text:
Just found this. Check it out, looks decent
Step 1: The Parts
You will need:
-100k Potentiometer
-.1 micro farad capacitor
-220 micro farad capacitor
-10 micro farad capacitor --only necessary for the 200k boost loop
-LM386 microchip (or whatever it's called)
-Soldering Iron
-small circuit board
-headphone jack
-speaker --http://www.josepino.com/circuits/index.php?howto-speaker.jpc--make your own
-plastic cup
Note: I added a breadboard that I drew up from the circuit diagram, so if someone wanted to double-check it that would be nice
Step 2: Liberating the audio jack
Strip the wire
Strip the insulated wires, making sure that you don't strip them so far that the stripped part touches the non-insulated wire
EDIT: Don't twist the two (formerly) insulated wires together, it might hurt sound quality.
Those (formerly) insulated wires are the positive and the noninsulated are the ground.
Check out my instructable on headphone repair for more about headphone wires.
Step 3: Place everything on the board
Here's how I set up mine
Step 4: Make all the connections underneath
Do you knoweth of an amplifier circuit that only useth transistors but still pertaineth effectivity?
are you still looking for a transistor version of this??
Do you knoweth of an amplifier circuit that only useth transistors but still pertaineth effectivity?
Do you knoweth of an amplifier circuit that only useth transistors but still pertaineth effectivity?
can anyone help me to figure it out the problem
when selecting speakers look at the SPL number and the power handling (watts), match and balance them to the amp intended. hard to do when using preassemble speaker cabs. though when watts is generally all you are given.
I like the cup! I used a cutoff water bottle for a similar effect. ;-)
uncle frogy
Note that to use both op-amps in this chip, you'll need to buy twice the quantities of all of the resistors, capacitors, and potentiometers that are shown in the schematic. The Av term stands for gain. The potentiometer isn't shown in that schematic. You can use the 100K potentiometer just like in this instructable.
please help me!
your power source or Vcc as we call it should be connected to the sixth pin of the LM386 IC but on the bred board it is shown on pin7, just change the ports..
and little bit more info:
pin 7 is for bypass any high voltage between 4 to 18 volts will cause the output to be a bypassed one that is there wud be no amplification , you will het the same low power output you received from the mp3 player or you phone or whatever your source of signal was
its for bypass
you should have com\nnected the power port to pin 6 on the right hand side..
well you know that coz your speakers worked butthe schematics on the bread board are somewhat wrong
if not what device should i usefor this amplifier...
can you also teach me how to connect the audio plug..because i cannot understand the instructions...
thank you
10 W transistor
100-150W transistor
2x68W LM3886
the LM**** ones are going to be somewhat similar to this tutorial.
here's another one...
There's hundreds of these circuits on the web and with a little googling (just google: audio amp circuit) you can unearth many, many more than what I have listed above.


text:
A method of effecting a one-pot conversion of a tetrahydronaphthalic anhydride of formula (I):
The method comprises heating the tetrahydronaphthalic anhydride of formula (I) with a reagent mixture comprising ammonium nitrate so as to effecting concomitant dehydrogenation during the conversion.
1. A method of effecting a one-pot conversion of a tetrahydronaphthalic anhydride of formula (I):
to a nitrate salt of a benzisoindolenine of formula (II):
said method comprising heating said tetrahydronaphthalic anhydride of formula (I) with a reagent mixture comprising ammonium nitrate so as to effecting concomitant dehydrogenation during the conversion, wherein:
R1, R2, R3 and R4 are each independently selected from the group consisting of: hydrogen, hydroxyl, C1-20 alkyl, C1-20 alkoxy, amino, C1-20alkylamino, di(C1-20alkyl)amino, halogen, cyano, thiol, C1-20 alkylthio, nitro, C1-20 alkylcarboxy, C1-20 alkylcarbonyl, C1-20 alkoxycarbonyl, C1-20 alkylcarbonyloxy, C1-20alkylcarbonylamino, C5-20 aryl, C5-20 arylalkyl, C5-20 aryloxy, C5-20 arylalkoxy, C5-20heteroaryl, C5-20heteroaryloxy, C5-20heteroarylalkoxy and C5-20 heteroarylalkyl.
2. The method of
3. The method of
4. The method of
5. The method of
6. The method of
7. The method of
8. The method of
9. The method of
10. The method of
liberating the benzisoindolenine of formula (II) from the benzisoindolenine nitrate salt using sodium methoxide.
11. The method of
macrocyclizing said benzisoindolenine to form a naphthalocyanine of formula (III):
M is absent or selected from Si(A1)(A2), Ge(A1)(A2), Ga(A1), Mg, Al(A1), TiO, Ti(A1)(A2), ZrO, Zr(A1)(A2), VO, V(A1)(A2), Mn, Mn(A1), Fe, Fe(A1), Co, Ni, Cu, Zn, Sn, Sn(A1)(A2), Pb, Pb(A1)(A2), Pd and Pt;
A1 and A2 are axial ligands, which may be the same or different, and are selected from —OH, halogen or —ORq;
Rq is selected from C1-16 alkyl, C5-20 aryl, C5-20 arylalkyl, C1-20 alkylcarbonyl, C1-20 alkoxycarbonyl or Si(Rx)(Ry)(Rz); and
Rx, Ry and Rz may be the same or different and are selected from C1-20 alkyl, C5-20 aryl, C5-20 arylalkyl, C1-20 alkoxy, C5-20 aryloxy or C5-20 arylalkoxy.
12. The method of
13. The method of
This application is a Continuation of U.S. application Ser. No. 11/831,961 filed on Aug. 1, 2007 all of which are incorporated herein by reference.
The present application relates generally to an improved method of synthesizing naphthalocyanines. It has been developed primarily to reduce the cost of existing naphthalocyanine syntheses and to facilitate large-scale preparations of these compounds.
The following applications have been filed by the Applicant
The disclosures of these co-pending applications are incorporated herein by reference.
The following patents or patent applications filed by the applicant or assignee of the present invention are hereby incorporated by cross-reference.
We have described previously the use of naphthalocyanines as IR-absorbing dyes. Naphthalocyanines, and particularly gallium naphthalocyanines, have low absorption in the visible range and intense absorption in the near-IR region (750-810 nm). Accordingly, naphthalocyanines are attractive compounds for use in invisible inks. The Applicant's U.S. Pat. Nos. 7,148,345 and 7,122,076 (the contents of which are herein incorporated by reference) describe in detail the use of naphthalocyanine dyes in the formulation of inks suitable for printing invisible (or barely visible) coded data onto a substrate. Detection of the coded data by an optical sensing device can be used to invoke a response in a remote computer system. Hence, the substrate is interactive by virtue of the coded data printed thereon.
The Applicant's netpage and Hyperlabel� systems, which makes use of interactive substrates printed with coded data, are described extensively in the cross-referenced patents and patent applications above (the contents of which are herein incorporated by reference).
In the anticipation of widespread adoption of netpage and Hyperlabel� technologies, there exists a considerable need to develop efficient syntheses of dyes suitable for use in inks for printing coded data. As foreshadowed above, naphthalocyanines and especially gallium naphthalocyanines are excellent candidates for such dyes and, as a consequence, there is a growing need to synthesize naphthalocyanines efficiently and in high yield on a large scale.
Naphthalocyanines are challenging compounds to synthesize on a large scale. In U.S. Pat. Nos. 7,148,345 and 7,122,076, we described an efficient route to naphthalocyanines via macrocyclization of naphthalene-2,3-dicarbonitrile. Scheme 1 shows a route to the sulfonated gallium naphthalocyanine 1 from naphthalene-2,3-dicarbonitrile 2, as described in U.S. Pat. No. 7,148,345.
However, a problem with this route to naphthalocyanines is that the starting material 2 is expensive. Furthermore, naphthalene-2,3-dicarbonitrile 2 is prepared from two expensive building blocks: tetrabromo-o-xylene 3 and fumaronitrile 4, neither of which can be readily prepared in multi-kilogram quantities.
Accordingly, if naphthalocyanines are to be used in large-scale applications, there is a need to improve on existing syntheses.
In a first aspect, there is provided a method of preparing a naphthalocyanine comprising the steps of:
(i) providing a tetrahydronaphthalic anhydride;
(ii) converting said tetrahydronaphthalic anhydride to a benzisoindolenine; and
(iii) macrocyclizing said benzisoindolenine to form a naphthalocyanine
Optionally, the tetrahydronaphthalic anhydride is of formula (I):
Optionally, R1, R2, R3 and R4 are all hydrogen.
Optionally, step (ii) comprises a one-pot conversion from the tetrahydronaphthalic anhydride to a benzisoindolenine salt. This one-pot conversion facilitates synthesis of naphthalocyanines via the route described above and greatly improves yields and scalability.
Optionally, the benzisoindolenine salt is a nitrate salt although other salts (e.g. benzene sulfonate salt) are of course within the scope of the present invention.
Optionally, the one-pot conversion is effected by heating with a reagent mixture comprising ammonium nitrate.
Optionally, the reagent mixture comprises at least 2 equivalents of ammonium nitrate with respect to the tetrahydronaphthalic anhydride.
Optionally, the reagent mixture comprises urea.
Optionally, the reagent mixture comprises at least one further ammonium salt.
Optionally, the further ammonium salt is selected from: ammonium sulfate and ammonium benzenesulfonate
Optionally, the reagent mixture comprises a catalytic amount of ammonium molybdate.
Optionally, the heating is within a temperature range of 150 to 200� C.
The reaction may be performed in the presence of or in the absence of a solvent. Optionally, heating is in the presence of an aromatic solvent. Examples of suitable solvents are nitrobenzene, biphenyl, diphenyl ether, mesitylene, anisole, phenetole, dichlorobenzene, trichlorobenzene and mixtures thereof.
Optionally, the benzisoindolenine is liberated from the benzisoindolenine salt using a base. Sodium methoxide is an example of a suitable base although the skilled person will be readily aware of other suitable bases.
Optionally, the benzisoindolenine is of formula (II):
Optionally, the naphthalocyanine is of formula (III):
Optionally, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15 and R16 are all hydrogen.
Optionally, M is Ga(A1), such as Ga(OCH2CH2OCH2CH2OCH2CH2OMe); that is where Rq is CH2CH2OCH2CH2OCH2CH2OMe. For the avoidance of doubt, ethers such as CH2CH2OCH2CH2OCH2CH2OMe fall within the definition of alkyl groups as specified hereinbelow. Gallium compounds are preferred since they have excellent lightfastness, strong absorption in the near-IR region, and are virtually invisible to the human eye when printed on a page.
Optionally, step (iii) comprises heating the benzisoindolenine in the presence of a metal compound, such as AlCl3 or GaCl3 or a corresponding metal alkoxide. The reaction may be performed in the absence of or in the presence of a suitable solvent, such as toluene, nitrobenzene etc. When a metal alkoxide is used, the reaction may be catalyzed with a suitable base, such as sodium methoxide. Alcohols, such as triethylene glycol monomethyl ether or glycol may also be present to assist with naphthalocyanine formation. These alcohols may end up as the axial ligand of the naphthalocyanine or they may be cleaved from the metal under the reaction conditions. The skilled person will readily be able to optimize the conditions for naphthalocyanine formation from the benzisoindolenine.
Optionally, the method further comprises the step of sulfonating said naphthalocyanine. Sulfonate groups are useful for solubilizing the naphthalocyanines in ink formulations, as described in our earlier U.S. Pat. Nos. 7,148,345 and 7,122,076.
In a second aspect, there is provided a method of effecting a one-pot conversion of a tetrahydronaphthalic anhydride to a benzisoindolenine salt, said method comprising heating said tetrahydronaphthalic anhydride with a reagent mixture comprising ammonium nitrate.
This transformation advantageously obviates a separate dehydrogenation step to form the naphthalene ring system. The ammonium nitrate performs the dual functions of oxidation (dehydrogenation) and isoindolenine formation.
The isoindolenine salts generated according to the second aspect may be used in the synthesis of naphthalocyanines. Hence, this key reaction provides a significant improvement in routes to naphthalocyanines.
In general, optional features of this second aspect mirror the optional features described above in respect of the first aspect.
In a third aspect, there is provided a method of preparing a sultine of formula (V) from a dihalogeno compound of formula (IV)
The method according to the third aspect surprisingly minimizes polymeric by-products and improves yields, when compared to literature methods for this reaction employing DMF as the solvent. These advantages are amplified when the reaction is performed on a large scale (e.g. at least 0.3 molar, at least 0.4 molar or at least 0.5 molar scale).
Optionally, NaI is used to catalyze the coupling reactions when X is Cl or Br.
Optionally, a metal carbonate base (e.g. Na2CO3, K2CO3, Cs2CO3 etc) is present.
Optionally, the hydroxymethanesulfinate salt is sodium hydroxymethanesulfinate (Rongalite™)
Optionally, R1, R2, R3 and R4 are all hydrogen.
Optionally, the method comprises the further step of reacting the sultine (V) with an olefin at elevated temperature (e.g. about 80� C.) to generate a Diels-Alder adduct.
Optionally, the olefin is maleic anhydride and said Diels-Alder adduct is a tetrahydronaphthalic anhydride.
Optionally, the tetrahydronaphthalic anhydride is used as a precursor for naphthalocyanine synthesis, as described herein.
Optionally, the naphthalocyanine synthesis proceeds via conversion of the tetrahydronaphthalic anhydride to a benzisoindolenine, as described herein.
The invention will now be described in detail with reference to the following drawings, in which:
As an alternative to dicarbonitriles, the general class of phthalocyanines is known to be prepared from isoindolenines. In U.S. Pat. No. 7,148,345, we proposed the benzisoindolenine 5 as a possible precursor to naphthalocyanines
However, efficient syntheses of the benzisoindolenine 5 were unknown in the literature, and it was hitherto understood that dicarbonitriles, such as naphthalene-2,3-dicarbonitrile 2, were the only viable route to naphthalocyanines.
Nevertheless, with the potentially prohibitive cost of naphthalene-2,3-dicarbonitrile 2, the present inventors sought to explore a new route to the benzisoindolenine 5, as outlined in Scheme 2.
Tetrahydronaphthalic anhydride 6 was an attractive starting point, because this is a known Diels-Alder adduct which may be synthesized via the route shown in Scheme 3.
Referring to Scheme 2, it was hoped that the conversion of naphthalic anhydride 7 to the benzisoindolenine 5 would proceed analogously to the known conversion of phthalic anhydride to the isoindolenine 8, as described in WO98/31667.
However, a number of problems remained with the route outlined in Scheme 2. Firstly, the dehydrogenation of tetrahydronaphthalic anhydride 6 typically requires high temperature catalysis. Under these conditions, tetrahydronaphthalic anhydride 6 readily sublimes resulting in very poor yields. Secondly, the preparation of tetrahydronaphthalic anhydride 6 on a large scale was not known. Whilst a number of small-scale routes to this compound were known in the literature, these generally suffered either from poor yields or scalability problems.
The use of sultines as diene precursors is well known and 1,4-dihydro-2,3-benzoxathiin-3-oxide 10 has been used in a synthesis of 6 on a small scale (Hoey, M. D.; Dittmer, D. A. J. Org. Chem. 1991, 56, 1947-1948). As shown in Scheme 4, this route commences with the relatively inexpensive dichloro-o-xylene 11, but the feasibility of scaling up this reaction sequence is limited by the formation of undesirable polymeric by-products in the sultine-forming step. The formation of these by-products makes reproducible production of 6 in high purity and high yield difficult.
Nevertheless, the route outlined in Scheme 4 is potentially attractive from a cost standpoint, since dichloro-o-xylene 11 and maleic anhydride are both inexpensive materials.
Whilst the reaction sequence shown in Schemes 4 and 2 present significant synthetic challenges, the present inventors have surprisingly found that, using modified reaction conditions, the benzisoindolenine 5 can be generated on a large scale and in high yield. Hence, the present invention enables the production of naphthalocyanines from inexpensive starting materials, and represents a significant cost improvement over known syntheses, which start from naphthalene-2,3-dicarbonitrile 2.
Referring to Scheme 5, there is shown a route to the benzisoindolenine 5, which incorporates two synthetic improvements in accordance with the present invention.
Unexpectedly, it was found that by using DMSO as the reaction solvent in the conversion of 11 into 10, the reaction rate and selectivity for the formation of sultine 10 increases significantly. This is in contrast to known conditions (Hoeg, M. D.; Dittmer, D. A. J. Org. Chem. 1991, 56, 1947-1948) employing DMF as the solvent, where the formation of undesirable polymeric side-products is a major problem, especially on a large scale. Accordingly, the present invention provides a significant improvement in the synthesis of tetrahydronaphthalic anhydride 6.
The present invention also provides a significant improvement in the conversion of tetrahydronaphthalic anhydride 6 to the benzisoindolenine 5. Surprisingly, it was found that the ammonium nitrate used for this step readily effects oxidation of the saturated ring system as well as converting the anhydride to the isoindolenine. Conversion to a tetrahydroisoindolenine was expected to proceed smoothly, in accordance with the isoindolenine similar systems described in WO98/31667. However, concomitant dehydrogenation under these reaction conditions advantageously provided a direct one-pot route from the tetrahydronaphthalic anhydride 6 to the benzisoindolenine salt 12. This avoids problematic and low-yielding dehydrogenation of the tetrahydronaphthalic anhydride 6 in a separate step. Subsequent treatment of the salt 12 with a suitable base, such as sodium methoxide, liberates the benzisoindolenine 5. As a result of these improvements, the entire reaction sequence from 11 to 5 is very conveniently carried out, and employs inexpensive starting materials and reagents (Scheme 5).
The benzisoindolenine 5 may be converted into any required naphthalocyanine using known conditions. For example, the preparation of a gallium naphthalocyanine from benzisoindolenine 5 is exemplified herein. Subsequent manipulation of the naphthalocyanine macrocycle may also be performed in accordance with known protocols. For example, sulfonation may be performed using oleum, as described in U.S. Pat. Nos. 7,148,345 and 7,122,076.
Hitherto, the use of tetrahydronaphthalic anhydride 6 as a building block for naphthalocyanine synthesis had not previously been reported. However, it has now been shown that tetrahydronaphthalic anhydride 6 is a viable intermediate in the synthesis of these important compounds. Moreover, it is understood by the present inventors that the route shown in Scheme 5 represents the most cost-effective synthesis of benzisoindolenines 5.
The term "aryl" is used herein to refer to an aromatic group, such as phenyl, naphthyl or triptycenyl. C6-12 aryl, for example, refers to an aromatic group having from 6 to 12 carbon atoms, excluding any substituents. The term "arylene", of course, refers to divalent groups corresponding to the monovalent aryl groups described above. Any reference to aryl implicitly includes arylene, where appropriate.
The term "heteroaryl" refers to an aryl group, where 1, 2, 3 or 4 carbon atoms are replaced by a heteroatom selected from N, O or S. Examples of heteroaryl (or heteroaromatic) groups include pyridyl, benzimidazolyl, indazolyl, quinolinyl, isoquinolinyl, indolinyl, isoindolinyl, indolyl, isoindolyl, furanyl, thiophenyl, pyrrolyl, thiazolyl, imidazolyl, oxazolyl, isoxazolyl, pyrazolyl, isoxazolonyl, piperazinyl, pyrimidinyl, piperidinyl, morpholinyl, pyrrolidinyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, benzopyrimidinyl, benzotriazole, quinoxalinyl, pyridazyl, coumarinyl etc. The term "heteroarylene", of course, refers to divalent groups corresponding to the monovalent heteroaryl groups described above. Any reference to heteroaryl implicitly includes heteroarylene, where appropriate.
Unless specifically stated otherwise, aryl and heteroaryl groups may be optionally substituted with 1, 2, 3, 4 or 5 of the substituents described below.
Where reference is made to optionally substituted groups (e.g. in connection with aryl groups or heteroaryl groups), the optional substituent(s) are independently selected from C1-8 alkyl, C1-8 alkoxy, —(OCH2CH2)dORd (wherein d is an integer from 2 to 5000 and Rd is H, C1-8 alkyl or C(O)C1-8 alkyl), cyano, halogen, amino, hydroxyl, thiol, —SRv, —NRuRv, nitro, phenyl, phenoxy, —CO2Rv, —C(O)Rv, —OCORv, —SO2Rv, —OSO2Rv, —SO2ORv, —NHC(O)Rv, —CONRuRv, —CONRuRv, —SO2NRuRv, wherein Ru and Rv are independently selected from hydrogen, C1-20 alkyl, phenyl or phenyl-C1-8 alkyl (e.g. benzyl). Where, for example, a group contains more than one substituent, different substituents can have different Ru or Rv groups.
The term "alkyl" is used herein to refer to alkyl groups in both straight and branched forms. Unless stated otherwise, the alkyl group may be interrupted with 1, 2, 3 or 4 heteroatoms selected from O, NH or S. Unless stated otherwise, the alkyl group may also be interrupted with 1, 2 or 3 double and/or triple bonds. However, the term "alkyl" usually refers to alkyl groups having double or triple bond interruptions. Where "alkenyl" groups are specifically mentioned, this is not intended to be construed as a limitation on the definition of "alkyl" above.
Where reference is made to, for example, C1-20 alkyl, it is meant the alkyl group may contain any number of carbon atoms between 1 and 20. Unless specifically stated otherwise, any reference to "alkyl" means C1-20 alkyl, preferably C1-12 alkyl or C1-6 alkyl.
The term "alkyl" also includes cycloalkyl groups. As used herein, the term "cycloalkyl" includes cycloalkyl, polycycloalkyl, and cycloalkenyl groups, as well as combinations of these with linear alkyl groups, such as cycloalkylalkyl groups. The cycloalkyl group may be interrupted with 1, 2 or 3 heteroatoms selected from O, N or S. However, the term "cycloalkyl" usually refers to cycloalkyl groups having no heteroatom interruptions. Examples of cycloalkyl groups include cyclopentyl, cyclohexyl, cyclohexenyl, cyclohexylmethyl and adamantyl groups.
The term "arylalkyl" refers to groups such as benzyl, phenylethyl and naphthylmethyl.
The term "halogen" or "halo" is used herein to refer to any of fluorine, chlorine, bromine and iodine. Usually, however, halogen refers to chlorine or fluorine substituents.
Where reference is made herein to "a naphthalocyanine", "a benzisoindolenine", "a tetrahydronaphthalic anhydride" etc, this is understood to be a reference to the general class of compounds embodied by these generic names, and is not intended to refer to any one specific compound. References to specific compounds are accompanied with a reference numeral.
Chiral compounds described herein have not been given stereo-descriptors. However, when compounds may exist in stereoisomeric forms, then all possible stereoisomers and mixtures thereof are included (e.g. enantiomers, diastereomers and all combinations including racemic mixtures etc.).
Likewise, when compounds may exist in a number of regioisomeric or tautomeric forms, then all possible regioisomers, tautomers and mixtures thereof are included.
For the avoidance of doubt, the term "a" (or "an"), in phrases such as "comprising a", means "at least one" and not "one and only one". Where the term "at least one" is specifically used, this should not be construed as having a limitation on the definition of "a".
Throughout the specification, the term "comprising", or variations such as "comprise" or "comprises", should be construed as including a stated element, integer or step, but not excluding any other element, integer or step.
The invention will now be described with reference to the following drawings and examples. However, it will of course be appreciated that this invention may be embodied in many other forms without departing from the scope of the invention, as defined in the accompanying claims.
Sodium hydroxymethanesulfinate (Rongalite™) (180 g; 1.17 mol) was suspended in DMSO (400 mL) and left to stir for 10 min. before dichloro-o-xylene (102.5 g; 0.59 mol), potassium carbonate (121.4 g; 0.88 mol) and sodium iodide (1.1 g; 7 mmol) were added consecutively. More DMSO (112 mL) was used to rinse residual materials into the reaction mixture before the whole was allowed to stir at room temperature. The initial endothermic reaction became mildly exothermic after around 1 h causing the internal temperature to rise to ca. 32-33� C. The reaction was followed by TLC (ethyl acetate/hexane, 50:50) and found to be complete after 3 h. The reaction mixture was diluted with methanol/ethyl acetate (20:80; 400 mL) and the solids were filtered off, and washed with more methanol/ethyl acetate (20:80; 100 mL, 2�50 mL). The filtrate was transferred to a separating funnel and brine (1 L) was added. This caused more sodium chloride from the product mixture to precipitate out. The addition of water (200 mL) redissolved the sodium chloride. The mixture was shaken and the organic layer was separated and then the aqueous layer was extracted further with methanol/ethyl acetate (20:80; 200 mL, 150 mL, 250 mL). The combined extracts were dried (Na2SO4) and rotary evaporated (bath 37-38� C.). More solvent was removed under high vacuum to give the sultine 10 as a pale orange liquid (126 g) that was found by 1H NMR spectroscopy to be relatively free of by-product but containing residual DMSO and ethyl acetate (
The crude sultine from above (126 g) was diluted in trifluorotoluene (100 mL) and then added to a preheated (bath 80� C.) suspension of maleic anhydride (86 g; 0.88 mol) in trifluorotoluene (450 mL). The residual sultine was washed with more trifluorotoluene into the reaction mixture and then the final volume was made up to 970 mL. The reaction mixture was heated at 80� C. for 15 h, more maleic anhydride (28.7 g; 0.29 mol) was added and then heating was continued for a further 8 h until TLC showed that the sultine had been consumed. While still at 80� C., the solvent was removed by evaporation with a water aspirator and then the residual solvent was removed under high vacuum. The moist solid was triturated with methanol (200 mL) and filtered off, washing with more methanol (3�100 mL). The tetrahydronaphthalic anhydride 6 was obtained as a fine white crystalline solid (75.4 g; 64% from 10) after drying under high vacuum at 60-70� C. for 4 h.
The sultine was prepared from dichloro-o-xylene (31.9 g; 0.182 mol), as described in Example 2, and then reacted with maleic anhydride (26.8 g; 0.273 mol) in toluene (300 mL total volume) as described above. This afforded the tetrahydronaphthalic anhydride 6 as a white crystalline solid (23.5 g; 64%).
Urea (467 g; 7.78 mol) was added to a mechanically stirred mixture of ammonium sulfate (38.6 g; 0.29 mol), ammonium molybdate (1.8 g) and nitrobenzene (75 mL). The whole was heated with a heating mantle to ca. 130� C. (internal temperature) for 1 h causing the urea to melt. At this point the anhydride 6 (98.4 g; 0.49 mol) was added all at once as a solid. After 15 min ammonium nitrate (126.4 g; 1.58 mol) was added with stirring (internal temperature 140� C.) accompanied by substantial gas evolution. The reaction temperature was increased to 170-175� C. over 45 min and held there for 2 h 20 min. The viscous brown mixture was allowed to cool to ca. 100� C. and then methanol (400 mL) was slowly introduced while stirring. The resulting suspension was poured on a sintered glass funnel, using more methanol (100 mL) to rinse out the reaction flask. After removing most of the methanol by gravity filtration, the brown solid was sucked dry and then washed with more methanol (3�200 mL, 50 mL), air-dried overnight and dried under high vacuum in a warm water bath for 1.5 h. The benzisoindolenine salt 12 was obtained as a fine brown powder (154.6 g) and was found by NMR analysis to contain urea (5.43 ppm) and other salts (6.80 ppm). This material was used directly in the next step without further purification.
The crude nitrate salt 12 (154.6 g) was suspended in acetone (400 mL) with cooling in an ice/water bath to 0� C. Sodium methoxide (25% in methanol; 284 ml; 1.3 mol) was added slowly dropwise via a dropping funnel at such a rate as to maintain an internal temperature of 0-5� C. Upon completion of the addition, the reaction mixture was poured into cold water (2�2 L) in two 2 L conical flasks. The mixtures were then filtered on sintered glass funnels and the solids were washed thoroughly with water (250 mL; 200 mL for each funnel). The fine brown solids were air-dried over 2 days and then further dried under high vacuum to give the benzisoindolenine 5 as a fine brown powder (69.1 g; 73%).
Gallium chloride (15.7 g; 0.089 mol) was dissolved in anhydrous toluene (230 mL) in a 3-neck flask (1 L) equipped with a mechanical stirrer, heating mantle, thermometer, and distillation outlet. The resulting solution was cooled in an ice/water bath to 10� C. and then sodium methoxide in methanol (25%; 63 mL) was added slowly with stirring such that the internal temperature was maintained below 25� C. thereby affording a white precipitate. The mixture was then treated with triethylene glycol monomethyl ether (TEGMME; 190 mL) and then the whole was heated to distill off all the methanol and toluene (3 h). The mixture was then cooled to 90-100� C. (internal temperature) by removing the heating mantle and then the benzisoindolenine 5 from the previous step (69.0 g; 0.35 mol) was added all at once as a solid with the last traces being washed into the reaction vessel with diethyl ether (30 mL). The reaction mixture was then placed in the preheated heating mantle such that an internal temperature of 170� C. was established after 20 min. Stirring was then continued at 175-180� C. for a further 3 h during which time a dark green/brown colour appeared and the evolution of ammonia took place. The reaction mixture was allowed to cool to ca. 100� C. before diluting with DMF (100 mL) and filtering through a sintered glass funnel under gravity overnight. The moist filter cake was sucked dry and washed consecutively with DMF (80 mL), acetone (2�100 mL), water (2�100 mL), DMF (50 mL), acetone (2�50 mL; 100 mL) and diethyl ether (100 mL) with suction. After brief air drying, the product was dried under high vacuum at 60-70� C. to constant weight. Naphthalocyanatogallium methoxytriethyleneoxide was obtained as a microcrystalline dark blue/green solid (60.7 g; 76%); λmax (NMP) 771 nm (
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A microfluidic embryo handling device and method in which biological rotating of embryos is simulated. Fluid flow is used to move and position embryos without assistance of electrical stimulus or other means which may produce undesired heating of biological medium used as the fluid for transporting and position. Continuous or pulsed flow is maintained around an embryo or embryos in the device. The device provides an excellent simulation of biological conditions and may be used for culturing, sorting, testing, evaluating, fertilizing and other similar typical handling operations. An embryo may be parked at a desired location to carry out such a typical operation, while the microfluidic handling device maintains fluid flow around the embryo.
1. A microfluidic embryo handling device comprising:
an embryo transport network having a biological medium for movement of embryos inserted therein, said transport network including an approximate embryo scaled embryo fluidic channel to facilitate simulated biological rotating of individual embryos moving within said fluidic channel.
2. The microfluidic embryo handling device of claim 1, wherein said transport network is formed in a wafer and said embryo fluidic channel comprises a microchannel in said wafer.
3. The microfluidic embryo handling device of claim 1, further comprising:
a biological medium source for introducing said biological medium into said embryo transport network in a continuous flow manner; and
a control test embryo network fed said biological medium from said biological medium source, said control test embryo network being biologically isolated from said embryo transport network.
4. The microfluidic embryo handling device of claim 1, further comprising:
a gravity controlled biological medium source for introducing said biological medium into said embryo transport network in a continuous flow manner.
5. The microfluidic embryo handling device of claim 1, further comprising a formation in a path defined by said communication channel for holding an embryo at a desired location while maintaining flow of said biological medium past an embryo held at said desired location.
6. The microfluidic embryo handling device of claim 5, wherein said formation comprises a constriction.
7. The microfluidic embryo handling device of claim 6, further comprising:
a biological medium source for maintaining flow of said biological medium and for reversing flow of said biological medium to free an embryo held at said desired location.
8. The microfluidic embryo handling device of claim 1, wherein said fluidic channel has a flat bottom.
9. The microfluidic embryo handling device of claim 1, wherein said fluidic channel has a V-shaped bottom.
10. The microfluidic embryo handling device of claim 1, wherein said simulated biological rotating includes rotating and slipping.
11. The microfluidic embryo handling device of claim 1, further comprising:
a controlled biological medium source for introducing said biological medium into said embryo transport network in a continuous flow manner unassisted by electrical stimulus.
12. The microfluidic embryo handling device of claim 1, wherein movement of embryos in said embryo transport network is unassisted by electrical stimulus.
13. The microfluidic embryo handling device of claim 1, wherein said transport network is sealed from surrounding environment and said device further comprises an embryo entrance to said transport network and an embryo exit from said transport network.
14. The microfluidic embryo handling device of claim 13, wherein said embryo entrance comprises a hole penetrating a sealing member which seals said transport network, said hole maintaining separation between said biological medium and surrounding environment through surface tension.
15. The microfluidic embryo handling device of claim 14, further comprising a removable cover to seal said hole.
16. The microfluidic embryo handling device of claim 14, wherein said hole is funnel shaped.
17. The microfluidic embryo handling device of claim 14, wherein said hole is located in a midstream portion of said transport network.
18. The microfluidic embryo handling device of claim 13, wherein said embryo entrance comprises a well in fluid communication with said transport network.
19. The microfluidic embryo handling device of claim 13, wherein said embryo entrance comprises a hanging drop.
20. The microfluidic embryo handling device of claim 1, wherein said fluidic channel comprises an embryo compartment defining a culturing station for an embryo and said device further comprises smaller than embryo diameter fluid flow channels for moving fluid through said embryo compartment.
21. A microfluidic embryo handling device, comprising:
a fluid path for moving an embryo inserted therein by fluid flow unassisted by electrical stimulus;
surfaces defining at least a part of said fluid path and spaced to promote rotating of said embryo as it moves in said fluid path.
22. A method of handling embryos comprising steps of:
moving an embryo through exclusive use of fluid flow;
rotating said embryo as said step of moving is executed.
The present invention generally concerns handling of embryos. The invention also concerns handling of oocytes (prefertilized embryos). Embryo, as used herein, therefore encompasses oocytes as well as fertilized embryos. The invention more specifically concerns microfluidic handling of embryos for culturing, manipulation, and analysis.
Technology assisted reproduction techniques in which embryos are handled independently from their mammalian biological source are growing in importance and frequency of use. Such techniques have great direct benefit to persons unable to have babies through unassisted sexual reproduction. The agricultural industries also increasingly rely upon such assisted reproduction techniques. Embryo manipulation is used in livestock reproduction to control such things as the faster genetic evolution of cattle and permitting the genetic characteristics of a single exceptional cow or bull to be passed on to far greater numbers of offspring than would be possible through unassisted sexual reproduction.
Livestock embryo manipulation is becoming more routine due to the development of gene manipulation, cloning, and in vitro fertilization (IVF) techniques. The overall goal of embryo manipulation in livestock is to increase production efficiency, especially with regard to reproduction, milk production or production of specific milk components, lean tissue growth with reduced fat content and decreased susceptibility to specific diseases. Embryo transfer is also used to introduce or rescue valuable germplasm and propagate rare breeding animals such as endangered exotic species.
Expense and relatively low success rates place significant burdens on the use of these assisted reproduction techniques for humans as well as livestock. In human reproduction such expense and failure adds emotional as well as economic burdens. In addition, safeguards against failures often result in unwanted or unmanageable multiple births, as well as additional stored embryos which require maintenance and additional difficult decision making at some later point in time. Expense is the primary concern in livestock reproduction.
Failure rates in reproduction techniques as well as testing and other embryo handling techniques are attributable primarily to the significant handling and manipulation of embryos in executing these techniques. Animal reproductive technologies have advanced in recent years, but the physical tools used in animal reproduction have not changed significantly. Fine-bore glass pipets are still one of the basic tools of the embryologist. Using standard petri dishes, procedures such as in vitro maturation of eggs (IVM), in vitro fertilization, and embryo culture (EC) require picking up and placing individual eggs and embryos several times for each procedure.
Such handling and movement from one petri dish to another provides significant potential for damage or contamination. Perhaps more important, though, is the failure of a stationary embryo in a petri dish to simulate the corresponding natural biological reproduction condition. Some efforts have been made to move embryos in petri dishes via agitation of the dish, but this is a haphazard approach. Expense is also created here due to the relatively large amount of biological medium required for the manual petri dish conventional embryo handling methods. Bovine embryos are individually handled with pipets and large, expensive manipulators. Large quantities of biological medium including growth agents for human embryo culturing renders the corresponding in vitro procedure even more expensive. Livestock growth factors, for example, have costs exceeding $200 per 50 μg.
Such static culture systems also fail to allow for changing the milieu in the culture medium as the embryo develops. Current culture systems with flowing medium have culture chambers as small as 0.2 to 0.5 ml. However, the culture volumes are greater than needed and medium is replenished too quickly. The endogenous growth factors that enhance development are diluted out and washed away. The large volumes of medium required substantially increase costs when expensive growth factors, such as IGF-II ($200 per 50 μg) are used. In addition, known systems cannot track individual embryos.
Thus, there is a need for an improved embryo handling device and method which addresses problems in known embryo handling techniques. An improved embryo handling device and method should provide for an improved simulation of natural conditions. It should also provide a building block upon which larger and/or more powerful and accurate instruments may be based, such as embryo culturing systems, embryo analysis systems, embryo storage systems, and similar systems.
These needs are met or exceeded by the present microfluidic embryo handling device and method. The invention simulates biological rotating of embryos. An embryo fluidic channel moves an embryo inserted therein with fluid, and is sized on the same scale as the particular type of embryo or embryos to be handled. The sizing and fluid communication produces a simulated biological rotating of embryos. In addition, the fluid flow with and around the embryo or embryos prevents stagnation, reducing the likelihood of the embryo or embryos developing "bed sores".
The invention also permits the biological medium fluid to be altered gradually, having significant advantages compared to repeatedly manually transferring an embryo from one medium to another medium in a pipet or petri dish. Gradual changes avoid the shock from sudden changes in local environment. The microfluidic system of the invention further permits the co-culturing of an embryo with other embryos, co-culturing of an embryo or embryos with cells upstream of the embryo(s), and maintenance of a separate control culture that shares a common biological medium with a subject embryo(s) thereby ensuring that test embryos see the same environmental conditions as the subject embryo(s).
Other objects, features and advantages of the invention will be apparent to artisans who read the detailed description and reference the accompanying drawings, of which:
FIG. 1 shows a cross section of a preferred microfluidic embryo handling device constructed in accordance with the present invention;
FIG. 2(a) is a top view showing a preferred narrow microfluidic channel constriction for embryo positioning;
FIG. 2(b) is a cross-sectional view of an alternate preferred shallow microfluidic channel constriction for embryo positioning;
FIG. 3 is a perspective view of a preferred gravity flow driven microfluidic culturing and testing device constructed in accordance with the present invention;
FIG. 4 is a block diagram of an embryo analysis device constructed in accordance with the present invention;
FIGS. 5(a)-5(c) illustrate preferred embryo microfluidic channel insertion and removal structures in accordance with the present invention; and
FIGS. 6(a)-6(b) illustrate a preferred culturing device constructed in accordance with the present invention.
The present invention provides a microfluidic embryo handling device which reduces stress to embryos handled outside their natural biological host. The device and method reproduce simulated biological rotating of an embryo through fluid assisted movement in a channel that encourages embryo slipping and rotating. Rotating, as used herein, may include complete rotation or partial rotation. Partial rotation might also be referred to as a rocking motion.
Referring now to FIG. 1 shown is a cross-section of a microfluidic embryo handling device 10 including a embryo transport network 12 formed at least in part by a generally embryo scale channel 14. An embryo 16 in the channel 14 will move with fluid flow in the channel 14, while the close dimensions of the channel cause the embryo 14 to move with a simulated biological rotating motion. In biological hosts, developing embryos in their initial stages of development move toward the uterus to which they will attach with a rotating and slipping motion. The microfluidic channel 14 produces a simulation of such motion.
Sizing of a channel is important to establish the biological rotating. Height is the critical dimension, and it has been found that heights up to about three times the diameter of an embryo induce the rotating. This ratio may be determined to vary somewhat because fluid flow also plays a role, but the three to one maximum ratio has been found to produce the rotating. It will be appreciated that the channel width is less important. The width may be selected arbitrarily. Thus, if embryos are to be kept in single order, then the width would be less than twice the embryo diameter. If more embryos are desired, larger width channels may be used.
Networks of the channels 14 provide a means to culture embryos, as well as to move and place embryos to desired locations. During its initial stages of development, the size of most mammalian embryos remain generally constant during the first few days after fertilization. Thus, the size of the channels 14 provide no impediment to culturing an embryo therein. Advantageously, the embryo 16 may be kept moving and/or may have a continuous or pulsed fluid flow passed around it to avoid potential detrimental biological effects on the embryo 16.
A preferred exemplary construction of a device 10 including a channel is also illustrated in FIG. 1. The microfluidic channel 14 may be formed by any suitable micromachining technique into a suitable material, such as a silicon wafer 18. The material chosen must be capable of being sterilized and should not pose a biological threat to embryos. The channel(s) 14 of the device are sealed through a cover 20. Forming the cover of glass or other transparent material allows convenient visual monitoring of embryos in channel(s) 14. A bonding agent 22 bonds the cover 20 to the wafer 18. Additionally, the material of the cover could be formulated to shield harmful radiation from the embryo(s) in channel(s) 14.
Unlike other cells that tend to float in a fluid medium, the relatively large and heavy embryos sink to the bottom of the microfluidic channels 14. Typical mammalian preimplantation embryos of interest are 90 to 180 μm diameter spheres. In each embryo, a membrane surrounds each cell (blastomere) and the zona pellucida, a glycoprotein membrane or shell, surrounds the entire cell mass. The cells divide several times during the first few days after fertilization, the volume of the embryo remains constant and an egg may be fertilized and cultured to a blastocyst in the same device constructed based upon the principles of the invention. The blastocyst is the final stage before an embryo implants in the uterus.
Also important to production of such a device and similar devices is the ability to handle individual embryos, or small numbers of embryos. Positioning embryos to given locations, moving to alternate locations, and maintaining constant or changing biological conditions around the embryo(s) are abilities provided by basic principles of the present invention, and permit the construction of fertilization, culturing, testing, and other devices which rely on some or all of those abilities. For continuous movement of an embryo through a culture period of time, long channels may be created, or a loop may be formed. Alternately, a parking of an embryo may occur at a culturing station like those shown in FIGS. 6(a) and 6(b). A compartment or channel of limited size may also be used to roll an embryo back and forth therein by changing fluid flow, as will be further discussed with respect to FIGS. 6(a) and 6(b).
Accurate positioning of individual embryos is provided by the invention through the use of constrictions, preferred examples of which are shown in FIGS. 2a and 2 b. FIG. 2(a) is a top view of a cross section of a narrow constriction 24 formed in a microfluidic channel 14. There are many reasons such an accurate positioning may be desirable in an embryo handling device 10. Analysis instruments built into the device may require an embryo to be precisely positioned at electrodes, a photodetector, the focal point of a microscope, or other similar sensing device. Transporting an embryo to the constriction 24 permits such required positioning without resort to feedback systems. An embryo 16 is freed from the constriction 24 simply by reversing the flow of biological fluid medium 30. Even when held at the constriction, an embryo 16 experiences a flow of biological fluid medium around it since fluid 30 will flow past it and through the constriction 24. This is advantageous since an embryo in stagnant fluid has an increased potential to develop "bed sores", a suspected but yet unproven explanation for low success rates in embryo handling technology.
Sidewall portions 28 a, 28 b of the microfluidic channel 14 constrict it at a desired location to prevent passage of an embryo 16 therethrough. The constriction 24 does not completely close the microfluidic channel 14 so that fluid biological medium 30 may pass an embryo 16 positioned at the constriction 24. FIG. 2(b) shows a side cross-section of an alternate shallow constriction 26 where the fluid biological medium 30 is similarly able to pass when an embryo 16 is positioned at the constriction. Other shapes of constriction are also possible. Generally, any shape which prevents passage of an embryo 16 while simultaneously allowing fluid flow through the constriction, e.g., asymmetric shapes and comb-like fibers, is acceptable to position embryos in a device 10 according to the invention. It is preferred that the constriction be sized such that positioning of an embryo prevents the embryo from passing without an increased pressure from the fluid pressure used in a device 10 to move embryos. Constriction length should also be kept small enough to avoid fluid control problems since the constriction portion of a microfluidic channel will have much higher fluidic resistance per unit length than unrestricted portions of the microfluidic channels 14.
A culture and test device 31 including a constriction like that shown in FIG. 2(a) for positioning an embryo is illustrated in FIG. 3. The device 31 has fluid flow in a network 32 of microfluidic channels 14 driven by gravity based upon levels of fluid 30 in a plurality of fluid reservoirs 34. Any suitable means for driving fluid 30 is contemplated as being compatible with the general principles of the invention, e.g. pumping, but the gravity method illustrated in FIG. 3 is preferred for its simplicity and efficiency. Directions of flows are controlled simply by levels of fluid in reservoirs 34. Thus, for example, an embryo 16 held at a constriction 24 for culturing or examination by a suitable instrument is positioned by first setting fluid levels to cause its travel from inlet port 36 to constriction 24, and is released when fluid flow is reversed through the construction 24. Removal of the embryo 16 is accomplished by causing fluid flow to move it to exit port 38.
During movement through the microfluidic channels 14 of the network 32, the embryo(s) roll and slip to simulate natural movement of embryos toward a uterus in a mammalian host, as discussed above. This desirable manner of moving may be aided by a suitable surfactant such as BSA (bovine serum albumin). The surfactant will help to promote some slippage of the embryo as it rolls.
FIG. 3 also illustrates an additional advantage of the invention, in the provision of a parallel additional microfluidic handling and culturing device 31 a. The additional device 31 a has a structure similar to that of device 31, but may have fewer or even a single microfluidic channel. Ideally, the structure is the same. The important feature of the device 31 a is that it shares a common fluid source with inlet port 36 and outlet port 38 of primary device. Embryo(s) handled in the device 31 a are isolated biologically from embryo(s) in primary device 31, but experience the same biological conditions through sharing the same fluidic source, pressure and/or the same biological medium condition. In an exemplary use, the additional device 31 a therefore might form an important control culture in which development or lack of development of test embryo(s) could confirm suitability or unsuitability of conditions created in the primary device 31.
FIG. 4 is a block diagram of an embryo analysis device. In the FIG. 4 device a network 32 of microfluidic channels 14 moves embryos to one or more analysis stations 40 a, 40 b or 40 c. Embryos are positioned at a given analysis station through constrictions like those described above. The analysis stations may include any instrument capable of obtaining information concerning an embryo, with the constriction being formed to position embryos at the proper sensing point for the particular instrument used in an analysis station. Embryos are moved out of the device through one or more exit ports 38 a, 38 b, which might alternately lead to a culturing station in the form of a parking area for an embryo, an additional length of microfluidic channel 14, or a microfluidic channel loop for continuous movement of an embryo during culturing.
Inlet and outlet ports used in devices of the invention may comprise any conventional manner or structure for embryo insertion or removal. However, additional preferred structures for insertion and removal are shown in FIGS. 5(a)-5(c). In FIG. 5(a), a well 42 which is in fluid communication with a microfluidic channel 14 is used. Fluid in the well 42 preferably also comprises a gravity feed which helps drive microfluidic flow in the channel 14. An embryo 16 is placed in the well 42 and moves into the channel 14 with biological medium, or simply sinks unaided into the channel 14 if no flow condition is created. A second similar well 42 may be used to remove an embryo using a pipet 44 or similar device, which might also be used for insertion. In FIG. 5(b), a hanging drop 46 at the end of a channel 14 is used for insertion and removal. The hanging drop 46 is held by surface tension. After embryo insertion, fluid may be added at that point, or the embryo may be sucked in by fluid flow in the device. Alternately, the device may be inclined to promote embryo movement away from the hanging drop 46. In FIG. 5(c), a funnel shaped hole 48 in direct communication with channel 14 is used for insertion and removal. The funnel shape aids positioning of a pipet 44 or similar device. Surface tension at a small diameter hole 48 will prevent fluid from leaking out, but the pressure in channel 14 must not exceed the point that would defeat surface tension and cause fluid to leak out. Inserted embryos will sink into the channel 14, while removal may be accomplished by drawing fluid from hole 48 when an embryo approaches. Of course, any of the FIG. 5 techniques may be combined with each other or conventional techniques for insertion and removal in a given handling device. In addition, the wells 42 or holes 48 may be covered by a removable cover or flap as protection against contamination and/or evaporation.
Referring now to FIGS. 6(a) and 6(b), an embryo culturing device 50 according to the present invention is shown. Fluid medium flow in the culture device 50 is in either direction between a medium inlet 52 and a medium outlet 54. The device includes a number of traps or compartments 56. As best seen in FIG. 6(b), the traps 56 comprise deep regions separated by shallow regions 58. Fluid flow between inlet 52 and outlet 54 is over shallow regions and through deep regions to move embryos back and forth within the deep region compartments 56. Embryos are inserted and removed through access holes 60, which may be formed by any of the preferred methods in FIGS. 5(a) through 5(c). In the device 50, artisans will thus appreciate that embryos may be moved back and forth within compartments 56 to simulate biological rotating, may experience the same medium conditions as other embryos within the culture, and may be easily removed and inserted. Though FIGS. 6(a) and 6(b) show a top loading embodiment for placing embryos within the compartment, the device will also work in a bottom loading arrangement, essentially inverted from that shown in FIGS. 6(a) and 6(b). In such a bottom loading arrangement, the embryos will still be held in the deep portions but cannot pass the shallow portions. An alternate embodiment might comprise a gap in place of shallow constructions where embryos cannot pass through the gaps but fluid flow may occur therebetween and the depth of the gaps may be the same as those of the embryo holding compartments.
Prototype devices like that shown in FIG. 3 have been produced and tested. Typical prototypes are described here for the sake of completeness. Artisans will appreciate that the manner of fabricating the prototypes may be accomplished by any other convention microfabrication techniques. Artisans will also appreciate that production device manufacturing may differ significantly, and that specific numerical dimensions and conditions of the prototype devices do not limit the invention in the breadth described above.
In typical prototype channels, a pressure gradient of 1 Pa/mm causes the medium to flow on the order of 10−10 m3/s (100 nl/s), with an average speed of 1 to 2 mm/s. Under these flow conditions the embryos roll along the bottoms of the channels; traveling at speeds ranging from ⅓ to Į that of the fluid that would otherwise be in the same region of the channel. By manipulating the pressure at the wells connected to the ends of the channels, the embryos can be transported to (and retained at) specific locations including culture compartments and retrieval wells. Embryos fill a considerable portion of the channel, thereby greatly altering the flow of medium. The flow of medium through the channels is laminar.
Networks of prototype microfluidic channels have been fabricated in a device like that shown in FIG. 3 by etching trenches in 3-inch <100> silicon wafers, and then bonding glass covers to form channels. Typical channel networks contain several branch microfluidic channels that intersect near the center of the device. The branches, which range from 1.5 to 2.5 cm in length, are 160 to 200 μm deep and 250 to 350 μm wide at the top. A first step in producing prototype devices involves patterning silicon nitride (SiN) coatings on using conventional photolithography techniques. The microfluidic channels are anisotropically etched with a potassium hydroxide (KOH) solution. Access holes in the glass covers are drilled, either conventionally using carbide tipped bits or ultrasonically. Glass covers are bonded to the wafers using UV curable epoxy (NOA 61, Norland Products, Inc, New Brunswick, N.J.) or Pyrex 7740 covers are anodically bonded to the wafers using 500V in a 450░ C. environment. The nitride coatings are removed using buffered oxide etchant (BOE) before anodic bonding. Glass wells are bonded to the glass cover at the end of each branch of the channel network with either an epoxy (Quick Stick 5 Minute Epoxy or 5 Hour Set Epoxy Glue; both from GC Electronics, Rockford, Ill.) or a silicone adhesive (RTV 108 and RTV 118 from General Electric Co., Waterford, N.Y., or Sylgard« Brand 184, Dow Corning Corp., Midland, Mich.).
In the prototype devices, constrictions like those in both of FIGS. 2(a) ("narrow") and 2(b) ("shallow") have also been fabricated and tested. Channels with "narrow" constrictions, as shown in FIG. 2(a), can be fabricated using a single mask and etching operation. Channels with the "shallow" constrictions, as shown in FIG. 2(b), require two masks and two etching operations.
All the component materials of the prototype devices except the five minute epoxy were tested for embryo biocompatibility. In applying the present invention, artisans will appreciate that alternate materials may be used from those selected for the prototype devices, but biocompatibility must always be established through prior data and/or testing. Although many materials are known to be compatible with or toxic to certain cells, little work has been done to investigate the compatibility of materials used in micro fabrication with embryos. The materials selected may also vary depending upon the type of mammal from which the specific embryos to be handled are taken.
In prototype testing, two-cell mouse embryos (B6SJL/F2) were randomly assigned to and cultured on the substrata, in medium M16 (Sigma, St. Louis, Mo.) with bovine serum albumin (BSA; 4 mg/ml; Sigma), covered with mineral oil (Sigma). All embryos were cultured at 37░ C. in a 5% CO2 in air atmosphere for 96 h. Developmental rates of embryos were examined every 24 h. The percentage of embryos that reached the blastocyst stage for each material was compared with the percentage from the control group. Mouse embryos that reach the blastocyst stage, the latest possible stage before embryo transfer, are probably not developmentally hindered. While the absence of negative effects is not guaranteed unless the embryos are also transferred to recipient mice and monitored until the offspring are born, tests are commonly concluded at the blastocyst stage for practical and economic reasons. Most of the materials tested proved to be compatible with the mouse embryos, including silicon wafers, SiN coatings, NOA 61, and RTV 118. Some materials, such as the 5-minute epoxy, have not been tested since it is only used in conceptual devices to demonstrate mechanical and fluidic principals of the invention, and would likely not be used in production devices.
Tests were run to examine several aspects of the prototype devices. Different tests required devices with different channel configurations. In all the tests, a halogen bulb via optical fibers illuminated the channel, which was viewed under a stereomicroscope. A graduated cylinder and a stopwatch were used to determine flow rates. Since the fluid is incompressible, the average fluid velocity in any section of channel is just the flow rate divided by the cross-sectional area.
Measurements of the rate of travel of the embryo for a given flow rate occurred in a simple straight channel, 29 mm long, 162 μm deep, and 160-380 (bottom—top) μm wide. The pressure gradient was varied and the speed of the embryo was measured for each setting. The channels were filled with phosphate buffered saline (PBS), with and without BSA. Flasks of the medium were connected to the channel. By adjusting the heights of the flasks, using micrometer head translation stages, the pressure difference was finely tuned to within 0.05 Pa. The flasks were connected to each other by tubing between the tests to zero the pressure head. The microfabricated prototype devices were cleaned in a hydrogen peroxide/ammonium hydroxide/deionized water solution and new pipet tips were adhered with epoxy before the tests were conducted. All the tests using PBS without BSA were conducted before those with BSA. Once the channels were filled with medium the mouse embryos were placed in the inlet well, at the channel entrance.
Tests were run to observe the influence of channel size and shape on the transport of embryos. For these tests, a device was fabricated with one long, circuitous channel with 11 sections each at one of four depths: 140, 164, 194, and 210 μm. At each depth the channel has 2 or 3 different widths. Widths, measured at the surface of the wafer, range from 275 to 480 μm. In the narrowest segments, the embryos were geometrically constrained to travel on a V-groove while in the other regions along a flat-bottomed channel. The speed of travel and rotating characteristics were observed and compared for different segments.
Observations of embryos at constrictions occurred in several devices, with both narrow and shallow type constrictions. Embryos were actually directed to specific constrictions. Altering the height of the medium in each well, by adding or subtracting fluid, tailored the pressure gradients in each branch of the channel network. Pressure heads were adjusted by a 1 to 8 mm (10 to 80 Pa).
Just as embryos placed in medium sink to the bottom of the container, embryos placed in microfluidic channels settle to the bottom. In all the tests, when the medium flowed, the embryos rolled and slid along the bottom of the channel in the direction of flow. Often they also remained in contact with one of the side walls of the channel. In initial tests without any surfactant in the medium (phosphate buffered saline) the embryos appeared to roll without slipping along the bottoms of the channels. Embryos slid or rolled with slip along the bottoms in later tests when the medium contained BSA (4 mg/ml).
Tests revealed that the rate of travel of an embryo in a channel depends upon the velocity of the medium. Sometimes they stick to the bottom of the channel when the velocity of the fluid around them is below 50 μm/s. For both media, PBS and PBS/BSA, a pressure gradient of 0.16 Pa/mm drives the flow through the channel at an average velocity of approximately 380 μm/s. The embryos rolled at 187-250 μm/s, 49 to 66% of 380 μm/s. As the medium flows more quickly, the embryos roll faster, slipping as they roll. The actual speed of travel and the tendency to stick varies from one embryo to the next One embryo has been observed to travel 25% quicker than another at the same time in the same channel, in almost the same path line. In the observed range, 150 to 1000 μm/s, the velocity is linear with pressure gradient.
Results from testing the effects of channel size and shape match a priori predictions. For a given flow rate, the average fluid velocity and embryo speed is greater in a channel with smaller cross-sectional area. In contrast, for a given pressure gradient, the average fluid velocity and embryo speed is greater in a channel with larger cross-sectional area. In both cases, embryos travel slower on V-grooves than on flat-bottomed channels. Embryos are also more likely to become wedged and stuck in a V-groove than on a flat-bottomed channel.
Fluid under electroosmotic flow also caused embryos to roll through channels. An embryo rolled along the channel bottom at approximately 10 μm/s due to the pressure driven trickle flow. Switching on the voltage caused the mouse embryo to roll along the channel bottom 20 μm/s faster, at approximately 30 μm/s, toward the well with the negative electrode. With the voltage polarity reversed, the embryo rolled at approximately 10 μm/s in the reverse direction. No surfactant, such as BSA was used so there was little or no slipping. Electric assistance was determined to be undesirable due to its heating of the medium.
Computational fluid dynamics modeling using Fluent/UNS 4.2 (Fluent, Inc., Lebanon, N.H.) and 2-dimensional finite element analysis of prototype microfluidic channels with constant cross-section using Quickfield (Tera Analysis, Inc., Tarzana, Calif.) verified the observed flow rates and flow patterns. The embryo was modeled as a rigid sphere. Recall that the embryo does not appear to deform under typical conditions. To analyze the laminar flow, 1 or 2 mm sections of channel were meshed into 10,000 to 30,000 tetragonal elements. Once verified, computer modeling was used to determine flow velocity profiles, design constrictions with lower pressure drops, to observe forces on embryos retained at constrictions, and to analyze electrically driven flows in similar channels. However, analysis incorporating adhesion of the embryo to the channel walls and distortion of the embryo would be significantly more complex and was not attempted.
As discussed above, in the straight channel tests of embryo velocity, the medium had an average velocity of 380 μm/s under a pressure gradient of 0.16 Pa/mm. Finite element analyses determined the centerline velocity to be 815 μm/s under these conditions. When traveling in the channel, the embryo was tangent to the bottom and one wall. Consider a 100 μm diameter circle tangent to the bottom and one side of the channel. The average velocity of the fluid traveling through this circle when the embryo is not present is 480 μm/s. However, the embryos rolled at only 187-250 μm/s, 39-52% as quickly, in both PBS and PBS/BSA media. The velocity profile encourages the embryo to roll forward and along the wall, which confirms visual observations. In sum, embryos roll at ⅓ to Į the speed at which fluid would flow in the same region of the cross section.
The constrictions greatly increase fluidic resistance in the channels. Standard analytical formulas can help approximate the resistance, but the cross-sectional shapes of the constrictions vary with position. Three-dimensional models of the constrictions were analyzed before masks were designed and wafers were etched. The information gained from the finite element analyses led to optimally-sized constrictions. The shallow constrictions, sized individually for the geometry of the device, balance the need for minimal flow resistance and robust fabrication. Typical constrictions have a minimal depth of 20 μm.
Studies of the placement of the embryos at the traps reveal lateral forces on the order of 10−8 to 10−7 N force the embryo to the side and part way up the ramp at the entrance to the shallow constriction.
The tests revealed several interesting characteristics of microfluidic transport, such as variations in velocity between embryos and the tendency to roll along the bottoms of the channels, often tangent to a side wall. However, the testing did reveal several other issues, Electrical control of fluid flow was investigated initially, but the high voltages harm the embryos in several ways. Even with the embryos in sections of channel away from the electric fields, the applied energy heats up the medium (Joule heating) beyond physiological temperatures and the electrolysis products alter the pH. Note that an embryo requires about 0.029 Osmol, i.e., a relatively high conductivity. Also, EOF is degraded in channels with surfactant, but the embryos survive better in medium with a surfactant, such as BSA.
Microfluidic transport free of electrical assistance offered through gravity fed devices like that in FIG. 3, or through pumped fluid pressure devices, offers an important advantage. The medium can be easily altered with time to meet the changing requirements of the developing embryos. Gradually changing the composition of the medium avoids inducing stresses upon the embryo from the abrupt environmental changes that often accompany transfer from one petri dish to a second dish with a different medium. The microfluidic handling of embryos by the invention is not physically harsher than transfer with pipets and definitely less damaging than many techniques in conventional practice including some which pierce the outer membrane.
It is anticipated that control of fluid flow, and therefore embryo positioning, in handling devices like that shown in FIG. 3 will be handled through programmed control instruments for largely automated devices. Alarms and warnings may be incorporated based upon sensed conditions within an embryo handling device of the invention. In similar fashion, monitoring of embryos with conventional instruments applied to a handling device of the present invention. Artisans will generally recognize that the microfluidic embryo handling device thus forms a basic building block upon which many useful devices may be based, and that such devices will incorporate the essence of the present invention.
While various embodiments of the present invention have been shown and described, it should be understood that other modifications, substitutions and alternatives are apparent to one of ordinary skill in the art. Such modifications, substitutions and alternatives can be made without departing from the spirit and scope of the invention, which should be determined from the appended claims.
Various features of the invention are set forth in the appended claims.
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Regional Groups
VSA members also participate as members of regional superintendent groups. There are five such groups. They are:
Champlain Valley
Winooski Valley
Each region elects a representative to the Board of Trustees. Terms of office are two years with Champlain and Winooski Valley groups electing in odd-numbered years and the Northeast, Southwest and Southeast regions electing in even-numbered years.
Each regional group meets regularly and elects its own officers and establishes its own operational procedures.
Contact current regional presidents listed below for information on when their respective group meets.
Champlain Valley: Elaine Pinckney, Chittenden South Supervisory Union and Judy DeNova, Chittenden Central Supervisory Union
Northeast: Steve Urgenson, Orleans Central Supervisory Union
Winooski Valley: David Bickford, Orange-Windsor Supervisory Union
Southeast: Ron Stahley, Windham Southeast Supervisory Union
Southwest: Joan Paustian, Rutland Southwest Supervisory Union
For more information concerning the Vermont Superintendents Association regional groups, contact Jeff Francis, Executive Director.


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Car flips, driver taken to hospital
Oct. 04, 2013 @ 05:10 PM
A single-car crash Friday on Raleigh Road near Henderson flipped a car on its side and sent the driver to the hospital with what appeared to be non-threatening injuries.
Responders had to cut the roof off of the silver Chevrolet Cobalt in order to safely remove the driver. According to State Trooper Joseph Thomas, the northbound Cobalt flipped after leaving the roadway to the right and hitting a ditch in the yard across from First Street.
"She was pinned inside," Thomas said.
Thomas said that witnesses he interviewed indicated the Cobalt crashed on its own.
"Witnesses said they saw only one vehicle," he said.
Contact the writer at firstname.lastname@example.com.


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Researchers in the news
Could this be cure for negative campaigning?
The St. Catharines Standard, June 10, 2015
Brock University researchers win Ontario government funding competition
97.7 htz fm , June 9, 2015
Are school dress codes sexist and oppressive?; Thanks to social media, girls in skimpy clothes are the new champions of women's rights
Globe and Mail, June 9, 2015
A Group Of Teens Are Trying To End "Oppressive" Dress Codes In Toronto
Buzzfeed, June 8, 2015
FIFA and the double-edged sword of corporate sponsorship
Globe and Mail, June 5, 2015
Part-time jobs a place for young workers to learn - and get hurt; Haskayne research show teenagers vulnerable to injuries, have little voice in their short-term workplaces
Globe and Mail, June 5, 2015
EDITORIAL: Kinder, gentler recess times
Niagara This Week, June 3, 2015
New research shows 'a little thirst isn't the end of the world'
CBC, June 3, 2015
Forgive and feel better
United Church Observer, June 1, 2015
WATERLOGGED; We tote water bottles to the gym, strap them to our waists, and sip from them at our desks.
Globe and Mail, June 1, 2015
Taking on school dress codes: Teen rebels with a cause
Globe and Mail, June 1, 2015
Early 20th century immigrants faced discrimination
St. Catharines Standard, June 1, 2015
Accursed blackflies only doing their job; While they're eating you alive, think of the good those clouds of critters provide, keeping the food chain intact
Globe and Mail, May 30, 2015
Dress-code rebels send a message; Protests may be sign of social change, experts say
Times Colonist (Victoria), May 30, 2015
Addition of women's teams in FIFA 16 a huge boost for female sport
the roar, May 29, 2015
Toronto: city of losers
University of Western Ontario Gazette, May 29, 2015
Dress codes have valid purpose: administrators; Say attire must not deviate from school's mission
National Post, May 28, 2015
Also in the Edmonton Journal, Ottawa Citizen, Windsor Star
Seeking help in deciphering child thinking
The St. Catharines Standard, May 28, 2015
Brock prof seeks sleep study volunteers
Bullet News Niagara, May 29, 2015
Experts say teens' push against dress codes could be a sign of social change
weyburn review, May 29, 2015
What is Niagara's role on world stage?
The St. Catharines Standard, May 27, 2015
Recess play promotes empathy, combats bullying
Niagara This Week, May 26, 2015
Toolbox: How Important is Hydration?
pezcycling news, May 26, 2015
Canada AM | Video - Watch Canada's Most Watched National Morning News Program - Canada AM
canadaam.ctvnews.ca, May 23 2015
Also: CTV - CANADA AM, May 22, 2015
Daybreak May 23: Stephen Cheung - Daybreak Alberta - CBC Player
CBC, May 23, 2015
Is the Ontario Federation of Labour in trouble? by am900chml
soundcloud.com, May 23 2015
Are we over the size-zero model?
The Globe and Mail, May 22, 2015
Canada is a nation, experts say; PQ disagrees
National Post, May 22 2015
Seeking help in deciphering child thinking
Thorold Niagara News, May 21 2015
Coach Quietly Lays Off Its Canadian Part-Time Workers
Huffington Post Canada, May 21 2015
Average-Size Models Could Be Better For Business, Study Says
Huffington Post, May 14 2015
Fired workers caught in tangled web of loopholes; THIRD OF FOUR PARTS Ontario's outdated employment laws don't give anyone protection from wrongful dismissal
Toronto Star, May 18 2015
Sixty years of Eurovision
abc online, May 19 2015
Don't sweat feeling thirsty; A study finds that athletic performance, to an extent, is not unduly affected by dehydration
The Globe and Mail, May 15, 2015
How much do you need to drink – On hydration and performance
Canadian Running, May 19 2015
Hydration and performance: How much do you need to drink?
Triathlon Magazine Canada, May 19 2015
You can ride harder than you think when dehydrated
road.cc, May 20 2015
Study debunks water myth
The St. Catharines Standard, May 15 2015
Eurovision Song Entries Carry Hidden Political Messages -- Maybe
Groong, May 17 2015
School dress codes 'demeaning' to both sexes
CBC.CA News, May 14 2015
Walkerville Brewery recycles bourbon barrels for beer
Windsor Star, May 13 2015
What good are GO trains without regional transit?
The St. Catharines Standard, May 13 2015
Working fathers are going 'all in.' Inside the daddy wars
Chatelaine, May 12, 2015
Sports drinks don't help athletic performance, Canadian study suggests | HEALTH
Toronto Sun, May 12 2015
This article ran in more than a dozen publications all across Canada. Other media outlets include:
New Brunswick Telegraph-Journal, Edmonton Sun, Sudbury Star, Belleville Intelligencer, Orillia Packet and Times, Brockville Recorder and Times, Barrie Examiner, Welland Tribune, St. Catharines Standard, Owen Sound Sun Times, Northumberland Today, Pembroke Observer, Chatham Daily News, Kirkland Lake Northern News, simcoe.com, yorkregion.com, insidehalton.com, mykawartha.com, nsideToronto.com, Grande Prairie Daily Herald-Tribune, Fort McMurray Today, Stratford Beacon-Herald
Leading off: Down the drain
National Post, May 12 2015
Dehydration does not impair athletic performance: study
The Hamilton Spectator, May 12 2015
Articles also in: mississauga.com, Truro Daily News (NS).
Hydration not connected to performance for athletes: study - Latest Hamilton news, CBC Hamilton, May 12, 2015
Dehydration during sporting events doesn't hurt performance, study finds
newstalk 1290 cjbk - Mon May 11 2015
Is your young child forgetful? Brock University is looking to find out why.
am900chml, May 11, 2015
The daddy wars; Fathers are the unseen victims of the work-life crunch, a growing group argues. And it's time to fight back
Macleans, May 11 2015
Psychopaths feign emotion in order to take advantage of others - Study
sott.net, May 10 2015
Why the gender wage gap persists; Pay-equity legislation hasn't completely solved the differences, as some employers still favour men over women for promotions
Globe and Mail, May 8, 2015
Children needed by Brock researcher - Erie Media | Erie Media
Erie Media, May 8 2015
Brock researcher examines children's memory, seeks participants
1057 ezrrock, May 6 2015
Wages for U.S. retail workers begin to climb
Toronto Star, May 6, 2015
Book profiles hidden powers at city hall
Pelham News (and other Sun Media papers), May 6 2015
Asexuality: The Invisible Sexual Orientation That's Very Real
yahoo! News, May 4 2015
Brock University marketing professor Kai-Yu Wang published research that shows millennials, the fashion industry's target consumers, find average size models just as, or even more attractive than the high fashion size zeros. Aired on CHCH TV.


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A/C repairs cause sticker shock as coolant costs soar
Price of R-22 Freon skyrockets as it's phased out
SAN ANTONIO - When Kim Crawford called her air conditioner repair company to top off her Freon because her unit was leaking, she was shocked at the price.
"I got new Freon, and it was twice as expensive as it was just a year ago," she said.
Her old air conditioner uses R-22, the same type of Freon most air conditioners more than two years old use. Now the coolant is one hot commodity. The cost is triple what it was just six months ago.
Air conditioning companies are now having to pass along the increased cost to their customers.
"A lot of customers are surprised," said Ryan McLean of Shafer Services.
Refrigerant leaks are common problems with aging air conditioners. But where it used to be no big deal to add a pound or two, now it's a big costly deal.
"You find it anywhere from $250 to $500 is what people are charging to put in two to three or four pounds of Freon," McLean said, depending on what the company paid for it.
Why the price jolt?
The Environmental Protection Agency is phasing out R-22 because it depletes the ozone. The regulation that goes back to 1987 calls for 90 percent of R-22 to be phased out by 2015 and to be virtually obsolete by 2020.
The government has not yet announced how much R-22 will be available this year, causing the sudden spike in prices. "The price tripled overnight," McLean said.
A newer Freon type, R-410-A, is more environmentally friendly.
Homeowners can determine if their air conditioner is affected by checking the sticker on the unit. It will have R-22 written on it.
Units that take the R-410-A have a pink sticker.
The phase-out means homeowners are left to weigh their options: repair or replace?
"Once you reach a point where you're having to put Freon in all the time, it's a good choice to go ahead and replace the equipment.
Crawford has been delaying the expense of a whole new air conditioner, but expects the time has just about come.
"It's kind of like an old car. You put into it and put into it. Just buy a new car," she said. "It becomes silly to keep putting money into it."
For a list of recent stories Marilyn Moritz has done, click here.
Copyright 2012 by KSAT.com All rights reserved. This material may not be published, broadcast, rewritten or redistributed.


text:
Build Your Royalty Free Music Library
When you buy royalty free music from SmartSound, it's always available for you to customize and download directly from our web site or by installing it on your workstation for access with our Sonicfire Pro software.
Save Time With Our Patented Software
Our Professional Soundtrack Creation Technology automatically delivers professional music edits instantly. Adjusting the length, arrangement and instrumentation of every SmartSound track is instantaneous and 100% professional!
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SmartSound technology (Sonicfire Pro) allows you to express your creativity far beyond regular royalty free music. Since our music is totally customizable for length, mood and instrument mix, you can create over 20 million different soundtracks to find that perfect fit for your project.
Impress Your Audience
Our royalty free music library is comprised of a hand-picked and professionally curated collection of music. Our music comes from top musicians, film composers, indie bands and independent artists. No sifting through thousands of crowd-sourced tracks. Every SmartSound track is a winner!
Use Legal Music
SmartSound offers one of the most generous worry-free music licenses in the industry. Put SmartSound in your (or your clients) productions and play it anywhere for any amount of time at no additional cost. (*If you need to manufacture more than 10,000 pieces of physical media, you'll need to contact us for approval...that's it!)
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Add light touches of accompaniment with featured solo instruments. Sophisticated piano, blues guitar, tender oboe, and more, give Solo Simplicity a diverse range of musical vignettes. Ideal for intimate and personal moments needing sensitivity and a quiet touch.
(All tracks include unlimited access to Full, Broadcast and custom lengths with a commercial license)
Album Tracks (18 tracks on album)
- 1 After Hours
- 2 April's Dance
- 3 Black Water
- 4 Blue Water
- 5 Emaline
- 6 Embers of Amber
- 7 Hometown
- 8 Magic Hat
- 9 Noble Talent
- 10 Rabbit Tracks
- 11 Rockin' Chair Blues
- 12 Root Bear
- 13 Shamrock Jig
- 14 Sweetwater Country
- 15 True North
- 16 Twilight Encounter
- 17 Urban Rhythm
- 18 Warm Wood
Search Criteria
Choose your license:
|Customizable with Sonicfire Pro and Quicktracks
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|Display at Film Festivals
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(one theater)
(unlimited theaters in one country)
|Assign Distribution Rights
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To view individual tracks select from the album track menu to the left


text:
Posted 2 years ago
By Post Staff
NEW YORK (AP) — A judge has ordered Mindy McCready to get help for mental health and alcoholism.
People magazine reports during an emergency hearing Wednesday, McCready admitted she's drinking too much because of the death of her boyfriend, songwriter David Wilson.
He died three weeks ago. McCready's two children have been put into foster care.


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|Baked Sweet Potato
I had planned for this sweet potato to be part of my Easter Ham post but I forgot to take pictures of the ham during cooking and pretty much ruined it, I had a catchy title and everything all worked out. Little did I know, that the humble little sweet potato would end up stealing show and becoming the true star of the meal.
I started by taking a nice sweet potato and slicing it about 3/4 way through the potato about 1/4 inch apart. Then I sliced a fresh green apple in to thin wedges and placed the wedges between the cuts in the potato.
|Sliced Sweet Potato & Green Apples
Place the potato & apples on sheet piece of foil, drizzle with a little vegetable oil, or if you feel like living dangerously use some butter. Then I topped with pecans, craisins and a mixture of brown sugar my BBQ Rub, 2 parts brown sugar to 1 part rub. Wrap up the package and place is on the grill or smoker.
|Sweet Potato Baking Package
I was smoking a ham at 250º, so I cooked the sweet potatoes for about 2 hours. Cooking it on the grill at 400 will take about an hour. Once it's done remove the sweet potato and dump the contents of the foil pouch on top, be sure to get all those nuts, craisins and the spicy brown sugar sauce on the potato.
|Sweet Potato Pack hot off the smoker
In retrospect the baked sweet potato was the highlight of the meal. The potato was soft and had absorbed all the flavors from the apple, craisins and brown sugar rub. The cooked green apples paired nicely with the potato and the pecans brought a contracting crunch to the party. By far the best flavor came from the craisins, during the cooking they plumped up with all those amazing flavors floating around in the bottom of the pouch. It was so dang good I forgot all about the ham pictures.


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- Home
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- Shoe Brands
- Report Shoes
Report Shoes1 Items Found
Report Harmoni Boot
$80.00 $20.00 - $28.00
Report Shoes
Belk Report Shoes
Report Shoes are great wardrobe staples for shoe lovers looking for quality and style. Step out of your house in a bold, eye-catching shoe. It's time to step up your style a few notches, and Report footwear is going to help you achieve a higher standard. The Report Shoes shop is filled with a variety of merchandise to cater to all of your fashionable needs. Find wedges, flats, sneakers, and sandals in this diverse collection of shoes. It is never hard to find something for your day to day look in this assemblage, and shoes are so comfortable that you can leave them on all day. Buy Report Shoes if you are looking to sport a fabulous pair of shoes. Never be afraid to experiment with your style. Report Shoes will give you the style and the comfort you deserve and enjoy.


text:
Do you have questions about this product?
get answers from real customers and in-house experts with AnswerBox.
Q:what is the term to lease 2 HOSHIZAKI HNC-150BA-R-S AND HOW MUCH
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A:We just launched a financing special that is good through the end of the year (12/31/2012)! It is a 13 month lease-to-own option valid on equipment purchases over $3,000.
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Yulee attractions are listed below. Find things to do in or near Yulee, FL for your upcoming individual or group travel for Yulee visitors. We also offer the great discounts on Yulee Florida hotel and motel rooms.
Reserving group travel? Save with Yulee Group Rates
Attractions + Things To See in Yulee
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Home 1902-3 Curved Dash Oldsmobile Return
Model R
Year - 1902-03
Production in 1902 - 2500 (approximate)
Production in 1903 - 3924 (approximate)
Serial Numbers in 1902 - 6425 thru 9999 (not sequential)
Serial Numbers in 1903 - 10000 thru 19999 (not sequential)
The 1902 Model R CDO is a continuation of the 1901 car with a brake installed inside the differential, somewhat ineffective, but making the car legal in France. Truss rods are fitted initially to the front and later to the rear axles and front & rear fenders added, maybe as an option. The body was all black except for the trim areas, painted a bright red. The red is more of a cherry color. Following a carefully study of several fairly original vehicles, the pin striping in the sketch is believed to be correct. The sketches show the design of the transmission band adjustment mechanism. An excellent picture of the chassis without a body is available and shows all the principle elements of the vehicle. At the time of production a continuing problem was the spring that connects the tiller to the steering arms. It failed several times on the cross country trip of Whitman and Hammond. It was changed yet again on the 1904 Model 6C.
Differences between 1902-03
There was a continuing effort by Oldsmobile to improve the CDO during 1902-03. For convenience the CDO Club has given the Model R a suffix denoting the year it was built, however these must be considered as very flexible dates, we believe the changes were ongoing through the entire three year period. Thus the CDO for 1901 has been called the "R1" and the differences between it and the next two years were quite extensive. However the differences between R2 (1902) and R3 (1903) are much more subtle. The R2 had rubber bumpers which hold the body to the frame. The R3 body is bolted directly to the "L" brackets which are riveted on the out side of the frame rails. The R2 has a split in the rear frame member which formed a hole for the top of the chain to pass through. The R3 has a rear frame section which is bent upwards over the top of the chain, no hole. The R2 does not have fins on the lower cylinder block, while the R3 has large fins cast into the cylinder block. The R2 uses a centrifugal water pump coupled to the end of the crankshaft, while the R3 has the gear pump connected to the camshaft. The R2 has the "screw together" differential housing, (carried over from 1901) while the R3 has either a pressed or cast differential housing. The R2 uses block chain and the R3 conventional roller chain. The R2 has a fibre block to hold the timer spring and the R3 a brass frame. The R2 has a decompression lever mounted to brackets riveted to the frame while the R3 uses a compression release pedal the worked up and down like a plunger, bolted to the floor boards.
The water tank is on top of the gas tank and the oiler mounted in the hole over the cylinder. The radiator has fins with 38 3/4 inch brass tubes with a capacity of 2 gallons. The water jacket is now removable and 4-1/8' long. The valves are 1-1/8" diameter with a stem diameter of 3/8".
Some models had wooden spoke wheels when the supply of wire wheels were exhausted. Wheels on the model R were wire and wooden, both clincher and tubeless. The wooden wheels were built with both 12 and 14 spokes and some metal artillery wheels were also offered in late 1903 as well as a poorly designed staggered wooden wheel manufactured by Pruden that caused a number of warranty problems.
Rear Axle
The rear axle continued with the "small" differential housing but now had a transmission brake installed. The foot brake operates on the transmission bands.
The gear change continues to be with a lever at the drivers right hand side. The operation of low speed is quite tricky, as it is meant to operate only when going from neutral to low and then to high. When downshifting, the mechanism should NOT engage low band. If this mechanism fails, it will eventually destroy the transmission.
Most of the CDO Model R cars were built with the "Mixer" carburetor. During 1903 this was replaced with a Holley design, which used a now conventional float to maintain the gasoline level.
Selden Patent Plate
The Selden Patent plates have been investigated by Tom Reese of Mound MN and the following are the patent numbers he has found on various cars:
Year Patent Plate Number Make
1903 1946 Cadillac
1903 2359 Elmore
1903 2363 Knox
1903 3560 Pope-Hartford
1903 3640 Pope-Tribuine
1903 7031 Orient Buckboard
1903 10131 Oldsmobile
1904 5975 Cadillac
1904 8121 Cadillac
1904 10218 Oldsmobile
1904 11098 Cadillac
1904 12394 Oldsmobile
1904 14000 Oldsmobile
1904 14260 Oldsmobile
1904 14960 Oldsmobile
1904 16295 Pope
1904 21082 Oldsmobile
1904 21274 Oldsmobile
1904 21620 Oldsmobile
1904 21624 Oldsmobile
1904 22613 Oldsmobile
1904 22618 Oldsmobile
There is no known reason the the 1903 Oldsmobile has a higher number than the
1904 Cadillac. It may be that blocks were assigned to the manufacturers.
Coulter Carriage Supply Company at PO Box 276 Cokedale CO (303 848 9678) has all the
material to add or repair the top on your 1901-03 CDO.
Items required are:
Qty Catalog # Description
2 1507 Top Props
2 1508 Top Props
2 1903 Wood Bows
8 1506 Prop nuts
1 1500 Flex Joints
1 1510 Shifting Rail
2 1502 Socket set
1 0307 Curtain window
This is an original picture of a CDO, the passengers were Allen and Eva Firestone.
It appears to be 1902 model with 12 spoke wheels, clincher rims, fenders, truss rods front & rear, no external brake and no visible radiator. It appears that the CDO has a horn rather than a bell, maybe a 1902 but could well of been taken a little later.
The lady is in the driving seat, named Eva Mae Firestone, but it doesn't appear that she is actually driving the car . She married Oscar Mummert, when she was a bit older of course. Her father is sitting next to her. His name is Abner Allen Firestone although he went by Allen. He is a first cousin of Harvey Firestone, founder of Firestone tires. In fact, Harvey attended Eva and Oscar's wedding and his son, Harvey Jr. drove them to the train station for their honeymoon.
The picture is reported to have been taken in Alliance, Ohio.
Updated 7 March, 2010


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When you come to the Academic Counseling and Advising Center (ACAC), you will first meet with an Academic Counselor to assess your academic progress. Together you will develop an individual improvement plan to assist in your academic progression. You will have on going contact with our staff of Academic Counselors, Coaches and Advisors until your grades improve.
Click here for more information on College Success Factors.
Students who are experiencing more long-term issues and difficulties, including but not limited to: major depressive disorder, anxiety disorders, eating disorders, learning disabilities, abusive relationships, suicidal ideation, and other difficulties that cannot be resolved through the problem-solving model will be referred to other resources for assistance. There may or may not be a cost associated with those services.


text:
The race for the District Three County Commission seat between Republican Chris Alexander and Democrat Tim Case proved to be the most clear cut local race out of the three taking place in Elbert County.
Chris Alexander handedly won his district with 1,023 votes compared to Tim Case's 580 total votes. Alexander claimed nearly 64% of the total vote, while case managed to just over 36%.
Alexander said he was grateful for the voters of District 3.
"I just would like to thank all the voters in District 3. I am encouraged by the victory, and I look forward to working for them", said Alexander.
Tim Case could not be reached last night for his comments on the election results.
Alexander will take over for current District 3 Commissioner Frank Eaves, who chose not to run for re-election after this most recent term.


text:
Resumen disponible en
FIG. 1 is a left side elevational view of a shoe sole showing my new design, the broken lines showing a shoe upper being for illustrative purposes only and forming no part of the claimed design;
FIG. 2 is a top plan view thereof;
FIG. 3 is a right side elevational view thereof;
FIG. 4 is a front elevational view thereof;
FIG. 5 is a rear elevational view thereof;
FIG. 6 is a bottom plan view thereof;
FIG. 7 is a cross-sectional view taken on line 7--7 of FIG. 6;
FIG. 8 is a cross-sectional view taken on line 8--8 of FIG. 6; and,
FIG. 9 is a cross-sectional view taken on line 9--9 of FIG. 6.
The broken lines are for purposes of illustration only and form no part of the claimed design.
Imagen original