to be clinically significant as the range of acceptable values for haemoglobin [male 14–18 g/dL; female 12–16 g/dL (32) spans at least a ± 20% range.
The main myelosuppressive changes were detected in the platelet count, with platelet levels falling for both the low dose ethanolic (1.2 mg kg−1) and high dose MET (13 mg kg−1) formulation treated groups (Fig. 5c). The nadir values for both groups were recorded on Day 7 with platelet nadir values of 56 and 79% of the control values recorded for the high dose MET (13 mg kg−1) and low dose (1.2 mg kg−1) ethanolic formulations respectively. For the ethanolic formulation treated animals, recovery was observed after 14 days while the higher dose MET nanoparticle treated group showed recovery after 21 days. In essence the administration of ten times the ethanolic dose as a MET formulation resulted in very little additional haematological toxicity when compared to the low dose ethanolic formulation.
Macrophage Uptake
Macrophages are a heterogeneous group of cell types, which are resident in a number of tissues, including the bone marrow and the liver; with one of their functions being the removal of particulate cellular debris (33). In order to understand the mechanisms underpinning the low liver, bone and bone marrow drug levels, we hypothesised that the main mechanism could lie with a reduced uptake of the MET nanoparticles by the macrophages in the liver and bone marrow. Previously we have shown that on intravenous injection, liver deposition is comparatively low for MET nanoparticles, with only 4% of the intravenous dose found in the liver 10 minutes after dosing (16), compared to peak levels of 67% of the intravenous dose of liposomes found in the liver 80 minutes after dosing (34). We hypothesised that the low liver uptake of MET nanoparticles could be due to the low uptake by liver macrophages and it is conceivable that the bone levels may also be reduced due to low bone marrow macrophage uptake.
The rate of macrophage uptake is thus slower for the MET nanoparticles when compared to the uptake by liposomes and it is thus possible that the reduced liver and bone and bone marrow drug levels and consequent relatively mild effect on the bone marrow of the dosage form could be the result of reduced macrophage uptake. The macrophage uptake data thus provides further proof that the MET nanoparticles appear not to be taken up by the reticuloendothelial system to an appreciable extent.
Brain Tumour Model
Brain Tumour Treatment
During treatment the animals' body weight was monitored and there was no significant difference between the body weights when all groups were compared (untreated, ethanolic and MET treated animals – Fig. 8b). This indicates that the high dose formulation did not cause gross toxicities and the MET formulation thus enabled the administration of a higher dose, with the lack of uptake by the bone marrow and liver likely to limit the toxic effects (15,28, 29, 30) that are normally observed with lomustine therapies.
The MET formulation presented as a nanoemulsion in which the oil droplets are presumably coated with the MET polymer and polysorbate 80 (Fig. 1). The size distribution is heterogeneous and comprises micelles as well as oil droplets. The distribution of the two amphiphiles—the MET polymer and polysorbate 80 between the micelles and the oil droplets is unknown. This formulation enables a higher dose to be administered as a bolus and this is in turn associated with a longer survival time (Fig. 8a), when compared to the administration of the lomustine ethanolic—polysorbate 80 formulation at the maximum bolus dose possible. However, it is the relatively reduced delivery of drug to the liver and bone (Fig. 4, Tables II and III) and the resultant minimal effect of the high dose formulation on myelosuppression (Fig. 5) that is the most interesting feature of the MET formulation. Lomustine chemotherapy is associated with dose limiting myelosuppression (15,28, 29, 30) and autologous stem cell rescue (15). Hepatotoxicity in dogs is also a feature of lomustine chemotherapy (31). Myelosuppression and hepatotoxicity are thus dose limiting adverse effects for lomustine and any formulation that minimises bone marrow and liver exposure would have a positive impact on treatment and allow patients to take fewer treatment breaks, as the tumour would presumably continue to grow during such treatment breaks allowing the supporting microenvironment to recover rapidly. Lomustine is normally given orally and we administered it intravenously in this study, using it as a model drug to test the bone marrow sparing potential of the intravenous MET formulation.
We have found that MET nanoparticles are taken up at a slower rate in vitro by mouse monocyte derived macrophage cell lines (Fig. 6) and while these are not tissue resident macrophages which have distinct functions, such as immune surveillance, removal of cell debris and iron processing (33), it is clear that these macrophages phagocytose liposomes more readily than when compared to the MET nanoparticles and we conclude that the surface chemistry of the MET particles must be responsible for their slower phagocytosis when compared to liposomes. Such a conclusion is not without precedent as the exposure of phosphatidyl serine on the surface of apoptotic cells enables these cells to be taken up by tissue resident macrophages expressing the phosphatidyl serine receptor (33).
It is possible that this reduced macrophage uptake is a key driver for the low bone and liver levels of the drug (Fig. 4). Low liver uptake of MET nanoparticles has been demonstrated in a number of studies (16,35). For example the coating of peptide nanofibres with the MET polymer resulted in a significant reduction in liver deposition (35), hence evidence is beginning to emerge in support of the hypothesis that the MET polymer coating diverts nanoparticles from the liver (Fig. 4), spleen (16) and bone marrow (Fig. 4) on intravenous injection. While it is clear that there are correlations between macrophage uptake (Fig. 6) and liver (Fig. 4) and spleen (16) deposition of MET nanoparticles, when compared to liposomes (34) (Fig. 6), we have not proven that the reduced macrophage uptake observed actually leads to reduced liver, spleen and bone marrow deposition of MET nanoparticles on intravenous injection, especially as tissue resident macrophages (which are proliferative and of embryonic origin) (33) were not used in our in vitro studies.
To further understand the advantages offered by the liver/ bone sparing MET formulation, which in essence targets the drug to the brain, one must consider the fact that dose intensification would allow the drug to overwhelm the enzyme repair systems that confer resistance to the therapeutic, such as the enzyme O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme (7). Dose intensification, when tried in the clinic, although beneficial, is usually confounded by the toxicity of the regimen, most notably haematological toxicities (36). In our studies, dose intensification with the MET formulation does not confer significant additional myelosuppressive effects when compared to the low dose ethanolic formulation and yet is therapeutically beneficial in this mouse intracranial tumour model. Dose intensification with the MET system definitely warrants clinical testing.
Dose intensification with chemotherapy may be achieved either by increasing the dose or by increasing the frequency of dosing. The resulting haematological toxicities will take the form of neutropenia, thrombocytopenia or a reduction in red blood cells (36,37). Neutropenia, may lead to fatal infections and is the most frequently occurring toxic effect encountered with cytotoxic drugs (37). Due to the short life span (6–14 hours) (38) of granulocytes, they are usually the first to be negatively affected during chemotherapy (38). However, in the current study, the white blood cells were not affected by the high dose MET formulation and neither were the total bone marrow cells (Fig. 5a and d). Platelets with a life span of 9–10 days in humans (39) and 4–5 days in the mouse (40) are usually the next susceptible cell type to be affected by chemotherapy after the granulocytes (41). The results of the current study are consistent with this platelet vulnerability, as all treatment groups show a significant drop in platelet counts after 7 days, although platelets counts did revert to baseline levels after 14 and 21 days for the low dose ethanolic (1.2 mg kg−1) and high dose MET (13 mg kg−1) formulations, respectively (Fig. 5c).
Toxic effects on the red blood cells are the last to be seen due to the longer life span of the erythrocytes with a life span of 100–120 days in humans (41,42) and 30–52 days in the mouse (40) and although there were minor drops recorded in the red cell counts and haemoglobin levels in our studies, these changes in red blood cell counts and haemoglobin levels amount to no more than a 20% change and thus are unlikely to be clinically significant.
Our findings show that lomustine dose intensification with the MET particle system improved the survival of intracranial tumour bearing mice and did not produce significant additional myelosuppressive effects as bone and bone marrow deposition of the drug was reduced with the MET system. Liver deposition was also reduced with the MET system and since myelosuppression and liver toxicity are features of lomustine therapy, such a dose intensification strategy warrants clinical testing.
Wellcome Trust Grant number: 083944/Z/07/Z Tertiary Education Trust Fund (TETFund, formerly Education Trust Fund), Nigeria - Academic Staff Training and Development Grant and Obafemi Awolowo University, Ile-Ife Nigeria (Grant administrator) – Funding for PhD studies for Funmilola A. Fisusi. The authors declare no conflict of interests.
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Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.


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I first came across Strange Names last year in Philadelphia at a show with fellow Minneapolis band, Carroll. I didn't know what to expect, but I was blown away by their live set, and couldn't wait to hear more. The brief songs I saw live stuck with me, I was out of luck they were unreleased, until now.
This week we see the release of Strange Name's debut full length album, Use Your Time Wisely. The album is almost like a flash back, something not of this era. Instead of hitting play on my computer, I feel like I should be popping the cassette into my walkman and dancing along in my leg warmers.
Basically, what I'm trying to say is that Strange Names have found a way to reinvent the wheel and bring us back the new wave/synth-pop of the 80s that we all know and love. With a sound similar to that of Flock of Seagulls or Simple Minds, they also found a way to add a modern day touch. Incorporating some harmonies at times, and set to the synth beats, it's all really something special to experience.
Strange Names have been teasing followers with several tracks off the album over the last several months. First released was the highly impressive track 'Ricochet', which thankfully was one of the songs stuck in my head from awhile back. 'Ricochet' gave us a perfect indication of what the rest of the album was going to be like, which is pure awesome.
This album is being released at a perfect time to gear up for summer. I foresee many road trips ahead that are set to Use Your Time Wisely. The album provides the perfect upbeat jams we need to bring us into the sunlight and fun times. With songs like 'Trespassing' or 'I Can't Control Myself', which you can't help but dance to. Or 'Supernatural Silence' or 'Brick City' that transport you into another atmosphere. You also have more rocking songs like 'Only Boy' to bring you out of the 80s vibes, and into something new.
Be on the lookout for Strange Names, because these guys are likely to take off soon.
I Can't Control Myself
Supernatural Silence
Only Boy
Where & Why
Brick City
Love & Truth (Is There a Place)
Overused Phrase
Words by: Carolyn Lederach

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|Stage 1 "Mama"
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I was sitting out on my deck and noticed a Junco bird walking right pass me. My eyes followed where she was heading. She flew right up into my planter stand which contained some yellow primroses, she was building her nest along with her mate.
I was really worried because other birds had built their nests in my flower containers on the deck before and the babies didn't make it. I was nervous but I couldn't stop her, she was on a roll bringing in straw, moss etc., etc.
I took pictures with each process, from building the nest till the babies where born. The painting where she is sitting on her nest, I was sitting pretty close to her and zoomed in with my camera. The painting that shows the babies born, I was able to get a shot from inside the house looking down at them.
I would whistle, Jesus Loves You every time I would walk out on the deck, they seemed to feel comfortable around me.
The babies did make it and flew away but I think that they are still around and feeding from the bird feeder.
After the nest was empty I saw the mother days later on the roof. I whistled that tune again to see if it was her, she flew down by me and sat on the chair to say hello. To my surprise she laid some more eggs about two weeks later, they made it too.


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Mute Planet
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Mute Planet is a website dedicated to travel, science, writing and photography. Through silent media, it explores and celebrates the many wonders of this amazing world we share.
Welcome to the Mute Planet 靜音星球
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Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7
Autumn/Winter 2017 Fashion Week began with a blast. A blast of snow and frigid temps courtesy of Winter Storm Niko. The biggest storm of the season caused seasoned fashionistas to reconsider their front-row frocks in favor of more appropriate cold weather attire.
Not this fashionista, however. I saw every show from the comfort of my PJ's all warm and cozy in my Hudson Valley home. Thank god for the live stream.
While the manfriend spent a good two hours plowing over a foot of snow out of our rather long driveway, I was immersed in the emergence of all the trends we'll be fawning over in fall.
The 70's, crushed velvet, cold-shoulders, bohemia, pink, lace, and a stampede of military inspired looks to outfit an army of resistance. All of this just from day one.
Here's a look at some of my favorite looks so far. Keep checking back throughout the week to see more.
Looks from Nicholas K, Tadashi Shoji, and Erin Fetherston
Looks from Colovos, Cinq à Sept, and Jeffrey Dodd
Looks from Marissa Webb, La Perla, and Cynthia Rowley
*All photos from VogueRunway.com
Follow the Stylaphile NYFW Favorites Board on Pinterest!


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The Health and Fitness Program on the College of Kinesiology is ideal for students curious about wellness, private coaching, teaching, or being a health and fitness educator. This can be a quickly growing subject and graduates are in high demand. The Michigan Benefit" lies not only in the quality of our choices, but additionally in the energy of all of the highly ranked, interdisciplinary packages across U-M's campus — medicine, public health, training, and business, to name a number of — and a world-class athletics program. Arrive early. Arrive to your appointment quarter-hour early to be sure you can get checked in and fill out any required paperwork before your scheduled appointment. Warmth grill pan to medium-excessive. Season chicken with paprika, salt and pepper. Grill until cooked via, four-6 minutes per aspect. Put aside until cool sufficient to handle, then reduce into 1-inch slices.
Health care can kind a major part of a country's financial system. In 2008, the health care trade consumed a mean of 9.0 % of the gross home product (GDP) across probably the most developed OECD international locations.2 The United States (sixteen.0%), France (11.2%), and Switzerland (10.7%) had been the top three spenders. Now for an additional accountable pleasure to reconsider, unhealthy meals. We all love unhealthy meals particularly after a lengthy day within the medical heart it's nice to remedy your self to a draw back. Current studies have found that even one meals that's high in physique fats can do hurt to our bloodstream.
We're happy that you're coming to Macalester! This is a community that values holistic health and wellness. The Health & Wellness Heart (HWC) is here to assist you in realizing your private health and to contribute to the effectively-being of the group. A technique this is accomplished is to require some information from you before your arrival to campus. These new pupil requirements should be accomplished and submitted to HWC by June twenty fifth, 2017. NOTICE: Health insurance coverage waive out/choose in will probably be obtainable on or around July 5, 2017. Info on the insurance web site prior to July fifth will reflect last year's coverage (which is similar to this yr's coverage) and will not will let you waive/enroll.
ohh its good thought…Right here i've one suggestion for you. our is without doubt one of the greatest for making medication in Indian methodology in line with WHO tips. How does relate to understanding? It is easy. In the event you allow your physique to behave as if you happen to're worn out, you will not carry out near in addition to you'll for those who power your self to behave as if you're not close to so drained. And it would not need to be something main. Simply little issues will do.
Any pocket book can be utilized as a prayer journal however I have selected some stunning journals made specifically for holding monitor of your prayers. Non-Verbal Leaning Dysfunction (NVLD or NLD) is beneath investigation in the State of Michigan at Michigan State University. The Departments of Psychiatry and Psychology have joined to assemble in cooperation to have a look at NVLD and its influence on our nation's youth.


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How do you get a secretion out of a bunch of girls?
Last night I came home with probs the dryest eyes I've ever been in, and requested a donation of tears from my housemates.
I don't think it was too much to ask either. When they talked about it earlier, torrid tales of sadness were exchanged and eyes were scrunched up. All I've been able to get is the odd snivel from one or more of them.
I've tried the old "onions under the eyes trick" but it failed. I've tried repetitive poking in the eye and suchlike and got nowhere. I need some new tactics. Help.


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Conn Hallinan
Diego Garcia: The "Unsinkable Carrier" Springs a Leak
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College of Humanities & Behavioral Sciences
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The Department of Foreign Languages and Literatures (FORL) at Radford University offers instruction in seven languages: Arabic, Chinese, French, German, Latin, Russian and Spanish. Why do Radford University students choose to learn a language and its target culture in our department? Our classes are comparatively small, and, through individual and hands-on classes, we can provide more individual attention to our students who establish close dynamic relationships with their instructors. Students also build meaningful friendships with other students studying our languages.
FORL study teaches students core skills rooted in the humanities that are transferable to myriad careers: critical thinking and careful close analysis of texts, research based on persuasive written & oral communication and a cross-cultural and cross-historical understanding.
Foreign language study at Radford University can also set students apart from the others by complimenting a second major. In the 21 century, the ability to understand, speak, read and write in world languages, in addition to English, is critical to success in fields related to business, medical, criminal justice, research and international relations. The United States needs more people to speak languages other than English and to become familiar with the other cultures in order to provide social and legal services for a changing population.
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Accomplished poet, translator and essayist Rosa Alice Branco will give a Portuguese poetry reading and host a discussion on her work "The Cattle of the Lord" at 7 p.m. on April 17 in the College of Humanities and Behavioral Sciences 1006.


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Built in 1837 and still the county's oldest remaining public building, Essex County Jail was designed by Eastern State Penitentiary architect John Haviland and served as the area's main prison for over a century. After undergoing a few renovations and additions over the years, it closed when a new jail was completed in 1970, and it's been abandoned ever since. [x]
The Newark Landmarks committee rejected a plan to demolish the existing buildings to make a park, due to its designation on the National Register of Historic Places, but the roof is collapsing in places and a number of the tiny cells are occupied by drug addicts. The Warden's House recently collapsed entirely, and rehabilitation at this point would require some very creative engineering and demolition of sections of the building.
But, considering so many decades of abandonment, its condition is still pretty impressive.


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The Detroit Red Wings and Tomas Tatar have agreed terms on a new contract. The Slovakian winger, 26, has signed a four-year deal worth $5.3 million AAV, ensuring that he stays a part of the organisation for the foreseeable future. The deal also includes a varied no-trade clause (full during year two/most of year three, limited during year four).
The agreement signals a positive end to discussions, which at times seemed destined for a rather bitter end. The parties had entered salary arbitration on Thursday with both seeming to be a long way apart in terms of an acceptable deal. Tatar had reportedly rejected a $5 million, five-year deal earlier in the summer, seeking additional years and a salary closer to $6 million. They also went in to the hearing Thursday with Tatar requesting $5.3 million and Detroit offering closer to $4.1. Had the arbitrator made a ruling, the contract would have been limited to one year given that this is Tatar's last year as a Restricted Free Agent. The player had reportedly told Slovakian news outlets that a one-year contract agreed in this way would signal his last year as a Red Wing.
Fortunately for all concerned, it hasn't gone that far and Detroit can use Tatar as a cornerstone on which to start the rebuild (which is happening, regardless of how many times Ken Holland denies it). Although quite a 'streaky' player, Tatar has put up solid numbers since being drafted 60th overall by the Red Wings in the 2009 Draft. He has topped the 20-goal mark in three of his six seasons and led all Red Wings with 25 goals last season. He ranked third on the team in points with 46 and only Thomas Vanek (15.2%) had a better shooting percentage than Tatar (15.1%).
With his new deal signed, Tatar can get back to preparing for the new season, and one that could see him reach some personal career milestones:
- If he remains healthy, Tatar could play in his 400th career regular season game for the Red Wings (currently 345)
- Tatar needs one goal to reach 100 regular season goals.
- He needs five assists to reach 100 regular season assists.
- He is six points shy of reaching 200 career regular season points.
- Tatar currently has 49 points on the power play, one more would take him to 50.
Detroit still has work to do
With Tatar now signed, Detroit must focus their attention on signing another RFA in forward Andreas Athanasiou. The Tatar deal leaves them with just under $1 million cap space remaining – taking in to account Johan Franzen is on LTIR. One potential option for Detroit is a 48-hour buyout window that has opened as a result of Tatar electing to go to salary arbitration. The window is open from Today (Monday) and will close on Wednesday. Although they are unlikely to use it, the Red Wings could easily free up some cap space and a roster spot by buying out a contract during this window. The favorite for this would be defenseman Jonathan Ericsson. He is currently earning $4 million per season, and quite frankly it's a contract that Detroit should get out of if they can. They have some good young options on the blue line, a fact they should give some serious consideration to.


text: May 27, 2013 Where are your shoes? May 27, 2013/ Julie Lenard On a street corner in Philly: A man stands on the corner, shoeless. A car pulls up: "Hey, where are your shoes?" the passengers call. "I just have to eat my dinner first!" he replies. You just can't make this stuff up. How do you like my super sleuth photography??


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Simple Math
A hawk flies over stubbled fields.
Before I can calculate
the distance between us,
he arrives at your door.
Kip Knott
Kip Knott has authored three chapbooks of poetry: The Weight of Smoke (Bottom Dog Press), Whisper Gallery (Mudlark), and Everyday Elegies (Pudding House). Whisper Gallery can be accessed online at http://mudlark.webdelsol.com/mudlark26/contents.html.


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Home > ANNOUNCEMENT
- Zhang Dong was appointed Vice President of CTA2011-9-20
- Shi Peihua resigned as Vice-president of CTA2011-1-17
- Dai Bin was appointed President of CTA.2010-7-28
- Du Jiang resigned as President of CTA2010-4-30
- Liu Xiaojun resigned as Vice-president of CTA2010-4-30
- Shi Peihua was appointed Vice-president of CTA.2008-6-6
- Dai Bin was appointed Vice-president of CTA.2008-2-29
- Sun Gang was appointed Honorary President of CTA. 2008-2-29
- Liu Xiaojun was appointed Vice-president of CTA.2008-1-8
- Du Jiang was appointed President of CTA. 2008-1-8
- CTA established by approval of the State Commission Office for Public Sector Reform2008-1-8
- Total 11 page 1 of 1 First Previous Next Last


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Adopt Me: Dakota
This sweet little pony has not had a good start in life. She came to HorseWorld in August 2015 as part of a huge group of feral horses – this included stallions and mares with foals. Dakota was very underweight and had a foal at foot that seemed to be around 2-3 months old, due to her demeanour and appearance we estimated she was an older girl.
We were able to separate Dakota and her foal but she was extremely nervous of people so we left them to settle in a little herd of mares and youngsters. Her foal was weaned successfully but poor Dakota was struggling to keep weight on and had bouts of severe diarrhoea. A faecal egg count showed a huge worm burden so she needed careful treatment for worms so her digestive system wasn't aggravated.
We always suspected that Dakota was pregnant and in the spring of 2016 she gave birth to a fine colt foal – but very sadly he died at birth. Although Dakota was distraught to start with she then began to pick up and recover physically. It was very sad to lose the baby but for Dakota it meant her body had a chance to recover.
We had to have her sedated using a dart gun so we could attend to her feet and whilst she was sedated the vet examined her and estimated that she is only 2-3 years old.
Dakota can now have a head collar on and be led. She regularly sees the farrier without sedation and has been treated by the dentist. She has made great progress and is much more trusting of people.


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The Outside Influence-March 25, 2002
Dan Glamour here. I have this column called the Outside Influence. You might have heard of it.
Since I will be in Cleveland this week on Thursday. I will have a report on WWF Smackdown for Friday. I am going to try to do two columns a week. One for Tuesday and one for Friday. I hope you all enjoyed my Wrestlemania XVIII column and again I apologize for the lateness of the column.
Tonights RAW starts off with Linda McMahon, thats not a good sign and good luck trying to get on WWF.com. Everyone and their brother will be going to WWF.com to see who else is picked. I suggest that instead of wasting your time on WWF.com, go to Lords of Pain to find out who went where.
Mr. Perfect thinks that he will be the number one pick? Not after losing to Tazz, hes not. Mr. Perfect should be lucky and glad that is he not the Road Dogg.
Jake, whos not a regular wrestling fan, brings up a good point. Oh by the way the Rock was Vinces first pick. Wouldnt it be cool to have a wrestler named JESUS? The front of the JESUS shirt would say Get Ready and the back of the shirt would say for CHRIST. JESUS finisher could be the Sacrifice or the Crucifix.
Do they get WWF Smackdown in Canada? SHUT UP ROCK!!!
WOOO!!! Ric Flair picked the Undertaker; nobody on the Internet saw that coming.
Jake needs to watch wrestling more often. WWF Forcible Entry sounds pretty derogatory. I mean why dont they just call it WWF Rape. It might be a mistake for a porno on DVD. Classic stuff Jake, keep it up.
Like I have said in the past, Vince is only as good on the microphone as the people whom he is interacting with. Kurt Angle made Vince McMahon bearable. Nice job on the part of Ric Flair for picking the NWO. So now all he has are a whole bunch of slow big guys and X-Pac. Vince picks up Chris Benoit.
Ric Flair picked Kane? Again all Flair has are a whole of big slow guys and X-Pac.
Vince picked Hulk Hogan, again nobody on the Internet saw that coming.
Thank God, that Ric Flair picked Rob Van Dam and Billy and Chuck are going to WWF Smackdown. In case you are reading this for the first time, we dont get UPN or WWF Smackdown here in Wheeling, West Virginia.
Rock and Hogan vs. the NWO, would everyone in the room please shut up about the conspiracy theories. Hogan is not going to turn on the Rock.
Kane makes the save for The Rock and Hulk Hogan? Looks like someone is getting a push. X-Pac needs to put some pants on too. Thats the first time that Ive seen him since the return.
Hey Ric and Vince, slow down on the picks. Ric picked Booker T and the Big Show. Damn it Ric, quick picking big slow guys. Vince then picked Edge and Rikishi.
Well it looks like its back down to Ohio Valley Wrestling for Rico. Lita looked hurt after the Jeff Hardy and Billy match. Lita is one of the few women that can actually wrestle too. Say hello to Mark Henry for me.
Todd was there when King Kong Bundy big splashed Little Beaver at Wrestlemania II.
Damn it!!! They split up the Dudley Boys!!! How could you do that shit?!? The Tag Teams in the WWF are lacking already and now the Dudley Boys get split up. Thats bullshit!!!
Man, I wonder if Brock Lesnar could do that kind of shit to the late Yokozuna? Brock picked up Rikishi like he was nothing.
They are really picking these guys quick. Thank goodness, Ric Flair was able to pick Brock Lesnar. Does Vince McMahon think we are all idiots? Vince picked Mark Henry, so I guess that means Rico wont get to say hello to him when he is fired. Vince also ended picking Maven, the hardcore champion. Oh well, I wont miss Maven but I will miss the Hardcore Title.
Hey, Kurt Angle vs. Rob Van Dam, it is time for a bathroom break. I mean there has been so much WWF action this evening already.
I nearly had a heart attack when Chris Jericho and Triple H hit each other with the title belts. Stephanie was a second away from becoming WWF Champion. I really thought that it was a good match. I mean it had a lot of suspense build up. And now Stephanie will be off WWF television for a while too.
So tonights RAW is getting a Watchable rating. The draft was interesting but the Split is going to be a terrible idea. Good main event too. Be sure to check out WWF.com to see which of the jobbers end up on either RAW or Smackdown.
Say Stephanie, if you arent busy next Monday night .
Peace Out,
Dan Glamour
© 2002
The Outside Influence
Disclaimer: This site is not affiliated with any professional wrestling promotion or wrestling related website. Images and names are property of WWE (World Wrestling Entertainment).


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Welcome Class of 1954
September 9, 1994
Meadow Woods Country Club
Dinner Dance:
September 10, 1994
Meadow Woods Country Club
Cocktail Hour
6p.m. - 7:30p.m.
7:30p.m. - 9p.m.
9p.m. - 1a.m.
Cocktail Hour
A.T. McCrary
Victor McMenamy
Introduction by Master of Ceremonies
Larry Cooksey
Glenda (Sanders) Meizer
Merlin and the Meriettes
Merlin Johnson
Barbara Crain
Wanda (DeBow) Bailey
Mariann (Nolta) McDonal
Door Prizes
Vincent McMenamy
(DJ - Expressions)
40th Reunion Committee
Centralia Gang
For this reunion the committee did not have the traditional chairman and committee. We all did a little bit of this, a little bit of that, and some a large bit of that. Following is a list of the committee and a brief breakdown of all the bits, both little and large.
Duane and Helen Niekrenz - treasurer, music, decorations, photographer; carried a large box full of 1954 class information to every meeting.
Norma (Bundy) Turner - reservations, decorations, booklet, any mistakes in the booklet made by the computer.
David and Arleen Cross - decorations, worked with printer on booklet; any mistakes in booklet made by computer.
Jane (Fehrmann) Edmonson - decorations, kept check on what Helen and Duane did with all the checks; never offered to carry large box.
A. T. McCrary - head joke teller, decorations, said "no problem" on an average twelve times per meeting.
Gerry (Eller) VanZandt - wrote letter #1, decorations, menu, "balloon lady", whoops, sorry, "balloon person".
Suzie (Shreve) Sprehe - decorations, menu, laughed most at Fred Bauer's stories.
Pat (Stonecipher) Kuhn - wrote letter #2, dedication page, decorations, bought all the lovely gifts with our lovely money.
Barbara (Long) Linder - our May Queen of 1954 and the perfect choice to give out our door prizes at the picnic, plus she laughed at everyone's jokes.
Fred Bauer - an "out-of-towner" who, when in town, came to our monthly Thursday night meeting at The Green Grill. The experience changed the color of his hair.
Millicent Webster - "in-town" instigator, publicity, coordinator at large, used telephone a whle bunch. Favorite phrase, "Do we have to pay tax?"
Other members of the "Centralia Gang" who started with us but could not always attend the meetings were:
Dorothy Coleman
Dale Cook
Shirley (Davis) Yuhasz
George Marcum
Mary Anna (Minton) Nollman
Roger Sutherland
Doris (Johnson) Dearing
The "Out-of-Town Gang"
Pat and Dick Jones - "out-of-town" instigators, decorations, "tracer of lost persons".
Marilyn (Sprehe) Howell - "out-of-town" instigator, decorations, "tracer of lost persons".
Glenda (Sanders) Meizer - memorial, memorial page, decorations.
Other "tracers of lost person" - Carolyn (Carlson) Young, Phyllis (Philipp) Tolliver, and all the anonymous classmates who helped with names and addresses.
Coca-Cola Bottling Co. of Southern Illinois, Inc.
Ron Barrow
Lora Timmons
Norma (Bundy) Turner - Magnets handcrafted by and courtesy of Norma.
Mickey Sprinkle
Bill and Phyllis Doan
Kim Randolph
The Green Grill
A special thanks to all who attended the 40th Reunion of the Class of 1954 and helped make it such a great success!


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Urban Innovator
Joel Marcovitch
Growing Toledo's Jewish Community
Toledo's Jewish population is a lot smaller than it was at its peak, but the community of 2,200 is extremely involved and striving to make the entire city a better place, says Joel Marcovitch, CEO of the Jewish Federation of Greater Toledo.
"I look around at the Jews we have here and they're still very much involved in the betterment and social justice in our city and making sure the community is taking care of itself," he says. "I think it's an incredibly generous community."
The Jewish Federation of Greater Toledo oversees numerous Jewish organizations and programs within the city, from the Senior Adult Center to Gan Yeladim Preschool. The federation also has partnerships with three area congregations, Etz Chayim, Congregation B'nai Israel and Temple Shomer Emunim.
Originally from London, Marcovitch moved to Toledo to work at the Federation in 2012. Before that, he held various positions with Hillel, an organization focused on Jewish life at universities.
Marcovitch and his wife Erin have a 22-month old daughter and are expecting another baby in August: "We're committed to growing this community, literally through ourselves," he said with a laugh.
Of course, Marcovitch has plans to grow Toledo Jewish community in other ways, too. One strategy focuses on marketing to young families in large cities like Chicago or San Francisco, who are likely paying high rent for a small space and spending a lot of time in traffic. The Federation is working with local human resources directors and the Toledo Regional Chamber of Commerce to figure out how to help them get jobs or move their businesses to the area.
"A stronger Toledo bodes well for a strong Jewish community, so if Toledo gets all the things it needs to do right to build itself and become a powerhouse like we believe it can be, then it will bode well not only for the Jewish community but for the entire community," Marcovitch says.
Jewish families are attached to Toledo for the same reason any other family is, Marcovitch says, including museums and the Toledo Zoo. The amount and quality of Jewish cultural events play a part too.
Though the community is small, Marcovitch says they raise about $1.1 million each year. That money is used for the Federation's many community programs, including Jewish family services and the Jewish Historical Society.
Marcovitch is proud that the Federation has programs and events for every life stage. It starts from birth to age six, when children receive a book each month meant to inspire Jewish bedtime stories. After that, they attend kids-focused programming at local festivals, and then they move on to Jewish summer sleepaway camp. Young adults in college can be a part of Hillel and then be a part of Young Jewish Toledo. The offerings continue on to the Federation's "amazing senior program" that features educational programs, exercise classes and more.
"It's a tremendous asset that the Federation offers something for you at every phase of your life," Marcovitch says.
There's a special emphasis on children and young people in the Jewish community in order to instill a sense of Tzedakah, or , Marcovitch says. That means teaching them to give back to the Jewish community and the community as a whole.
"They're our legacy," Marcovitch says. "They're our future. The Jewish community is closely looking at the next generation of Jews to take over in leadership positions. We're training them to think globally, act locally."
The Federation tries to reach non-Jewish kids in the community, too, with Diversity Days, where 600 kids hear about the Holocaust from a survivor. The program has become so popular that in 2017 the venue is moving from the Toledo Lucas County Public Library to the Valentine Theatre in hopes of reaching even more kids.
Overall, the Federation's goals are to make Jewish life in Toledo the best it can be, and to take care of those in the Jewish community who are in need.
"We have a long-standing commitment in this community to make sure that Jewish life will always be here," he says. "It will always be relevant, it will be cutting-edge, and not only will we be leading the Jewish community here, but we will do our utmost in order to help lead and advise and help with the greater, non-Jewish community of Toledo as well."
The Jewish Federation of Greater Toledo receives calls from other Federations across the country trying to learn how to better help their, often larger, Jewish communities. Marcovitch says he and the Federation staff are always proud to receive such calls.
"Since I've arrived, I've always wanted Toledo to be a shining example of how to do Jewish in a community," he says.
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As Northamptonshire County Council makes preparations to deal with wintry weather over the coming months – the authority is giving people the chance to name 10 of its 26 gritting lorries, with winning entries painted on to the trucks.
The competition runs from today until 29 September and is open to schools, classes or individuals. The winning entries will have the name painted onto the gritter.
In Northamptonshire, a total of 1,198 miles of road receives precautionary gritting – the same distance as between London and Rome. This service is carried out by Northamptonshire Highways.
Cllr Michael Clarke, county council cabinet member for transport, highways and environment, said: "Keeping the county moving over the winter months is a huge task and the positive comments that we have received from our residents show that we are improving our service each year.
"There's never any telling just what sort of winter we're going to get – we just have to be ready to tackle what nature throws at us.
"The gritting team do a tremendous job keeping the county moving during the winter, with 24 hour monitoring between the months of October and April."
Entries should be emailed to email@example.com or tweet @NNhighways and include:
The weather during the period from October to April is monitored by Northamptonshire Highways, and the county's roads are salted, when required, subject to the weather conditions.
Weather forecasts are received from the Meteo Group and local knowledge of the network used to work out what the likelihood of frost or snow is.
If necessary, the situation will be monitored on an hour-by-hour basis before making decisions to send crews out to salt the roads. There are also seven weather stations so that road conditions can be monitored.
Northamptonshire Highways plan the service to ensure villages are linked to priority routes.
A total length of approximately 1,198 miles of road receives precautionary salting, just under half of the total network in the county.
Priority is given to those roads carrying the highest volume of traffic and where the risk of accidents is greatest.
In addition, certain local roads such as those serving industrial areas, shopping centres and some key roads in towns and rural areas are also salted.
In prolonged, adverse weather conditions - that is where the forecast conditions are unlikely to improve for at least 48 hours and the temperature is not expected to rise above freezing within that period - an additional 359 miles of roads will be treated, provided that roads on the precautionary salting network do not require treatment.
In the event of snowfall, roads will be cleared according to their priority status and need.
Gritting updates are available on Twitter at @NNhighways.
Subscribe to Northamptonshire County Council's monthly e-newsletter - your brief of the month's key campaigns, news headlines, upcoming events and important consultations.


text:
Provide a true and fair view
The annual financial statement provides a true and fair view of the financial position, results of operations and cash flows.
The organization prepares its annual financial statement in accordance with Swiss GAAP FER accounting standards and applies Swiss GAAP FER 21 – Accounting for charitable non-profit organizations.
Fundraising and general advertising expenses as well as administrative expenses are calculated and reported using the methods published by Zewo.
The annual financial statement is audited by independent and professionally qualified auditors.
The organization has its annual financial statement audited in accordance with Swiss GAAP FER by independent, professionally qualified auditors:
The auditor produces a written report on the results of the audit. Depending on the type of audit, the report contains:
*According to Swiss GAAP FER 21, organizations are classified as small if they meet two of the following criteria: balance sheet total < CHF 2 million/turnover < CHF 1 million/FTE <10.
Any investments made by the organizsation are sustainable and in accordance with investment regulations.
If financial investments and the value of any investment property exceed CHF 2 million, the organization puts investment regulations in place.
These regulations consider the risk tolerance and liquidity requirements of the organization, the income objectives of its financial investments and the purpose of the organization. The regulations also address social and ecological factors as well as good governance criteria.


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Introduction to Business Statistics
When the consulting solution is aided by a learning tool, we need a powerful vehicle to analyze and derive mathematical models for predictable outcomes in our business processes.
Traditional software solutions available in the market are provided for the customer while the value add that MGBS offers is long term mentoring and support for such software use. MGBS also has its in-house tools and templates that can be downloaded from the net with minor support and mentoring for the same.
This package provides participants with a strong foundation in mathematical and statistical methodology and experience in its applications. The knowledge of statistical computing packages and their application provides a significant advantage over competition in the Human Resource market.
Course Contents:
MGBS is Authorized Distributor:
Statistical and Process Management Software for Six Sigma and Quality Improvement
Excel Add-in Software for Lean Six Sigma, Statistical and Graphical Data Analysis
Download Course Overview


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2017-10-08 11:04:03
Valtteri Bottas had the chance
Mercedes driver Valtteri Bottas in Japan finished fourth, one second away from the podium.Valtteri Bottas "I had a chance I used it, but failed to overtake Riccardo. He was very fast, and I was just one lap for overtaking.Second place in the championship is a good goal, but I would like to fight for the victory in the championship. Yes, this year I am ready to fight for the Vice-championship, I'm focused on the last race, but next year I'm going to fight for a title".
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Mosaic Wine Group has learned that San Antonio artist Gilbert Duran has filed a complaint with the American Civil Liberties Union after the Stone Oak Property Owners Association forced restaurateur Damien Watel to build a wall blocking Duran's Fork sculpture from view.
The POA had claimed that the Fork was an authorized form of advertisement.
Watel and Duran contended that it was a work of art but were forced, nonetheless, on legal grounds to conceal the work from view from the street.
The complaint was reported today on the website of the San Antonio Express News, MySanAntonio.com.