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Patent application title: BODIPY Structure Fluorescence Dye for Neural Stem Cell Probe
Young-Tae Chang (Singapore, SG)
Young-Tae Chang (Singapore, SG)
Seong-Wook Yun (Singapore, SG)
Duanting Zhai (Singapore, SG)
Kit Mun Cheryl Leong (Singapore, SG)
IPC8 Class: AC09B5700FI
USPC Class:
435 71
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving antigen-antibody binding, specific binding protein assay or specific ligand-receptor binding assay
Publication date: 2013-09-19
Patent application number: 20130244251
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The present invention is directed to a fluorescence compound represented
by structural Formula (I), with specificity to neural stem cells: I or a
pharmaceutically acceptable salt thereof. The variables for structural
Formula (I) are defined herein. Also described are methods for detection
of neural stem cells, comprising using a compound of structural Formula
(I) or pharmaceutically acceptable salts thereof. Compounds of structural
Formula (I) can detect and separate neural stem cells without
immunostaining, providing a much shorter and more convenient method for
detection of neural stem cells.
1. A compound represented by structural Formula (I) or pharmaceutically
acceptable salts thereof: ##STR00164## wherein: R is
(C6-C10)aryl, (C5-C10)heteroaryl,
(C6-C10)aryl(C2-C6)alkenyl or 2-4 member polycyclyl,
wherein each 2-4 member polycyclyl optionally and independently contains
1-2 ring heteroatoms selected from oxygen, nitrogen and sulfur; and
wherein R is optionally substituted with 1-4 substituents independently
selected from (C1-C6)alkyl, halo(C1-C6)alkyl,
hydroxy(C0-C6)alkyl, (C6-C10)aryl,
halo(C6-C10)aryl, hydroxy(C6-C10)aryl,
(C1-C6)alkoxy, halo(C1-C6)alkoxy,
halo(C6-C10)aryl(C1-C6)alkoxy, halogen, amino,
nitro, (3 to 9 membered)heterocyclyl, (C0-C6)alkyl(C6-C
10)aryl(C0-C6)alkoxy, (C5-C 10)heterocycle,
--OCF3, --B(OH)2, cyano(C1-C6)alkylene amino,
(C2-C6)alkenyl(C1-C6)alkoxy, (C1-C6)sulfoxy
or --N(CH3)(C1-C6)OH.
2. The compound of claim 1, wherein: R is (C5-C10)heteroaryl, optionally substituted with 1-4 substituents independently selected from (C5-C10)heteroaryl, (C6-C10)aryl, halo(C6-C10)aryl, (C1-C6)alkoxy, (C1-C6)alkyl, halo(C1-C6)alky, hydroxy(C0-C6)alkyl, or (C0-C6)alkoxy(C6-C10)aryl.
3. The composition of claim 1, wherein R is aryl.
4. The compound of claim 3, wherein R is phenyl.
5. The compound of claim 4, wherein R is optionally substituted with 1-4 (C1-C6)alkoxy substituents.
6. The compound of claim 4, wherein the compound is represented by structural Formula (II): ##STR00165##
7. A method for detection of a neural stem cell (NSC), comprising: a) staining said neural stem cell with a compound, forming a dye-stained neural stem cell by binding said compound to a marker protein of said neural stem cell, wherein said compound is of structural Formula (I) or pharmaceutically acceptable salts thereof: ##STR00166## wherein: R is (C6-C10)aryl, (C5-C10)heteroaryl, (C6-C10)aryl(C2-C6)alkenyl or 2-4 member polycyclyl, wherein each 2-4 member polycyclyl optionally and independently contains 1-2 ring heteroatoms selected from oxygen, nitrogen and sulfur; and wherein R is optionally substituted with 1-4 substituents independently selected from (C1-C6)alkyl, halo(C1-C6)alkyl, hydroxy(C0-C6)alkyl, (C6-C10)aryl, halo(C6-C10)aryl, hydroxy(C6-C10)aryl, (C1-C6)alkoxy, halo(C1-C6)alkoxy, (C3-C8)cycloalkyl, halo(C6-C10)aryl(C1-C6)alkoxy, halogen, amino, (C1-C6)alkoxy(C6-C10)aryl(C1-C6)alkoxy, nitro, (3 to 9 membered)heterocyclyl, (C0-C6)alkyl(C6-C 10)aryl(C0-C6)alkoxy, (C5-C 10)heterocycle, --OCF3, --B(OH)2, cyano(C1-C6)alkylene amino, (C1-C6)alkoxyamino, (C6-C10)aryl(C2-C6)alkenyl, (C2-C6)alkenyl(C1-C6)alkoxy, (C1-C6)sulfoxy or --N(CH3)(C1-C6)OH; b) incubating product of step a) to form a said incubated dye-stained stem; c) analyzing said incubated dye-stained stem cell by a flow cytometry and FACS; and d) subjecting said dye-stained neural stem cell to two-dimensional SDS-PAGE (2DE) fluorescence scanning to detect fluorescence signal, wherein the presence of a signal is indicative of the presence of said neural stem cell.
8. The method of claim 7, wherein: R is (C5-C10)heteroaryl optionally substituted with 1-4 substituents independently selected from (C5-C10)heteroaryl, (C6-C10)aryl, halo(C6-C10)aryl, (C1-C6)alkoxy, (C1-C6)alkyl, halo(C1-C6)alky, hydroxy(C0-C6)alkyl, or (C0-C6)alkoxy(C6-C10)aryl.
9. The method of claim 7, wherein R is aryl.
10. The method of claim 9, wherein R is phenyl.
11. The method of claim 10 wherein R is optionally substituted with 1-4 (C1-C6)alkoxy substituents.
12. The method of claim 11, wherein the compound is represented by structural Formula (II): ##STR00167##
13. The method of claim 7, wherein said neural stem cell is a human or a mouse neural stem cell.
14. The method of claim 7, wherein said marker protein is a fatty acid binding protein 7 (FABP7).
[0001] This application claims the benefit of U.S. Provisional Application No. 61/416,808, filed on Nov. 24, 2010. The entire teachings of the above application are incorporated herein by reference.
[0002] Development of fluorescent molecules and their application are indispensable techniques for the analysis of a variety of biological phenomena. During the past few decades, a number of fluorescent small molecules have been developed as reporters and chemosensors for use in biological analyses, which typically are elaborately designed to selectively detect a target substance or conjugated to biomolecules1. These fluorescent molecules employ an increase or decrease in their emission intensity in response to the surrounding medium or through specific molecular recognition events. Due to their simplicity and high sensitivity, fluorescent sensors have been widely utilized as popular tools for chemical, biological and medical applications. The most general strategy for fluorescent sensor design is to combine fluorescence dye molecules with designed receptors for specific analytes, expecting that the recognition event between receptor and analyte will lead to a fluorescence property change of the dye moiety. Although many fluorescent sensors have been successfully developed through this approach, each individual development requires a major effort in both the design and synthesis of the sensors. Also, the sensor's scope of application is limited to the selected specific analytes that the sensor was rationally designed for, so-called Analyte Directed Sensors. Combinatorial dye library synthesis offers one of the most promising alternatives as Diversity Directed Sensors, once an efficient synthetic route can be developed for a diverse set of dyes.
[0003] Neural stem cells (NSC) generate the nervous system, promote neuronal plasticity and repair damage throughout life by self-renewing and differentiating into neurons and glia2,3. Beneficial effects of NSC engraftment into the affected brain areas in several brain diseases have been demonstrated by animal experiments4,5. NSC also has great potential for drug screening and efficacy testing significantly reducing the time and efforts needed in drug discovery. The conventional methods for the isolation and characterization of NSC depend on their behavior in a defined culture medium such as neurosphere formation and immunodetection of marker molecules. These methods, however, are time-dependent and involve the use of antibodies which may render the cells unsuitable for further experimental and therapeutic applications. Therefore, a need exists to develop novel chemical compounds that are useful for detection of neural stem cells.
[0004] A novel chemical structure with fluorescence emission and specificity to neural stem cells is described. This scaffold is compatible with a range of chemical functional groups, and can be bioconjugated to proteins as well as other macromolecules of interest, such as carbohydrates and lipids. One of the compounds, named as compound of designation (CDr3), selectively stains both human and mouse neural stem cells (NSC) by binding to a NSC marker protein fatty acid binding protein 7 (FABP7).
[0005] The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.
[0006] FIG. 1A shows the selective staining of NS5 by CDr3. Nuclei of all E14, NS5, D-NS5 and MEF were visualized by Hoechst 33342 (DAPI); but only NS5 was selectively stained by CDr3 (TxRd). Top panel, bright field (BF) images; middle and bottom panels, fluorescent images obtained with DAPI and Texas Red filter set. Scale bar, 50 μm.
[0007] FIG. 1B shows the chemical structure of CDr3.
[0008] FIG. 1C shows the flow cytometry dot plot images of E14, NS5, D-NS5 and MEF incubated with CDr3. DMSO was added for unstained control cells. The images of each type of cells were overlaid. Segregated NS5 cells by CDr3 are marked.
[0009] FIG. 1D shows selective staining of NS5 by CDr3. Mixed primary mouse brain cells (PC) cultured for 2 weeks in vitro were incubated with CDr3 and Hoechst 33342. The images of living cells are shown in bright field (BF) and fluorescence (DAPI and Texas Red) panels. The primary brain cells with various morphologies were not stained by CDr3, while NS5 treated in parallel was stained. The images of the same cells were acquired after immunocytochemical staining (ICC) with antibodies to neuron-specific class III β-tubulin (Tuj1; TxRd channel) and astrocyte-specific glial fibrillary acidic protein (GFAP; FITC channel). Scale bar, 100 μm.
[0010] FIG. 2A shows the identification of CDr3 binding protein. A protein lysate of CDr3-stained NS5 was separated by 2DE. The major fluorescent spot was marked with a circle (left panel). Many silver-stained protein spots were detected in a duplicate gel (right panel).
[0011] FIG. 2B shows the MS/MS fragment ion analysis of tryptic peptide (MVVTLTFGDIVAVR) SEQ ID No.: 1, indicated FABP7 as a binding target of CDr3. Only the main y-series of ion fragmentation was labeled in the spectrum. M* indicates oxidation at methionine residue.
[0012] FIG. 2C shows the fluorescence signals from EGFP and CDr3 overlap only in the cells expressing either mouse FABP7 or human FABP7 fused to EGFP. The fluorescence images were acquired on a Nikon Ti microscope using DAPI (Hoechst), FITC (EGFP) and Texas Red (Cdr3) filter sets. Scale bar, 50 μm.
[0013] FIG. 3A shows real-time RT-PCR analysis data of FABP7 expression in H1, ReNcell VM and ReNcell VM-differentiated neurons. Relative expression level of FABP7 to GAPDH is depicted.
[0014] FIG. 3B shows that the strong signal of FABP7 protein (14 kDa) was detected by Western blotting in ReNcell VM (lane 3) lysate, while it was not detectable in the lysates of H1 (lane 1) and ReNcell VM-differentiated neurons (lane 2). β-Actin (42 kDa) staining demonstrates consistent loading across sample lanes. Fluorescence scan showed CDr3-labelled FABP7 only in the lysate of ReNcell VM (lane 3) incubated with CDr3.
[0015] FIG. 3C shows the immunocytochemistry of FABP7 in H1, ReNcell VM and ReNcell VM-differentiated neurons (D-ReNcell VM). Only ReNcell VM was brightly stained by FABP7 antibody. Scale bar, 50 μm. Upper panel, nuclei staining with DAPI; lower panel, FABP7 staining with antibody.
[0016] FIG. 3D shows H1, ReNcell VM and ReNcell VM-differentiated neurons incubated with CDr3. Fluorescence signal was detected only in ReNcell VM. Upper panel, bright field image; lower panel, fluorescence image. Scale bar, 50 μm.
[0017] FIG. 4A shows differentiated E14 cell distribution in FACS after staining with CDr3. The CDr3-stained embryoid body cells were separated into CDr3bright and CDr3 dim populations.
[0018] FIG. 4B shows the higher expression of FABP7 in CDr3bright than in CDr3dim cells, which was determined by immunocytochemistry followed by flow cytometry. The primary FABP7 antibody was detected by an Alexa Fluor 488-conjugated secondary antibody.
[0019] FIG. 5 shows the higher expression of FABP7, Hes1, Musashi, Nestin and Pax6 in CDr3bright cells than in CDr3dim cells. The CDr3bright and CDr3dim cells were collected separately by FACS for gene expression analysis by real time RT-PCR and neurosphere assay.
[0020] FIG. 6A shows the flow cytometry dot plot images showing shift of stained
[0021] E14.5 fetal mouse brain cells to bright fluorescence (+) compared to control group (-).
[0022] FIG. 6B shows the numbers (left panel) and the sizes (right panel) of neurospheres for the cells sorted by CDr3, CD133 antibody, SSEA-1 antibody and Aldefluor compared to unsorted cells.
[0023] FIG. 7 shows the numbers (upper panel) and sizes (lower panel) of neurospheres cultured in the presence of CDr3 and DMSO used as a vehicle.
[0024] FIG. 8 shows number of NS5 cells (left panel) and percentage of BrdU-positive cells (right panel) cultured in the presence of CDr3 and DMSO used as a vehicle.
[0025] FIG. 9 shows the spectroscopic information of CDr3.
[0026] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
[0027] The invention pertains to a novel chemical structure with fluorescence emission and specificity to neural stem cells. This scaffold is compatible with a range of chemical functional groups, and can be bioconjugated to proteins as well as other macromolecules of interest, such as carbohydrates and lipids. One of the compounds, named as compound of designation (CDr3), selectively stains both human and mouse neural stem cells (NSC) by binding to a NSC marker protein fatty acid binding protein 7 (FABP7).
[0028] One embodiment of the invention is a compound represented by structural
[0029] Formula (I) or pharmaceutically acceptable salts thereof:
[0030] R is (C6-C10)aryl, (C5-C10)heteroaryl, (C6-C10)aryl(C2-C6)alkenyl or 2-4 member polycyclyl, wherein each 2-4 member polycyclyl optionally and independently contains 1-2 ring heteroatoms selected from oxygen, nitrogen and sulfur;
[0031] and wherein R is optionally substituted with 1-4 substituents independently selected from (C1-C6)alkyl, halo(C1-C6)alkyl, hydroxy(C0-C6)alkyl, (C6-C10)aryl, halo (C6-C10)aryl, hydroxy(C6-C10)aryl, (C1-C6)alkoxy, halo(C1-C6)alkoxy, (C3-C8)cycloalkyl, halo(C6-C10)aryl(C1-C6)alkoxy, halogen, amino, (C1-C6)alkoxy(C6-C10)aryl(C1-C6)alkoxy, nitro, (3 to 9 membered)heterocyclyl, (C0-C6)alkyl(C6-C10)aryl(C0-C6)alkoxy, (C5-C10)heterocycle, --OCF3, --B(OH)2, cyano(C1-C6)alkylene amino, (C1-C6)alkoxyamino, (C6-C10)aryl(C2-C6)alkenyl, (C2-C6)alkenyl(C1-C6)alkoxy, (C1-C6)sulfoxy or --N(CH3)(C1-C6)OH.
[0032] Another embodiment of the invention is a method for detection of a neural stem cell (NSC) comprising:
[0033] a) staining said neural stem cell with a compound, forming a dye-stained neural stem cell by binding said compound to a marker protein of said neural stem cell, wherein said compound is of structural Formula (I) or pharmaceutically acceptable salts thereof:
[0034] b) optionally incubating product of step a) to form a said incubated dye-stained stem (intensity of fluorescence can be increased by incubation for the period of time sufficient for achieving desired intensity, e.g., from about 1 hour to about 24 hours, but less or more time may be acceptable.);
[0035] c) analyzing said incubated dye-stained stem cell by a flow cytometry and FACS; and
[0036] d) subjecting said dye-stained neural stem cell to two-dimensional SDS-PAGE (2DE) fluorescence scanning to detect fluorescence signal, wherein the presence of a signal is indicative of the presence of said neural stem cell.
[0037] In one embodiment of the invention said method is applied in neural stem cell biology and regenerative medicine.
[0038] (a) pyridine, piperidine, 50° C., 48 h, then 80° C., 24 h, (b)H2, Pd/C, MeOH, RT, 6 h, (c) K2CO3, H2O/EtOH, reflux, overnight, (d) 2,2,2-trichloroethanol, pyridine, DCC, EA, RT, overnight, (e) 3,5-dimethyl-1H-pyrrole-2-carbaldehyde, POCl3, DCM, RT, 4 h, (f) DIEA, BF3OEt2, DCM, RT, overnight, (g) R--CHO, pyrrolidine, acetic acid, ACN, 85° C., 15 min.
TABLE-US-00001 CHART 1 Building block acid chlorides employed in the synthesis (R-CHO in Scheme 1). Code Structure 1 ##STR00004## 2 ##STR00005## 3 ##STR00006## 4 ##STR00007## 5 ##STR00008## 6 ##STR00009## 7 ##STR00010## 9 ##STR00011## 14 ##STR00012## 16 ##STR00013## 17 ##STR00014## 18 ##STR00015## 19 ##STR00016## 20 ##STR00017## 22 ##STR00018## 25 ##STR00019## 26 ##STR00020## 30 ##STR00021## 32 ##STR00022## 33 ##STR00023## 34 ##STR00024## 36 ##STR00025## 37 ##STR00026## 38 ##STR00027## 40 ##STR00028## 42 ##STR00029## 43 ##STR00030## 45 ##STR00031## 46 ##STR00032## 48 ##STR00033## 52 ##STR00034## 53 ##STR00035## 54 ##STR00036## 61 ##STR00037## 62 ##STR00038## 63 ##STR00039## 65 ##STR00040## 67 ##STR00041## 68 ##STR00042## 69 ##STR00043## 70 ##STR00044## 73 ##STR00045## 76 ##STR00046## 77 ##STR00047## 82 ##STR00048## 83 ##STR00049## 88 ##STR00050## 89 ##STR00051## 90 ##STR00052## 91 ##STR00053## 94 ##STR00054## 95 ##STR00055## 97 ##STR00056## 98 ##STR00057## 101 ##STR00058## 103 ##STR00059## 105 ##STR00060## 107 ##STR00061## 108 ##STR00062## 110 ##STR00063## 121 ##STR00064## 126 ##STR00065## 132 ##STR00066## 135 ##STR00067## 136 ##STR00068## 137 ##STR00069## 139 ##STR00070## 140 ##STR00071## 144 ##STR00072## 153 ##STR00073## 163 ##STR00074## 164 ##STR00075## 177 ##STR00076## 178 ##STR00077## 186 ##STR00078## 187 ##STR00079## 190 ##STR00080## 195 ##STR00081## 199 ##STR00082## 206 ##STR00083## 209 ##STR00084## 216 ##STR00085## 223 ##STR00086## 228 ##STR00087## 236 ##STR00088## 238 ##STR00089## 241 ##STR00090## 242 ##STR00091## 243 ##STR00092## 245 ##STR00093## 247 ##STR00094## 251 ##STR00095## 257 ##STR00096## 259 ##STR00097## 260 ##STR00098## 263 ##STR00099## 264 ##STR00100## 268 ##STR00101## 274 ##STR00102## 282 ##STR00103## 284 ##STR00104## 290 ##STR00105## 298 ##STR00106## 299 ##STR00107## 300 ##STR00108## 301 ##STR00109## 305 ##STR00110## 307 ##STR00111## 308 ##STR00112## 310 ##STR00113## 311 ##STR00114## 313 ##STR00115## 314 ##STR00116## 316 ##STR00117## 319 ##STR00118## 320 ##STR00119## 322 ##STR00120## 323 ##STR00121## 325 ##STR00122## 329 ##STR00123## 331 ##STR00124## 332 ##STR00125##
[0039] "Alkyl" means a saturated aliphatic branched or straight-chain monovalent hydrocarbon radical having the specified number of carbon atoms. Thus, "(C1-C6) alkyl" means a radical having from 1-6 carbon atoms in a linear or branched arrangement. "(C1-C6)alkyl" includes methyl, ethyl, propyl, butyl, pentyl and hexyl.
[0040] "Alkylene" means a saturated aliphatic straight-chain divalent hydrocarbon radical having the specified number of carbon atoms. Thus, "(C1-C6)alkylene" means a divalent saturated aliphatic radical having from 1-6 carbon atoms in a linear arrangement. "(C1-C6)alkylene" includes methylene, ethylene, propylene, butylene, pentylene and hexylene.
[0041] "Heterocycle" means a saturated or partially unsaturated (4-7 membered) monocyclic heterocyclic ring containing one nitrogen atom and optionally 1 additional heteroatom independently selected from N, O or S. When one heteroatom is S, it can be optionally mono- or di-oxygenated (i.e., --S(O)-- or -S(O)2--). Examples of monocyclic heterocycle include, but not limited to, azetidine, pyrrolidine, piperidine, piperazine, hexahydropyrimidine, tetrahydropyran, tetrahydropyran, morpholine, thiomorpholine, thiomorpholine 1,1-dioxide, tetrahydro-2H-1, 2-thiazine, tetrahydro-2H-1,2-thiazine 1,1-dioxide, isothiazolidine, or isothiazolidine 1,1-dioxide.
[0042] "Cycloalkyl" means saturated aliphatic cyclic hydrocarbon ring. Thus, "C3-C7 cycloalkyl" means (3-7 membered) saturated aliphatic cyclic hydrocarbon ring. C3-C7 cycloalkyl includes, but is not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
[0043] The term "alkoxy" means --O-alkyl; "hydroxyalkyl" means alkyl substituted with hydroxy; "aralkyl" means alkyl substituted with an aryl group; "alkoxyalkyl" mean alkyl substituted with an alkoxy group; "alkylamine" means amine substituted with an alkyl group; "cycloalkylalkyl" means alkyl substituted with cycloalkyl; "dialkylamine" means amine substituted with two alkyl groups; "alkylcarbonyl" means --C(O)-A*, wherein A* is alkyl; "alkoxycarbonyl" means --C(O)--OA*, wherein A* is alkyl; and where alkyl is as defined above and includes methoxy, ethoxy, propoxy, butoxy, pentoxy and hexoxy.
[0044] "Cycloalkoxy" means an cycloalkyl-O-- group wherein the cycloalkyl is as defined above. Exemplary (C3-C7)cycloalkyloxy groups include cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexoxy and cycloheptoxy.
[0045] Haloalkyl and halocycloalkyl include mono, poly, and perhaloalkyl groups where each halogen is independently selected from fluorine, chlorine, and bromine.
[0046] "Hetero" refers to the replacement of at least one carbon atom member in a ring system with at least one heteroatom selected from N, S, and O. A hetero ring system may have 1 or 2 carbon atom members replaced by a heteroatom.
[0047] "Halogen" and "halo" are interchangeably used herein and each refers to fluorine, chlorine, bromine, or iodine.
[0048] "Cyano" means --C≡N.
[0049] "Nitro" means --NO2.
[0050] As used herein, an amino group may be a primary (-NH2), secondary (--NHRx), or tertiary (--NRxRy), wherein Rx and Ry may be any of the optionally substituted alkyls described above.
[0051] The terms "haloalkyl" and "haloalkoxy" mean alkyl or alkoxy, as the case may be, substituted with one or more halogen atoms. The term "halogen" means F, Cl, Br or I. Preferably the halogen in a haloalkyl or haloalkoxy is F.
[0052] The term "acyl group" means --C(O)B*, wherein B* is an optionally substituted alkyl group or aryl group (e.g., optionally substituted phenyl).
[0053] An "alkylene group" is represented by --[CH2]z--, wherein z is a positive integer, preferably from one to eight, more preferably from one to four.
[0054] An "alkenylene group" is an alkylene in which at least a pair of adjacent methylenes are replaced with --CH═CH--.
[0055] The term "(C6-C10)aryl" used alone or as part of a larger moiety as in "arylalkyl", "arylalkoxy", or "aryloxyalkyl", means carbocyclic aromatic rings. The term "carbocyclic aromatic group" may be used interchangeably with the terms "aryl", "aryl ring" "carbocyclic aromatic ring", "aryl group" and "carbocyclic aromatic group". An aryl group typically has 6-14 ring atoms. A "substituted aryl group" is substituted at any one or more substitutable ring atom. The term "C6-14 aryl" as used herein means a monocyclic, bicyclic or tricyclic carbocyclic ring system containing from 6 to 14 carbon atoms and includes phenyl, naphthyl, anthracenyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like.
[0056] The term "heteroaryl", "heteroaromatic", "heteroaryl ring", "heteroaryl group" and "heteroaromatic group", used alone or as part of a larger moiety as in "heteroarylalkyl" or "heteroarylalkoxy", refers to aromatic ring groups having five to fourteen ring atoms selected from carbon and at least one (typically 1-4, more typically 1 or 2) heteroatoms (e.g., oxygen, nitrogen or sulfur). They include monocyclic rings and polycyclic rings in which a monocyclic heteroaromatic ring is fused to one or more other carbocyclic aromatic or heteroaromatic rings. The term "5-14 membered heteroaryl" as used herein means a monocyclic, bicyclic or tricyclic ring system containing one or two aromatic rings and from 5 to 14 atoms of which, unless otherwise specified, one, two, three, four or five are heteroatoms independently selected from N, NH, N(C1-6alkyl), O and S.
[0057] The term "2-4 member polycyclyl is a cyclic compound with 2-4 hydrocarbon loop or ring structures (e.g., benzene rings). The term generally includes all polycyclic aromatic compounds, including the polycyclic aromatic hydrocarbons, the heterocyclic aromatic compounds containing sulfur, nitrogen, oxygen, or another non-carbon atoms, and substituted derivatives of these.
[0058] The term "Alkenyl" means a straight or branched hydrocarbon radical having a specified number of carbon atoms and includes at least one double bond. The (C6-C10)aryl(C2-C6)alkenyl group connects to the remainder of the molecule through the (C2-C6)alkenyl portion of (C6-C10)aryl(C2-C6)alkenyl.
[0059] Another embodiment of the present invention is a pharmaceutical composition comprising one or more pharmaceutically acceptable carrier and/or diluent and a compound disclosed herein or a pharmaceutically acceptable salt thereof.
[0060] "Pharmaceutically acceptable carrier" and "pharmaceutically acceptable diluent" means non-therapeutic components that are of sufficient purity and quality for use in the formulation of a composition of the invention that, when appropriately administered to an animal or human, typically do not produce an adverse reaction, and that are used as a vehicle for a drug substance (i.e. a compound of the present invention).
[0061] Pharmaceutically acceptable salts of the compounds of the present invention are also included. For example, an acid salt of a compound of the present invention containing an amine or other basic group can be obtained by reacting the compound with a suitable organic or inorganic acid, resulting in pharmaceutically acceptable anionic salt forms. Examples of anionic salts include the acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide salts.
[0062] Salts of the compounds of the present invention containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base. Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceutically acceptable cation, which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts made from physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, N,N'-bisdehydroabietylamine, glucamine, N-methylglucamine, collidine, quinine, quinoline, and basic amino acids such as lysine and arginine.
[0063] 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) has superior advantages to be one of the more versatile fluorophores, such as high quantum yield, tunable fluorescence characteristics, high photo stability, and narrow emission bandwidth. Since the first discovery of BODIPY dyes in 1968, chemical modification for BODIPY scaffold has been well explored. Thus great numbers of BODIPY dyes have been used to label biomolecules. A large numbers of sensors and markers based on BODIPY scaffold have also been developed. But BODIPY-based library synthesis has been rarely reported due to the synthetic challenge.
[0064] Here we disclose our invention of a novel diversity oriented fluorescence BODIPY active ester compound library synthesized via solution phase synthetic method and one of the compounds which has been identified as neural stem cell selective imaging probe.
[0065] To develop fluorescent imaging probes which selectively detect NSC, we have screened in-house-generated 3,160 Diversity Oriented Fluorescence Library (DOFL) compounds in E14 mouse embryonic stem cell (mESC), E14-derived NS5 NSC, differentiated NS5 into astrocyte (D-NS5) and mouse embryonic fibroblast (MEF). For high throughput screening, the 4 different types of cells were prepared side by side in 384-well plates and incubated with 0.5 or 1.0 μM of compounds. After 24 hr incubation, the bright field and fluorescence images of the cells were acquired on an automated imaging microscope system ImageXpress Micro® and the fluorescence intensity of the stained cells was analyzed using MetaXpress® image processing software. Through the followed secondary and tertiary screenings CDr3 has been identified as the hit molecule that stains NS5 most selectively and brightly (FIG. 1).
[0066] The intrinsic fluorescence property of our compounds makes it possible to track the target without any modification from the stained living cells through all processes for identification once it binds strongly to the target molecules. When we subjected CDr3-stained NS5 lysate to two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE (2DE)) for a fluorescence scanning, a major spot of approximately 15 kDa was detected (FIG. 2A).
[0067] Matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS) and MS/MS analysis allowed us to identify the protein spot as FABP7 (FIG. 2B). Among the currently known 9 mammalian FABPs that play pivotal roles in transporting and trafficking of lipids in various tissues, FABP7 is particularly expressed in the central nervous system and a well-known marker of radial glial cells which play as NSC in the brain6. To confirm that FABP7 is the specific binding target of CDr3, we cloned both human and mouse FABP7 genes and fused them to EGFP constructs for expression in HEK293 cells. We incubated the cells with CDr3 and observed the signals of EGFP and CDr3 overlap in the cells that express either human or mouse FABP7 fused to EGFP (FIG. 2C).
[0068] With this result, we attempted to test CDr3 on a commercial ReNcell VM human NSC line derived from the ventral mesencephalon region of human foetal brain tissue. We first examined the expression level of FABP7 by real time RT-PCR and found a 540-fold higher level of FABP7 mRNA in ReNcell VM than in H1 human ESC. This expression was dramatically down-regulated (20-fold) by differentiation into neurons (FIG. 3A). Western blot analysis demonstrated a similar observation in protein expression levels with a strong FABP7 band at 14 kDa detected in ReNcell VM lysate while no FABP7 was detected in the lysates of H1 and ReNcell VM-derived neurons (FIG. 3B). In accordance with the Western blot data, ReNcell VM were strongly stained by FABP7 antibody while H1 and ReNcell VM-derived neurons were not stained (FIG. 3C). We then incubated the 3 types of cells with CDr3 to determine whether living ReNcell VM could be distinguished by the compound among others. As expected from the FABP7 expression analysis data, CDr3 selectively stained ReNcell VM in living cell cultures (FIG. 3D).
[0069] Next, we were interested to test whether CDr3 can be used for the isolation of NSC from the heterogeneous populations of cells. According to the protocol described by Bibel et al.7, we induced differentiation of E14 to generate FABP7-positive radial glial cells. The embryoid bodies generated from E14 in the absence of leukemia inhibitory factor were treated with retinoic acid for 2 days until dissociation into single cell suspension. The cells were stained with CDr3 and the CDr3bright and CDr3dim cells were collected separately by FACS. Each cell population was stained with FABP7 antibody and analyzed by flow cytometry. The overlay plot showed well-separated 2 clusters demonstrating that the cells isolated by CDr3 from the heterogeneous embryoid body cells were FABP7 expressing cells (FIG. 4). For the NSC isolation from the brain tissue, we incubated the E14.5 fetal mouse brain primary cells with CDr3, and the CDr3bright and CDr3dim cells were collected separately by FACS for gene expression analysis by real time RT-PCR and neurosphere assay. The expression levels of FABP7, Hes1, Musashi, Nestin and Pax6 in CDr3bright cells were more than 3-fold higher than in CDr3dim cells, while the differences of Hes5, Sox2 and Bmi1 expression levels were less than 2-fold (FIG. 5). In a neurosphere assay the CDr3bright cells grew to generate 26.8±2.5 neurospheres while CDr3dim cells generated only 0.25±0.5 neurospheres per well. As a separate study, we sorted the fetal mouse brain cells using CD133 antibody, SSEA-1 antibody, Aldefluor (BODIPY-aminoacetaldehyde) as well as CDr3 by FACS for neurosphere assay (FIG. 6A). We observed a big difference between all marker positive and negative cells in the numbers of generated neurospheres. Among the markers, largest number of neurospheres was generated in CDr3 positive cells followed by SSEA-1, Aldefluor and CD133 positive cells (FIG. 6B), while the sizes of neurospheres were all similar (FIG. 6C). This result shows highest correlation between neurosphere forming ability of the NSC and FABP7 level among the tested NSC markers.
[0070] Finally, we determined whether CDr3 affects NSC proliferation by culturing NS5 and neurosphere in the presence of CDr3. Total numbers of NS5 cells grown for 6 hr and 48 hr and the percentage of BrdU positive cells pulse-labeled in the CDr3-containing medium were not different from those of cells grown in DMSO only-containing medium which was used as a control (FIG. 7). In accordance with the result of experiment with NS5 cells, the number and size of neurospheres generated in the presence of CDr3 were not different from control (FIG. 8).
Materials and Methods
Cell Culture and Differentiation
[0071] E14 was maintained on gelatin-coated dishes in high-glucose DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.1 mM non-essential amino acids, 0.1% β-mercaptoethanol and 100 U/ml leukemia inhibitory factor (LIF, Chemicon). For differentiation, the cells were detached from their culture plates using 0.25% trypsin with 1mM EDTA solution (Invitrogen) and sub-cultured in non-adherent bacteria culture dishes in the E14 media but without LIF. Subsequently, 90% of the EBF media was changed on a daily basis for a total of 4 days and then retinoic acid (Sigma) was added to the final concentration of 1 μM. On day 6, the embryoid bodies were harvested and dissociated in 0.05% trypsin with 0.2 mM EDTA solution for 3 min at 37° C. to obtain a single cell suspension. NS5 was maintained in Euromed-N medium supplemented with 100 μg/ml Apo-transferin (Sigma), 5.2 ng/ml Sodium Selenite (Sigma), 19.8 ng/ml progesterone (Sigma), 16 μg/ml Putrescine (Sigma), 25 μg/ml insulin (Sigma), 50.25 μg/ml BSA (Gibco), 10 ng/ml bFGF (Gibco), 10 ng/ml EGF (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco) and 2 mM L-glutamine (Gibco). For differentiation of NS5 into astrocyte, the medium was changed to NS5 maintenance medium containing 5% FBS but without FGF and bEGF. MEF was maintained in the same media as used for E14 but without LIF. H1 was maintained in a feeder-free condition on matrigel-coated dishes in MEF-conditioned medium containing Knockout DMEM/10% serum replacement (Gibco), 0.1 mM MEM non-essential amino acids (Gibco), 1 mM L-glutamine (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 8% plasmanate (NUH pharmacy), 12 ng/ml LIF, and 10 ng/ml human recombinant Basic Fibroblast Growth Factor (bFGF; Gibco). ReNcell VM (Millipore #SCC008) was maintained on laminin-coated dishes in ReNcell NSC Maintenance Medium (Millipore #SCM005) containing 20 ng/ml bFGF and 20 ng/m EGF. For neural differentiation, ReNcell VM were seeded on PLO/Laminin-coated plates and cultured for up to 3 weeks in media comprising a 1:1 mix of N2-DMEM/F12 and B27-Neurobasal media supplemented with 0.1 mM MEM non-essential amino acids and 1 mM L-glutamine, all obtained from Gibco/Invitrogen. For mixed primary brain cell culture, the brains of neonatal mouse pups were cut into small pieces and digested in 0.25% trypsin with 1mM EDTA solution (Invitrogen) for 30 min at 37° C. before neutralization with FBS. After washing with PBS by centrifugation and resuspension, the tissues were triturated using a 10 ml pipette fitted with 1 ml tip and the suspension was filtered through a strainer with 40 μm nylon mesh. The obtained single cells were plated on 35 mm cell culture dishes in OptiMEM-GlutaMAX® containing 10% FBS. Unattached cells and cell debris were removed the next day by replacing medium. One-half of the medium was replaced twice a week thereafter.
DOFL High Throughput Screening
[0072] DOFL compounds were diluted from 1 mM DMSO stock solutions with the culture medium to make final concentration of 0.5 μM or 1.0 μM. The 4 different types of cells plated side by side on 384-well plates were incubated with the compounds overnight at 37° C. and the nuclei were stained with either Hoechst33342 or DRAQ5 the next day before image acquisition. The fluorescence cell images of 2 regions per well were acquired using ImageXpress Micro® cellular imaging system (Molecular Device) with 10× objective lens and the intensity was analyzed by MetaXpress® image processing software (Molecular Device). The hit compounds which stained NS5 more brightly than other cells were selected based on the intensity data and manual screening of the raw images.
Live Cell Staining
[0073] The cells were incubated with 0.5 μM of CDr3 in Opti-MEM GlutaMAX® for 1 hr and, if necessary, subsequently with 2μM of Hoechst 33342 for 15 min at 37° C. Then the cells were rinsed in the maintenance medium for 1 hr and the medium was replaced again with fresh one before image acquisition. The staining and destaining times were prolonged when necessary. The bright field and fluorescence images were acquired on ECLIPSE Ti microscope (Nikon Instruments Inc) or A1R confocal microscope (Nikon Instruments Inc) using NIS Elements 3.10 software or on Axio Observer D1 using AxioVision v 4.8 software (Carl Zeiss Inc).
Flow Cytometry and FACS
[0074] The cells incubated with CDr3 were harvested by trypsin treatment, washed and resuspended in PBS. The fluorescence intensity of the cells was measured on a flow cytometry (BD® LSR II) or collected using a FACS machine (BD FACS Aria®). The data were analyzed and processed using FlowJo 7 software.
Two-Dimensional Gel Electrophoresis
[0075] CDr3-stained NS5 pellet was lysed in a lysis buffer (40 mM Trizma, 7M Urea, 2M thiourea and 4% CHAPS) premixed with 10 μl/ml Protease Inhibitor Cocktail (EDTA free, GE healthcare), 50 μg/ml DNase I and 50 μg/ml RNase A (Roche). The proteins of 0.2 mg and 1 mg were separated by 2DE for silver staining and fluorescent imaging, respectively. The fluorescence image of gels was acquired using a Typhoon 9400 scanner (GE healthcares) at excitation/emission wavelengths of 532 nm/610 nm with PMT at 500 v and a duplicate gel was stained using PlusOne® Silver Staining Kit (GE healthcare) according to the manufacturer's protocol. The fluorescently 18abeled protein spots were directly excised from the gel for in-gel trypsin digestion and peptide extraction.
MALDI-TOF/TOF MS and MS/MS Analyses
[0076] Tryptic peptide of 0.6 μl was spotted onto Prespotted AnchorChip target plate (Bruker Daltonics Inc) according to manufacturer's protocol. The peptide mass fingerprint and selected peptide MS/MS fragment ion analysis were carried out on UltraFlex III TOF-TOF (Bruker Daltonics Inc) with the compass 1.2 software package including FlexControl 3.0 and FlexAnalysis 3.0 with PAC peptide calibration standards. The peak lists of MS and MS/MS were submitted to in-house Mascot server (phenyx.bii.a-star.edu.sg/search_form_select.html) through BioTools 3.2 with the database of SwissProt 57.8 (509,019 sequences) allowing peptide mass tolerance of 100 ppm and 0.5 Da with maximum 1 missed cleavage and considering variable modifications of carbamidomethyl at cysteine (C) and oxidation at methionine (M).
Chemical Synthesis
[0077] All reactions were performed in oven-dried glassware under a positive pressure of nitrogen. Unless otherwise noted, starting materials and solvents were purchased from Aldrich and Acros organics and used without further purification. Analytical TLC was carried out on Merck 60 F254 silica gel plate (0.25 mm layer thickness) and visualization was done with UV light. Column chromatography was performed on Merck 60 silica gel (230-400 mesh). NMR spectra were recorded on a Bruker Avance 300 NMR spectrometer. Chemical shifts are reported as δ in units of parts per million (ppm) and coupling constants are reported as a J value in Hertz (Hz). Mass of all the compounds was determined by LC-MS of Agilent Technologies with an electrospray ionization source. All fluorescence assays were performed with a Gemini XS fluorescence plate reader.
[0078] Synthesis of compound 1: The intermediate 1 in the Scheme 1 was synthesized as reported previously8.
[0079] Synthesis of CDr3: 1, (20 mg, 0.047 mM) and 3,4-dimethoxybenzaldehyde (16 mg, 0.094 mM) were dissolved in acetonitrile (4 ml), followed by the addition of the mixture of pyrrolidine (23.6 μl, 0.282 mM) and acetic acid (16.1 μl, 0.282 mM). The reaction was heated at 85° C. for 15 min and then cooled down to r.t. The resulting crude mixture was concentrated under vacuum and purified by normal-phase column chromatography (eluting system: hexane/ethyl acetate (6:1) to render CDr3 as purple solid (15 mg, 56% yield).
Characteristics of CDr3
1H and 13C NMR Spectra on CDr3
[0080] 1H NMR (300 MHz, CDCl3): 2.28 (s, 3H), 2.96 (t, J=7.5 Hz, 2H), 3.40 (t, J=7.5 Hz, 2H), 3.92 (s, 3H), 3.97 (s, 3H), 4.78 (s, 2H), 6.30 (d, J=3.9 Hz, 1H), 6.71 (s, 1H), 6.85 (d, J=3.9 Hz, 1H), 6.86 (d, J=8.1 Hz, 1H), 7.03 (s, 1H), 7.12 (d, J=1.8 Hz, 1H), 7.16 (dd, J=1.8, 8.4 Hz, 1H), 7.29 (d, J=16.2 Hz, 1H), 7.48 (d, J=16.2 Hz, 1H).
[0081] 13C NMR (75.5 MHz, CDCl3): 11.3, 23.7, 29.6, 33.0, 55.9, 56.0, 56.1, 74.0, 94.9, 109.6, 110.4, 111.1, 116.2, 116.6, 121.6, 122.1, 122.2, 126.7, 129.2, 133.6, 139.1, 143.0, 149.3, 150.8, 171.0.
[0082] ESI-MS m/z(C25H24BCl3F2N2O4) calculated: 571.1 (M+H)+, found: 551.1 (M-F).
Fluorescence Property Measurement
[0083] 10 μM solutions in DMSO was prepared and measured absorption and 2 μM solutions in DMSO was prepared and measured absorption emission of library compound. Spectrum of CDr3 is shown in FIG. 9.
Neural Stem Cell Isolation from Fetal Mouse Brain Cell Suspension
[0084] E14.5 fetal mouse brains were trypsinized in 0.25% trypsin with 1 mM EDTA solution (Invitrogen) for 30 mins at 37° C. before neutralization with FBS. The tissues were triturated sequentially with a 10 ml pipette followed by a 1 ml blue tip and a 0.2 ml yellow tip attached to the 10 ml pipette until the cell suspension flows through smoothly. The tissue suspension was washed 3 times with PBS by repeated resuspension and centrifugation and filtered through a 40 μm strainer. The cells were stained using CDr3, CD133 antibody, SSEA-1 antibody or Aldefluor as described below and FACS sorted. The FACS sorted bright and dim cells of 2% at each end and unsorted (randomly harvested) cells were plated in a DMEM/F12 medium containing 10 ng/ml bFGF, 20 ng/ml EGF and B27 without vitamin A (Invitrogen) at a density of 10,000 cells/well in 6-well plate to grow forming spheres. The number and size of neurospheres generated from each group were measured after 6 days culture.
[0085] Dissociated cells were incubated with 0.5 μM of CDr3 in neurosphere media for 1 hr and then spun down for resuspension in compound free neuropshere media for 30 minutes. The cells were then spun down and resuspended in PBS for FACS. For control group, the cells were incubated with 0.05% DMSO instead of CDr3.
CD133 Antibody
[0086] Dissociated cells were blocked in 1% BSA for 30 min and then incubated with CD133 antibody (1:500) for 1 hr. The cells were washed with PBS by centrifugation and resuspension and then incubated with Alexa fluor 488-conjugated anti-rat IgG (1:1,000) for 1 hr. The stained cell sample was washed again before resuspension in PBS for FACS. For control group, primary antibody was omitted.
SSEA-1 Antibody
[0087] Dissociated cells were blocked in 1% BSA for 30 min and then incubated with SSEA-1 antibody (1:500) for 1 hr. The cells were washed with PBS by centrifugation and resuspension and then incubated with Alexa fluor 633-conjugated anti-mouse IgM (Invitrogen) for 1 hr. The stained cell sample was washed again before resuspension in PBS for FACS. For control group, primary antibody was omitted.
[0088] The cells were incubated with activated Aldefluor substrate (5 μl/ml) for 30 min at 37° C. The cells were then spun down and resuspended in Aldefluore assay buffer for FACS. For control, diethylaminobenzaldehyde, a specific inhibitor of ALDH was added (5 μl/ml) to the cells together with Aldefluore substrate.
Serial Neurosphere Assay
[0089] Neurospheres were generated from the fetal mouse brain cells prepared as described in above (Neural stem cell isolation). After expansion by 2 times of passages, the cells plated in triplicate in 6-well culture plates at a density of 3,000 cells per well and cultured in the presence of 1 μM CDr3 or 0.1% DMSO for 6 days. After 6 days, the numbers and sizes of neurospheres were determined. For serial assay, the neurospheres were further passaged in the medium containing 1 μM CDr3 or 0.1% DMSO.
NS5 Cell Proliferation Assay
[0090] NS5 were seeded into 96-well plates (Greiner) at a density of 1000 cells/well. The next day, DMSO and 1 mM DMSO stock of CDr3 was added into 32 wells for each to be diluted to 0.1% and 1 uM, respectively. At 6 hr and 48 hr time points, 1 ug/ml of Hoechst 33342 was added and incubated for 15 min for image acquisition using an ImageXpress Micro® and MetaXpress Imaging system (Molecular Devices). Hoeschst33342 and CDr3 signals were detected via DAPI and Texas red filters, respectively, and the images of a total of 4 areas were captured per well. Multi wavelength scoring analysis was then run to quantify the number of cells based on Hoechst 33342-stained nuclei image. For the quantification of pulse-labeled cells with BrdU, the cells were stained using FITC conjugated anti-BrdU antibody (BD Pharmingen®) according to the manufacturer's instruction. Total numbers of Hoechst 33342-stained and BrdU-labeled nuclei were counted by image based analysis using ImageJ-ITCN software.
[0091] 1. (a) Geddes, C. D.; Lakowicz, J. R. Topics in Fluorescence Spectroscopy, Vol. 9; Springer: New York, 2005. (b) Geddes, C. D.; Lakowicz, J. R. Topics in Fluorescence Spectroscopy, Vol. 10; Springer: New York, 2005.
[0092] 2. Okano, H. & Sawamoto, K. Neural stem cells: involvement in adult neurogenesis and CNS repair. Philos Trans R Soc Lond B Biol Sci 363, 2111-22 (2008).
[0093] 3. Falk, S. & Sommer, L. Stage- and area-specific control of stem cells in the developing nervous system. Curr Opin Genet Dev 19, 454-60 (2009).
[0094] 4. Shimazaki, T. Biology and clinical application of neural stem cells. Horm Res 60 Suppl 3, 1-9 (2003).
[0095] 5. Daadi, M. M., et al. Adherent self-renewable human embryonic stem cell-derived neural stem cell line: functional engraftment in experimental stroke model. PLoS One 3, e1644 (2008).
[0096] 6. Malatesta, P., et al. Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage. Development 127, 5253-63 (2000).
[0097] 7. Bibel, M. et al. Differentiation of mouse embryonic stem cells into a defined neuronal lineage. Nat Neurosci 7, 1003-9 (2004).
[0098] 8. Malan, S. F. et al. Fluorescent ligands for the histamine H2 receptor: synthesis and preliminary characterization. Bioorg Med Chem 12, 6495-503 (2004).
[0099] While this invention has been particularly shown and described with references to example embodiments thereof, it Will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
Sequence CWU
1114PRTMouse 1Met Val Val Thr Leu Thr Phe Gly Asp Ile Val Ala Val Arg1 5 10
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text:
When a person gets charged with a crime, they are taken under police custody so that they attend the court proceedings which are supposed to prove if the accused is guilty of the crime or innocent. However, there is the option of the defendant being released under the condition that they will attend all the court dates as set by the court itself. This release can be made sure of by paying a certain amount of money to the authorities that is decided by the court. This is called bail. A person can pay the bail money in cash, assets or in the form of a bond signed with the authorities.
The amount of the bail depends upon the degree of crime that has been committed and is decided by the court. This sum can even extend to large amounts which are impossible for the common folks to pay at most times. In this situation, bail bond companies are brought into use. The primary function of the bail bond companies is for them to sign a surety vouching for the availability of the accused during the days and times that the court has set to go about the court proceedings to determine the guilt or the innocence of the criminal defendant. Once the bond has been signed, the criminal defendant basically becomes the responsibility of the bail bond agency seeing that it is them that are going to be accountable in case the defendant does not show up for the trial. In that case, these bail bond companies have to pay the entire sum of the bail while the accused is absconding.
Since bailing someone out is a matter of quite a sum of money, it becomes a huge blow to the financial status of the bail bond companies. A company with so much to lose is always expected to walk into any situation with a backup plan. In this case, the backup plan is bounty hunters.
America is one of the few countries that still have the concept of county hunters still going. The man job of bounty hunters is quite similar to what has always been showcased to the public while showing incidents of the Wild West with people on posters wanted in exchange of a certain amount of money (usually a large sum, depending on how wanted the criminal was). However, the current world has a more sophisticated version of it where these bounty hunters are actually trained in order to track down people and bring them to the court or to the bail bond companies.
The bail bond companies charge a non refundable fee in exchange for their services which is almost always a certain small percentage of the bail money. Once a bounty hunter is employed, they are also paid a certain sum from the money that is acquired from the defendant that has been caught to be brought to justice by the court of law for the crime they committed.

subset: commoncrawl


text:
Microsoft: No Nintendo, Xbox is More Popular
Seemingly in disagreement with Nintendo's claims of one week ago, Microsoft issued a release late Tuesday citing data from The NPD Group that Xbox, not GameCube, had the best-selling console launch ever. Moreover, Microsoft states that "Halo" for Xbox is outselling "Luigi's Mansion," which Nintendo had crowned the most popular launch title to date. According to NPD, gamers are purchasing 2.4 games per Xbox sold, as opposed to 1.9 with each GameCube.
"Xbox sold out as soon as we launched, and we're selling systems as fast as we can produce them. More than 100,000 units a week are being delivered to retailers," said Microsoft's Chief Xbox Officer Robbie Bach. Similarly, Nintendo has planned for weekly shipments of 125,000 additional GameCubes through the holidays. Both companies expect to ship close to 1.5 million units by the end of the year.
But at this point it may be impossible to deem a clear victor. The reports rely heavily on market data polled from select retailers, not hard numbers. Whatever the final tally may be however, it is clear both Microsoft and Nintendo pulled off record-breaking sales despite a dismal economic forecast. And perhaps most importantly, gamers cannot get enough of either console.


text:
Is LinkedIn Premium Worth the Investment for Your Business?
More than two hundred million professionals from around the world enjoy the services of social networking website LinkedIn. Of these, 39 percent pay for the site's premium services. But should you be one of them?
Target Your Searches
Free members can search LinkedIn for basic criteria, but things get really interesting for premium members. Business and Business Plus members can search by company size, seniority level, interests, and Fortune 1000 status. The latter probably won't concern small and medium business owners, but the first three will ensure you're speaking to executives rather than interns at companies in your professional sphere.
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Small and medium business owners probably can't justify the expense of the Executive or even the Business Plus levels, but LinkedIn's Business membership offers just enough incentive to upgrade.
If you do decide to upgrade, make sure that your profile is as professional as it can be. Run through our Ultimate Guide to the Perfect LinkedIn Profile.
If you're looking for additional help with LinkedIn, such as consulting or profile building, please contact us.
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By LL Wrighter
LL Wrighter, a graduate of Journalism and Communications at the University of Florida, is a tech and social media writer who also enjoys to dabble in DIY and budget stories.
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text:
I'm guessing you have an awesome life – one that tons of people are jealous of, even. But it kinda feels like you're falling apart inside. You don't feel like yourself, physically or mentally.
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I'll email you full notes after each session that include key points from our discussion, specific homework assignments, and personalized nutritional recommendations.
You'll finish the program with a totally different perspective on food and life, a lighter physical body, clear skin, great digestion, and a calm-cool mindset.
Read what other clients have said about working with me here, and click the pink box below to schedule a no-pressure Breakthrough Session with me.


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Labour-In-Vain Rd, Stansted TN15 7NY, UK – 01732 822696
Wedding reception.. absolutely amazing, staff so friendly and fun. (Even the waitress was dancing with my daughter). Food was excellent, wine exquisite and the bedroom was so comfortable. Would really recommend.
Been 4 times now and will book again as it's a great place. Food is excellent and portion size superb! Service exceptional and ambience excellent Starts with the first class flowers on entry and continues throughout you food. Rare find.
31 High St, Borough Green, Sevenoaks TN15 8BT, UK – 01732 882650
Not the biggest of restaurants but they are extremely well organised, very attentive and the service was excellent. There's an extensive menu which makes choosing quite different! Both our starters and main courses were superb and not expensive. Recommend it to anyone whether you're local or visitors like us!
Nice atmosphere, good value
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Regional Features
Special 2008 World Junior Championships
Check back to this page! It will be updated!
7/13/08 USA Sweeps 4x400 Relays; Nunley Grabs Silver in 110 Hurdles
The Athlete/Coach Relationship
The Athlete's Viewpoint. By Chris Lukezic. Chris Lukezic, Georgetown '06, has run 3:33.28 for 1,500 meters and 3:54.46 for the mile. My mother wasn't exactly thrilled that day in October 2000 when she discovered that I had carved numbers into the drywall next to my bed. It wasn't just a bunch of numbers, though.
Big and Buff---but Feminine!
By Jessica Hillam (Idaho State, 2007) / Summer 2007
Jessica Hillam (Idaho State, '07) has been a thrower (shot,discus and hammer) since her high school days in Idaho Falls. Here's her personal take on some of the problems girl throwers encounter, and how she has succcessfully dealt with them.
Matt Tegenkamp: Before and After that Pre Two-Mile
Summer 2007 / Courtesy of kimbia.net
Matt Tegenkamp broke the American Record for 2-miles, running 8:07.07 at the Prefontaine Classic. Here he recalls the three spring races that led up to the Pre two- mile, and then discusses that American Record race as he experienced it. (courtesy of Kimbia.com, used by per- mission).
BEFORE: June 4, 2007
Jenn Stuczynski and Her Coach Talk Vaulting
Summer 2007
The pole vault is still a recent event in women's track and field. Stacy Dragila was the first to better 15 feet, in 1999, and she went on to win the first Olympic gold medal in the event in 2000. She raised the American record to 15-10 in 2004.
Copyright © 2017 American Track & Field Athlete a Fortius Media Group, LLC Publication


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Thomas Lahusen
Professor of History
Affiliated Faculty, Centre for European, Russian, and Eurasian Studies
Room 111N, 1 Devonshire Place
Thomas Lahusen (PhD, Lausanne, Switzerland) is Professor at the Department of History and Centre of Comparative Literature. His research interests focus on the multi-faceted experiences of "real socialism" and other forms of "totalitarian" culture during the twentieth century. His publications include How Life Writes the Book: Real Socialism and Socialist Realism in Stalin's Russia (1997) and the following co-edited collections: Late Soviet Culture: From Perestroika to Novostroika (1993), Intimacy and Terror: Soviet Diaries of the 1930s (1995), Socialist Realism without Shores (1997), What Is Soviet Now? Identities, Legacies, Memories (2008), and a number of special journal issues, including "Aube Rouge: Les Années Trente en Extrême-Orient soviétique" (Revue d'etudes slaves, 1999), "Harbin and Manchuria: Place, Space, and Identity" (South Atlantic Quarterly, 2001), "Harbin: Histoire, Memoire et Difference" (Revue d'etudes slaves, 2001). He has also co-directed several documentary films, including The Province of Lost Film (2006-08); Komsomolsk mon amour (2007); The Photographer (2008); The Interim Country (2010-11), In Search of Roubakine (2012), all produced by Chemodan Films (www.chemodanfilms.com). He is presently working on a monograph devoted to the history of Soviet film exhibition and on two documentary films. The first is devoted to the state-wide effort in the People's Republic of China to use digital media for education and entertainment in geographical areas and social settings where positive state presence has declined; the second deals with the "memory industry" relative to the remembrance of former foreign residents of North-East China, including Japanese, Russians, Poles, and the Jewish diaspora.
Research interests
Twentieth Century History (political, social and cultural). History of North East China
Awards and distinctions
Distinguished Professor Eurasian Cultural History, 2013-2018
Distinguished Professor Eurasian Cultural History, 2009-2013
Canada Research Chair in History, Arts and Culture (Tier-1), 2002–2008
Selected Publications
Socialist Realism without Shores (Duke UP, 1997)
How Life Writes the Book: Real Socialism and Socialist Realism in Stalin's Russia (Cornell UP, 1997)
"Harbin and Manchuria: Place, Space, and Identity." Special issue of TheSouth Atlantic Quarterly, 2001)
What Is Soviet Now? Identities, Legacies, Memories (LIT Verlag, 2008)
Soviet Cultural History
Problems of Real Socialism
Memory, Trauma, and History
Displacements: A Journey from St. Petersburg to Los Angeles


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Daisy's Absolut Collaboration
ADDING yet another string to her illustration bow, this month, Daisy de Villeneuve is collaborating with Absolut Vodka on its latest project - the House of Masquerade, a temporary fashion space set to open on Redchurch high street in Shoreditch on October 23. A vintage clothing boutique and cocktail bar in one, for five nights only, the space will play host to a fashion wonderland, offering shoppers the chance to come in and play dress-up - there'll even be a dedicated stylist on hand to help - while chilling out in the elegant surround. And it doesn't stop there - de Villeneuve will also be lending her creative hand to a limited edition run of journals which, featuring one of her distinctive designs, will be sold exclusively through the House of Masquerade - the proceeds from which will be donated to the Terrence Higgins Trust. "I am so pleased to support Terrence Higgins, one of my favourite charities," says de Villeneuve and adding: "I'm a big fan of vintage."
House of Masquerade will be open to the public from October 24 until October 27, from 5-11pm.


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Mental health support service launched for children
21st April 2016
Figures suggest at least three children in every UK classroom are suffering from a mental health issue.
Read All ►
School should start later say experts
10th September 2015
UK scientists say starting school at 10am could have huge benefits for teenagers.
Read All ►
Head teachers worried about pupils' mental health
20th July 2015
A survey suggests head teachers worry more about pupils' mental health than any other issue.
Read All ►
School absence guidance should be withdrawn
28th November 2014
Calls to withdraw school absence guidance has been backed by doctors.
Read All ►
Emergency asthma inhalers for schools
22nd October 2013
The government has announced schools should keep a spare inhaler and spacer for emergencies.
Read All ►
iPhone converted into microscope
14th March 2013
iPhone microscope finds worm infections in schoolchildren in Tanzania.
Read All ►
Schoolgirl left with burns after using sunbed
30th January 2013
15-year-old schoolgirl suffers severe burns after being allowed to use a sunbed at a gym.
Read All ►
Relationship education should be compulsory
20th December 2012
MPs call for compulsory relationship classes in schools.
Read All ►
Two cups of milk a day best for children
18th December 2012
Too much milk can affect the body's ability to store iron, Canadian researchers find.
Read All ►
Body image lessons for children
30th May 2012
MPs have recommended all school children should have body image lessons.
Read All ►


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Local News
Longtime Wayside Theatre actress visits old friends
Thomasin Savaiano, coaching drama students at the Independent School of Winchester this summer, took a breather to visit with old friends from Wayside Theatre days in Front Royal.
Included was a past chairman of the historic Middletown theater's board of directors, Jim Wink, who is owner of the Horseshoe Curve Restaurant in Bluemont, and other past members of theatre boards. The Wayside, then the second oldest theater in the Commonwealth, closed its doors after a 51-year run a few years ago. Savaiano and her husband, theater director Warner Crocker, relocated to Chicago after more than a decade in the Virginia location.
In those days Craig Laird of Royal Oak Computers on Main Street was the valued go-to communications technician for the theater and last weekend hosted a "welcome home" gathering for Thomasin and another from the theater company, Sarah Blackwell of Richmond. The party was held in his "Vine & Leaf" wine and tea bar at 477 South Street in Front Royal.
Thomasin returns annually from her Chicago home to the Winchester school situated at 769 Roundhill Road to coach students for plays, several of which will be staged Friday, August 9, curtain time 1 p.m. on the Independent School stage. Since last summer the school relocated to the Roundhill Road address which was the site of an old firehouse. Savaiano pronounced the new location "amazing" as she prepared the children for their stage appearances, and friends wished her well in the new surroundings.
Claire McDonald, head of the school, has said that she is "delighted that (Thomasin) happily spends her summer vacations in Winchester, introducing young people to the magic of live theater."
Local News
Gainesville: University Boulevard Lane Closure the Week of August 26th
GAINESVILLE – The right lane of southbound University Boulevard is scheduled to be closed between Route 29 and I-66 from 9 a.m. Monday, August 26, to noon on Friday, August 30. The closure is needed so that construction crews can install curbs and gutters and pave the entrance to the new University Boulevard (Gainesville) Park and Ride Lot.
The first portion of the lot will open this October with more than 960 parking spaces available for carpooling, vanpools, and commuter bus service during construction of the Transform 66 Project. When fully completed in late 2022, the facility will provide over 2,000 park and ride spaces with access to and from the I-66 Express Lanes from University Boulevard.
All work is weather dependent and will be rescheduled if inclement conditions occur.
Learn more about the I-66 Outside the Beltway Project and sign up for project updates and lane closure alerts at Transform66.org.
Local News
FRIBA promotes Town with 'Cardinal' ornaments, annual Taste of the Town
There's a fun new project in town featuring the beautiful Virginia state bird: the cardinal.
The Front Royal Independent Business Alliance (FRIBA) members with the assistance of American Business Alliance are partnering to help stimulate local business growth in town, and they are doing so with cute, kiln fired clay cardinals. The goal of their partnership is to continue to create unity within the community by celebrating the anniversary of the "Virginia is for Lovers" campaign. With the 50th anniversary of the campaign coming up, FRIBA wants to celebrate by creating clay Christmas ornaments in the shape of cardinals and invite the community to paint them for an $8 donation. The cardinals will be used to help fund community love letters to bring the community together and spread the love.
Arline Link with Explore Art and Clay at the Kiln Doctor and George and Karen McIntyre from The Apple House introduced us to the project.
Link also wants to spread the word about the Taste of the Town event. A family focused, non-alcoholic community event hosted on September 27 from 6-9 P.M. on East Main Street, Taste of the Town promises to bring the best culinary aspects of Downtown Front Royal to light. East Main Street will be closed off for the event and the night will promise hours of family friendly fun.
In addition, on August 22 at the Open House Middle of Main there will be another community event showing a series of films featuring the history behind the Save Your Town project.
To learn more about Cookie Cutter Cardinals you can contact Arline Link at the Explore Art and Clay at the Kiln Doctor, (540) 636-6016.
Watch Arline Link and George McIntyre walked us through the process of how the cardinals are made in this video:
Local News
Invitation to bid: WCPS A.S. Rhodes Elementary School Renovation
You are invited to bid on a general contract for Warren County Public Schools, A.S. Rhodes Elementary School Renovation for the Warren County School Board. This project generally consists of approximately 21,070 SF of renovations, including but not limited to new mechanical, electrical and plumbing systems. All bids shall be on a lump sum basis.
Bids shall be received until 2:00 P.M. local prevailing time on Thursday, September 19, 2019, to the attention of Melody Sheppard, Assistant Superintendent for Administration, in the Board Room of the Warren County Public School Administration Building, 210 N. Commerce Avenue, Front Royal, VA 22630, at which time they will be opened publicly and read aloud. Bids received after this time will not be accepted. All interested parties are invited to attend.
Drawings and specifications may be examined at the Office of the Architect/Engineer, the Warren County School Board office, and at the following locations:
Valley Construction News
426 W. Campbell Avenue
Roanoke, VA 24016
Alex Gugliada | The Blue Book Bldg. & Construction Network
800 E. Main Street
Jefferson Valley, NY 10535
Builders' Exchange, Inc.
3207 Hermitage Rd.
Richmond, VA 23227
DODGE Data & Analytics
3315 Central Avenue
Hot Springs, AR 71913
30 Technology Parkway South | Suite 100
Norcross, GA 30092-2912
Construction Journal
400 SW 7th Street
Stuart, FL 34994
Plans and specifications can be obtained via download from ShareFile upon receipt of a nonrefundable check in the amount of $25.00. Contact firstname.lastname@example.com for ShareFile access. Upon receipt of check and valid email address, OWPR will provide a password with instructions on accessing and downloading plans and specifications. Hard copies of plans and specifications can be obtained from OWPR upon receipt of a nonrefundable check in the amount of $400.00. Plans and specifications can be shipped for additional charge. Contact OWPR for further details.
Bids shall be accompanied by a bid guarantee of not less than 5% of the amount of the bid, which may be a certified check or cashier's check, or a bid bond of 5% of the amount of the bid, made payable to Warren County School Board. A performance bond, payment bond and a material and labor bond will be required and the cost shall be included in the bid price.
Bidders are required under Title 54.1, Chapter 11, Code of Virginia (1950), as amended, to be licensed as a "Class A Contractor" before submitting a bid of one hundred twenty thousand ($120,000) dollars or more; or to be licensed as a "Class B Contractor" before submitting a bid of ten thousand ($10,000) dollars or more but less than one hundred twenty thousand ($120,000) dollars; or be licensed as a "Class C Contractor" before submitting a bid of no more than ten thousand dollars ($10,000). Each Bidder will be required to give their State Registration Number on their proposal. All non-resident contractors and subcontractors bidding the work described herein shall register with the Department of Labor and Industry under the provisions of Section 40.1-30 of the Code of Virginia.
The Owner reserves the right to waive irregularities and to reject any or all bids.
A pre-bid conference will be held on September 12, 2019 beginning at 2:00 P.M. at the Warren County Public School Administration Building, 210 N. Commerce Avenue, Front Royal, VA 22630 with a tour of the school afterwards. The school is located at 224 W. Strasburg Rd., Front Royal, VA 22630. This will provide an opportunity to answer questions and explain any items requiring further clarification.
Bids shall be binding for a period of sixty (60) days from the date on which bids are opened.
Local News
Lucky Star Reunion packs Front Royal Brewing Co. with memories of times past
As former Lucky Star Lounge co-owner and oft-bartender Trevor Lipton said Saturday evening, August 17 at the Front Royal Brewing Company in introducing the newest incarnation of the first and last band to play at his establishment, this reunion was more than a reuniting of former clientele and staff of a fondly-remembered eating, drinking and entertainment establishment, and more like an extended family reunion.
And to the backdrop of headliner AfterSHOCK, formerly known as The Shockers, formerly known as EyeSoar – happily reunited with guitarist Pat Flowers and often guest singer Renee Flowers – the evening had exactly THAT feel, as old friends and acquaintances embraced and reminisced about good times, lost times, and times to come wherever and whenever they might be encountered.
The Lucky Star spanned a nine-year period between 2005 and 2013, at 205A East Main Street, not far from the site of the Front Royal Brewing Company-hosted reunion event – and a shout out to the staff for its handling of the large, occasionally-demanding and hungry, and thirsty on a steamy night, crowd. The entrees, appetizers and home-brewed craft beers kept the enthusiastic crowd fueled for more.
Thank you to Lucky Star founding partners Trevor and Lorena Lipton; absent out west but fondly-remembered co-owners Patrick and Shawn Patterson; Star "spiritual advisor" Roxanne Bauserman, family and friends; past and future Zombie Walkers; and everyone else for the memories and opportunity to reunite and fondly recall the bonding characteristics of a true English and early Americana-style pub and eatery – the kind where revolutions, or at least minor rebellions, are planned.
Local News
'Clear the Shelter' day sees 26 animals in 'forever' homes
The Julia Wagner Animal Shelter in Front Royal came within a whisker or two of its target on Saturday, August 17, by adopting out 26 animals on the annual "Clear the Shelter" Day, a result Executive Director Meghan Bowers said was "highly satisfactory."
Bowers had set a high watermark for the special day with extended hours and a full staff on duty. In an interim report to me when I dropped by in early afternoon, she told me of six adoptions.
At the end of the day, when another Examiner staffer, Roger Bianchini, had stopped in to take photos, the totals stood at 15 "forever homes" for dogs and 11 for cats, just short of the target of 30 overall. Adoption fees were excused for the event.
Looking backward, then forward, Bowers reported a net total of $16,794 for last month's "Waggin' for Dragons" popular fundraiser.
"It's not as much as previous years," she said, but a healthy total since the event was dropped last year and the number of competitors – eight boats, 160 paddlers – last month was down by half.
She expects "Waggin'" will be back to a full complement of competitors in future years.
Looking forward, Bowers announced "Putt for Paws" at the Bowling Green Country Club on October 11. Teams of four compete on the 18-hole golf course, followed by dinner and door prizes. Price of entry for another key fundraising event is $400 per team.
Then the Humane Society of Warren County, headed by Ellen Aders, will stage one of its biggest attractions: "Tales & Ales," a dinner – the 8th annual – with silent auction, cash prizes including $1,000 that goes to a lucky $35 entry ticket holder. "Tales & Ales, usually a sellout, is scheduled for November 16. More information on both these events will be reported here as they approach.
And remember, not all the shelter's animals were adopted – some are still waiting for that special, loving person to take them home too…
Local News
Fireball Arcade finally has its Grand Opening
Fireball Arcade has been open for about a year at 218 East Main Street in Front Royal, but is just now getting around to their Grand Opening.
On August 17th the Fireball Arcade held its Grand Opening with a Ribbon Cutting ceremony by the Front Royal Warren County Chamber of Commerce.
The Fireball is an 80's themed Pinball and Video Game Arcade. They also have a Birthday room rental available! Share your childhood arcade experiences with your children.
King Cartoons
Wind: 3mph SSW
Humidity: 87%
Pressure: 29.97"Hg
UV index: 1


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Why isn't the condition of the Highly Sensitive Person (HSP) more publicized? Nowadays, we're thankfully more aware of people with different ailments and conditions that once went unrecognized and were quickly dismissed. Kids who couldn't concentrate in school were considered … Continue reading
Category Archives: Favorites
Why is live theatre so disappointing? Once upon a time, I was a true, die-hard, theatre aficionado. I enjoyed the theatre so much that I even got degrees in it before going to library school and becoming a librarian. Since … Continue reading
Why do we have so many words for "excrement" in the English language? Rather recently, my fiance and I decided to enjoy the beautiful spring weather we've been having lately by going to a nearby wildlife reserve that we especially … Continue reading
Why is water so darn soothing? It covers 70% of the earth's surface and makes up about 60% of our bodies. It's both constructive and destructive. Without it, all life–plant and animal–would cease to exist. Water is an all-powerful force … Continue reading
Why do so many of us pretend to be something we're not? This post is in response to a follower of this blog who commented on Nosy People. It seems that TV nowadays is inundated with all these reality shows … Continue reading
Why do so many of us have trouble facing our birthdays? Yesterday was my fiance's birthday, and although he's normally a positive, go-with-the-flow, the glass-is-half-full kind of guy, he wasn't 100 percent chipper about it. You could say he had … Continue reading
Why is knitting just so darn relaxing? When I was a kid, both of my grandmothers taught me different knitting stitches to keep me occupied and out of trouble (my grandfathers taught me woodworking for the same reasons, but that's … Continue reading
Why do people like confessing to librarians? It's no secret that I'm a librarian, but sometimes when I'm sitting at the desk (or "the perch," as I like to call it) I feel people mistake me for a priest or … Continue reading
Why do we attribute Caesar salad to a Roman emperor? With the dog days of summer here, you might be like me and seek some lighter, cooler fare at the kitchen table. After all, who on a hot day wants … Continue reading
Since the 4th of July holiday is upon those of us who are in the U.S., I thought it would be fitting to write a post with a patriotic theme. Aside from the fireworks and barbeques, there are other things … Continue reading


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System Center Virtual Machine Manager Self-Service Portal 2.0 – Now Available!
Following on from my previous posts on Creating a Private Cloud Part 1 and Part 2, System Center Virtual Machine Manager Self-Service Portal 2.0 (SSP) is now available.
For those who have not read the two posts yet, briefly the System Center Virtual Machine Manager Self-Service Portal 2.0 (SSP) is a fully supported, partner extensible solution that enables you to dynamically pool, allocate, and manage compute, network and storage resources to deliver a private cloud platform in you datacenter. Installed with System Center Virtual Machine Manager 2008 R2 and SQL Server 2008 or 2008 R2 the self-service portal consists of three components:
- VMMSSP website component. A web-based component that provides a user interface to the self-service portal.
- VMMSSP database component. A SQL Server database that stores information about configured assets and other information related to tasks performed in the portal.
- VMMSSP server component. A Windows service that runs default and customized virtual machine actions that the user requests through the VMMSSP website.
To Learn more about the System Requirements Visit the SSP page on TechNet.
Key Features:
· Automation and Guidance: To assess, plan and design your private cloud foundation infrastructure
· Customer/business unit on-boarding: Automated workflows to onboard business unit IT departments onto to your virtualized shared resource pool
· Dynamic provisioning engine: To rapidly provision virtualized infrastructure in conjunction with System Center and Hyper-V
· Self-Service portal: To empower consumers of IT to request and provision infrastructure for their apps/services
· Partner Extensibility: Enable partners to expose their unique hardware capabilities through familiar Microsoft scripting technologies while providing variety and flexibility to IT
Want more info?
- Visit the official Microsoft System Center VMM Self-Service Portal 2.0 blog.
- Read my two blogs at Creating a Private Cloud Part 1 and Part 2,
- Visit the Put the Cloud to Work For Your Organization site
- Visit the new Cloud Centre on TechNet
Questions or Comments?
· Feel free to send your comments and feedback to firstname.lastname@example.com, or visit www.Microsoft.com/SSP.
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The first time I heard Bravestation was a couple years ago when they had just released their 2010 E.P. Their music caught me right away.
My exact quote on the subject: "If New Wave had started out in the Canadian wilderness, the result would probably resemble this Toronto-based band's dreamy, complex sound." I mean, just listen to this track, "White Wolves", from 2010:
Bravestation is back, this time with a full-length called Giants & Dreamers, and boy, is it appropriately titled. It's big, soaring, and- forgive the redundancy- dreamy.
From the new album, here's "Signs of the Civilized." I hope you're ready to get a little dark while dancing in the forest.
You can buy Giants & Dreamers starting today on their Bandcamp or on iTunes. Go! Do it! Do it now!


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The silent roads
Our Kickstarter campaign has already started!
"A devastating apocalyptic event has mysteriously annihilated most of the world's population. No one knows what happened and there are many theories circulating, from the great Malthusian collapse to a technological singularity. According to some legends, it´s the result of a Von Neumann Berserker probe, but nobody can be too sure. Pockets of depraved humans still exist, surrounded by wilderness, abandoned cities left in ruins, and the unknown."
We believe in talent and hard work. We believe in passion. We dream of coming up with brilliant ideas that will reignite the Golden Era. We believe in art and design. Each of our illustrations and concepts should become a work of art. We believe in crafting a unique gaming experience without the aid of engines or any other constraints.
Essentially, we are gamers, working for gamers!
We are also firm believers in the fact that no matter what has been utilized in the past, it can still be improved by using the same ingredients as a base and adding our own special touches to create something remarkable.