ZipSnip All Purpose Cutter
ZipSnip All Purpose Cordless Cutter
Say goodbye to your scissors!
The folks at ZipSnip recently sent me a ZipSnip Lithium cutter to review and is also offering one to one of my readers. Give away offered later this week.
The ZipSnip is a 3.6V Lithium-ion cordless cutter that can cut through all types of materials.
The ZipSnip felt comfortable in my hand, it’s light, weighing on ly one pound. The top mounted safety / power switch and lower trigger offer safety protection and are easily reached by using your thumb and forefinger and a cinch to use.
After charging the ZipSnip for 5 hours I tested it out in my shop and around the house.
I tested the ZipSnip on aluminum and nylon window screen, old t-shirts, leather, plastic packaging, corrugated board, rubber roofing, 14 ga. wire, a magazine and bamboo screening. [see video below]
The tool cut everything easily leaving a clean cut line. It also cut curves well.
One neat thing that I noticed is that you can cut up close to an obstruction or corner where ordinary scissor can not.
- Cutter is 1½-in.-diameter, hardened steel blade is self-sharpening and replaceable.
- A built-in cutting guide ensures precise straight or curved cuts.
- The blade operates at 260 rpm (no load). Its maximum cutting thickness is ¼ in.
- 3.6-volt, cordless cutter is powered by a 1.4 Ah LithiumTechTM battery. An indicator light on the back of the handle shows when the battery is fully charged. Lithium technology enables the cutter to hold a charge for up to 24 months.
- 3-5 hour charger is included.
I was impressed with the Zip Snip cutter, it works exactly as advertised. Having the cordless tool is a nice touch as is the fact that the batteries are Lithium -ion.
My only comment to the company would be to make this tools safety switch a bit more challenging to operate. I could see a youngster operating this tool without much fuss. Although my kids are older, I tested this theory with both my 10 and 12 year old. Both kids picked the Zip Snip up and operated it with out instruction.
Where To Buy:
ZipSnip 3.6V Lithium-ion Self-Sharpening Cordless Cutter (#RC2602, $39.99) is available at Menards, and regional hardware and home centers, and online at www.zipsnipcutter.com.
This review contains my opinion of a product. I take pride in providing my readers with an honest and objective information as well as a practical approach to using a product. I never accept payment in exchange for a positive review. Many of the tools and products are provided to for free by a manufacturer for review.


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domain: design


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Les Minquiers 3 Hour Exploration Trip
Join us on this exploratory RIB voyage to one of wonders of the natural world, at Les Minquiers; home to breathtaking scenery, including its infamous reef at over 300 km2 (larger than most British cities), which boasts hundreds of granite fangs, wildlife, geology and archaeology - more spectacular than anywhere else in Europe.
Duration: 2-3 hrs
Price: starting at £55
At high water (around 50 metres!), the stunning surroundings of the island are submerged except the charming Maitresse, with stone cottages and no permanent inhabitants. Minquiers is the perfect getaway for peace and tranquillity. During your 3-hour voyage, you will have approximately 1.45 to 2 hours ashore. Book today for this scenic voyage of a lifetime!
Island RIB Voyages
Albert Quay, Pontoon No5
You might also like
Take an adventure aboard our 12 seater RIB to destinations that thrill and relax whilst experiencing the beauty that Jersey, France and other islands have to offer. Indulge in delicious French cuisine in Carteret, wonder in the magnificence of our local dolphin and seal wildlife, swim in crystal clear waters and so much more.

domain: geography


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this is a pretty amazing video created by my friend Keith Turner. he spent a year on this, building on top of footage we got with a GoPro while surfing. he used Maya, Mocha and some After Effects and basically taught himself as he created it.
incidentally, he posted this about five days ago and is already at over 50,000 hits. =)

domain: artistic


Bristol City v Oxford United injury news as five ruled out and four doubts
and live on Freeview channel 276
Scott wasn’t included in the Robins match-day squad for their season opener with Preston because of a knock. The 19-year-old has come under a lot of transfer speculation recently but Nigel Pearson played down fears that his absence could be down to a departure being imminent.
Club captain Weimann played in Scott’s position but only lasted 20 minutes. The 32-year-old had an injury that flared up and he was replaced by Joe Williams. He is likely to be rested in the hope that he will instead be available for the trip to Millwall, rather than their match with Oxford.
Kal Naismith has returned to training this week and could push to be involved against Oxford. Ross McCrorie has an infection and Pearson has been wise to not place a timeframe on his return.
As for long-term absentees, Ayman Benarous and Rob Atkinson are still out. Benarous is a ahead of Atkinson in his recovery but is likely to need a string of appearances for the Under-21’s before being fit enough for the first-team.
Mark Sykes - who was one of the many to make the move from the Kassam Stadium to Ashton Gate could be given a start on Wednesday. January signing Harry Cornick started ahead of him to cope with Preston’s physical presence but now the Ireland star will be eyeing a starting opportunity to pick up from his good pre-season form.
Oxford United meanwhile suffered an opening day defeat to Cambridge United. Former Cardiff City winger Josh Murphy wasn’t in the match-day squad, which could suggest he’s not in contention for the match at Ashton Gate.
James Henry is dealing with what is being described as a ‘short-term’ issue and may be available.
Former Bristol City striker Sam Baldock meanwhile was not in Oxford’s squad, and despite being under contract does not have squad number for the season at the U’s.


The Melvins unveil new album title as well as announce reunion with original drummer!
When a band have been around as long as The Melvins have, it can often arouse suspicion when they choose to release a covers album so late in their career. Are they running out of ideas or merely taking a rest? Considering that their latest album, Everybody Loves Sausages (read our review here), was only released a couple of months ago and news has already broken that they’re working on their second album of the year, it would seem that the band’s “rest” is an extremely brief one.
The Melvins are one of the most tenacious bands in the American scene, whether it be because they’re constantly touring or that they’ve released around twenty albums since 1983, which is not something to be sniffed at.
The band’s frontman and guitarist Buzz Osborne has announced that not only do they have their next album in the works for a release later this year, but they’ve also re-united with their original drummer Mike Dillard. He has to say:
Tres Cabrones is as close as we’re willing to get to the Melvins 1983 line-up. The best part is it’s all new songs. I specifically wrote tunes that would be good for these guys to play and it worked out great. We had no interest in rehashing tunes we wrote 30 years ago and chose instead to simply create NEW songs. It worked out perfectly.
The band’s previous drummer Dale Crover has moved over to bass for Tres Cabrones, to make room for Dillard’s return. He has to say:
After 30 years we release a record with the (almost) original line-up of the band. I’ve always felt that drummer Mike Dillard was one of us. It’s great that he’s still a piece of the puzzle! Plus, I get to see what it’s like being the bass player in the Melvins! Tres Cabrones indeed!
The band are currently on their massive, country spanning 30th anniversary tour with Butthole Surfers. Fans of the classic Melvins sound will likely be very pleased by this news.
Tres Cabrones will be released on November 5th via Ipecac Recordings.


Sunday, July 13, 2008
Yesterday I spent the day at a flyball tournament. It was fun, our team crashed and burned, but we had expected a few hiccups, and we had entered for one day only knowing that our team the Muttniks might have some issues so we were okay with the outcome. Of course it would be nice to have a few more successes at our next one, but patience is a virtue!
At the end of the day a short meeting was convened regarding the subject of publicity for flyball clubs and their members. The Alberta Region (12) has in the last year been at a 20% drop off from the public for interest in the game. The region is short of height dogs, and spectators aren't attending any of the tournaments.
In some circles the sport of flyball has been referred to as a competitive sport with speed elitism at every turn. Indeed flyball dogs are being 'ordered' and shipped in from all over Canada and the United States to play the game, and Flyball rescue organizations are popping up more and more.
In order to stem the tide of this unfortunate perception,the owners of the groups want to work together to present a more public friendly image of the sport and of its players.
There are so many benefits to having your dog play the sport. Exercise, teamwork, team-building, personal goals, and socializing for you and your pet.
By using websites, social media, and the press I hope that this is the beginning of a new direction for all of the flyball clubs. For more on flyball, please see my article posted here in Pet Rescue Magazine.
Posted by Rachel Sentes at 6:26 PM


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|Publication number||US5109112 A|
|Application number||US 07/548,429|
|Publication date||Apr 28, 1992|
|Filing date||Jul 5, 1990|
|Priority date||Jan 19, 1989|
|Publication number||07548429, 548429, US 5109112 A, US 5109112A, US-A-5109112, US5109112 A, US5109112A|
|Inventors||John J. Siekierka, Hsuen-Yun Hung, Marie J. Staruch, Nolan H. Sigal, Richard A. Mumford|
|Original Assignee||Merck & Co., Inc.|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (1), Non-Patent Citations (9), Referenced by (36), Classifications (12), Legal Events (4)|
|External Links: USPTO, USPTO Assignment, Espacenet|
The instant case is a Rule 62 File wrapper continuation application of U.S. Ser. No. 07/369,576, filed Jun. 21, 1989, now abandoned, which in turn is a continuation-in-part application of Ser. No. 300,659, filed Jan. 19, 1989, now abandoned.
1. Field of the Invention
The present invention relates to a new homogeneous cytosolic binding (HCB) protein which binds FK-506 but not cyclosporine A, is stable to heating at 56° C. for 30 minutes, and has a molecular weight of about 10-12 kilodaltons. The (partial) amino terminal amino acid sequence of the HCB protein is: H2 N-Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gly-Asp-Gly-Arg-Thr-Phe-Pro-Lys-Arg-Gly-Gln-Thr-X-Val-Val-His-Tyr-Thr-Gly-Met-Leu-Glu-Asp-Gly-Lys-Lys-Phe-Asp (wherein X is undefined). The HCB protein has peptidyl-proline isomerase enzymatic activity.
2. Brief Description of Disclosures in the Art
In 1983, the U.S. FDA licensed cyclosporine, and extremely effective anti-rejection drug that revolutionized the field of organ transplant surgery. The drug acts by inhibiting the body's immune system from mobilizing its vast arsenal of natural protecting agents to reject the transplant's foreign protein.
As effective as the drug is in fighting transplantation rejection, it suffers drawbacks in causing kidney failure, liver damage and ulcers which in many cases can be very severe.
EPO Publication No. 0184162 to Fujisawa, describes a new macrolide immunosuppressant FK-506 which is reputed to be 100 times more potent than cyclosporine. The macrolide is produced by fermentation of a particular strain of Streptomyces tsukuhaensis. Also described is the closely related macrolide immunosuppressant FK-520, produced by S. hygroscopicus subsp. yakushimaensis.
FK-506 shares a number of immunosuppressive properties with the cyclic peptide, cyclosporine A, though 10-100 times more potent in this regard. These similarities suggest that both agents may share a similar mechanism of action at the biochemical level.
For example, Cyclosporine A is known to bind with the cytosolic protein, cyclophilin, as described in SCIENCE, Vol. 226, pp. 544-546 (Nov. 1984) by R. E. Handschumacher et al, which is thought to mediate the immunsuppressive effects of cyclosporine A in cells. Cyclophilin has also been shown to possess an enzymatic activity, which catalyzes the cis-trans isomerization of peptidyl prolyl bonds, as described in Nature. Vol. 337, pp. 473-475 and pp. 476-478 by N. Takahashi et al. and Fischer et al., respectively.
Further, the article by V. Warty et al in Transplantation Vol. 46, No. 3, pp. 453-455 (September 1988) present data suggesting that FK-506 also binds a cytosolic protein with an apparent molecular weight of about 18-19 kilodaltons which is similar to the protein that also binds cyclosporine.
What is needed in the art is the isolation and identification of that particular FK-506 binding protein in order to help delineate the FK-506 mechanism of therapeutic action in the cell.
We have identified a homogeneous cytoplasmic binding (HCB) protein for FK-506 in the neoplastic human T-cell line, JURKAT, using [3 H]FK-506. It is believed that this protein is ubiquitous and is present in a variety of normal human cells and tissues and particularly is abundant in neoplastic and transformed human T-cell lines, preferably the JURKAT cell line, which is readily available at the American Type Culture Collection, 12301 Parklawn Drive in Rockville, Md., under the designation "JURKAT TIB-152, Clone E6-1 Human Acute T-cell Leukemia, deposited by Arthur Weiss.
The HCB protein has a molecular weight of 10-12 kilodaltons (as determined by gel filtration), is stable to heating at 56° C. for 30 minutes and does not bind cyclosporine A (CsA). This newly discovered protein contrasts with the CsA binding protein, cyclophilin, described in the above-listed prior art, in which cyclophilin is described as being heat labile and having a molecular weight of 15-17 Kd.
In accordance with this invention there is provided a homogeneous cytosolic binding (HCB) protein having a specific binding affinity for FK-506.
Further characteristics of this HCB protein are that it has no specific binding affinity for cyclosporine A; has a molecular weight in the range of 10-12 kilodaltons; a specific binding activity of about 26 micrograms FK-506 per mg HCB protein; is stable at 56° C. for 30 minutes; is derived from a variety of sources including, for example, normal and neoplastic human T-cell lines; and in particular, the JURKAT cell line, and has the amino terminal amino acid sequence (partially sequenced): NH2-Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gly-Asp-Gly-Arg-Thr-Phe-Pro-Lys-Arg-Gly-Gln-Thr-X-Val-Val-His-Tyr-Thr-Gly-Met-Leu-Glu-Asp-Gly-Lys-Lys-Phe-Asp (wherein X is at yet undefined); and has an enzymatic activity, which catalyzes the cis-trans isomerization of proline-containing peptide bonds.
Also provided is the purified complex formed between a biologically useful ligand and the above-described HCB protein, wherein said ligand possesses a specific binding affinity for the HCB protein. The ligand can be an immunosuppressant, e.g., FK-506 type macrolide, or an antibody to the HCB protein.
Further provided is a method of determining the presence or quantity of a biologically useful ligand, eg. FK-506, having an affinity for the above-described HCB protein, in a sample, e.g. a body fluid of an immunosuppressed individual on FK-506 therapy, which comprises contacting said sample with the HCB protein, wherein said HCB protein preferably can be immobilized, for example, on a cyanogen bromide activated Sepharose type affinity column.
Furthermore is provided an embodiment of the method to purify a biologically useful ligand wherein said ligand is present in a sample comprising a fermentation broth, a biological fluid, e.g. human blood, or is a chemical entity that is a potential drug, and wherein said ligand is preferably an immunosuppressant, e.g. FK-506, or is an antibody to the HCB protein.
FIG. 1 illustrates inhibition of IL-2 secretion in JURKAT cells stimulated with PMA and ionomycin by FK-506 and [3 H]-dihydro FK-506.
FIG. 2 illustrates the fractionation of cytoplasmically associated [3 H] FK-506 by HPLC gel filtration.
FIG. 3 illustrates direct binding of [3 H] FK-506 to a low molecular weight component.
FIG. 4 illustrates the separation of JURKAT cyclophilin from [3 H] FK-506 binding activity by HPLC gel filtration.
FIG. 5 illustrates binding of [3 H] FK-506 to JURKAT cytosol as a function of [3 H] FK-506 concentration.
FIG. 6 illustrates the nature of the peptidyl-proline isomerase (PPIase) assay.
FIG. 7 illustrates in (A), the PPIase activity of the HCB protein of this invention, and (B), the PPIase activity of cyclophilin.
FIG. 8 illustrates in (A) the specific inhibition of HCB protein PPIase activity by dihydro-FK-506 and (B), the specific inhibition of cyclophilin PPIase activity by cyclosporine A (CsA).
The specification contains a more detailed description of FIGS. 1-8 in the section headed "Figure Legends".
We have demonstrated the presence of a heat stable, low molecular weight cytosolic binding protein for the potent immunosuppressive agent FK-506, in JURKAT cells. The HCB protein appears to be distinct from cyclophilin by a number of criteria including, molecular weight, heat stability, ligand specificity and amino acid sequence. This does not, however, rule out the possibility that the two binding proteins are related, perhaps as members of the recently identified cyclophilin family (B. Haendler et al., EMBO J. 6, 947 (1987)). These low molecular weight proteins may constitute part of a signal transduction pathway utilized during T-cell activation which is important for activation of select lymphokine genes (e.g. IL-2).
The potent immunosuppressive agent, cyclosporine A (CsA), has found wide clinical use in the prevention of allograft rejection and treatment of graft versus host disease (B. D. Kahan, Cyclosporin: Biological Activity and Clinical Applications (Grune & Stratton, Orlando, Fla., 1983). CsA appears to act specifically during T lymphocyte activation by inhibiting the transcription of a limited set of early T-cell activation genes (IL-2, IL-3, gamma-IFN, GM-CSF, TNF and c-myc) (J. F. Elliot et al., Science 226, 1439 (1984); M. Kronke et al., Proc. Natl. Acad. Sci. U.S.A. 81, 5214 (1984). A recently isolated macrolide, FK-506, obtained from Streptomyces tsukubaensis has been shown to possess very similar, if not identical, immunosuppressive properties both in vivo and in vitro, though being 10-100 times more potent than CsA in this regard (S. Sawada et al., J. Immunol. 139, 1797 (1987)). Studies on the biochemical nature of the immunosuppressive pathway affected by CsA have led to the purification and characterization of a specific CsA binding protein termed, cyclophilin (R. E. Handschumacher et al., Science 226, 544 (1984); M. W. Harding et al., J. Biol. Chem. 261, 8547 (1986)). Two isoforms of cyclophilin have been isolated which bind CsA with identical affinities and exhibit a structure-function profile consistent with playing a role in immunosuppression. Recently, it has been reported that FK-506 may bind to and function via a cytoplasmic receptor (V. Warty et al., Transplantation, 46, p. 453 (1988)).
FK-506 was isolated from a culture of Streptomyces tsukubaensis No. 9993. [3 H]-dihydro CsA was made by catalytic reduction of the MeBmt double bond in CsA with tritium gas in dimethylformamide solvent using 10% Pd/C catalyst at room temperature, 1 atmosphere pressure, in a shaker apparatus for an hour, followed by purification by reverse phase high pressure liquid chromatography on a Whatman Partisil OD3 column. The specific activity of the obtained [3 H]-dihydro CsA was 44 mCi/mg. [3 H]-dihydro FK-506 was prepared by catalytic reduction of the allyl double bound in FK-506 by contacting FK-506 in ethyl acetate solvent with tritium gas under 1 atmosphere pressure and room temperature in the presence of 10% Pd/C catalyst for 20 minutes followed by isolating and purifying the material by reverse phase high pressure liquid chromatography to yield the product which had a specific activity of 49 mCi/mg. Dihydro-[3 H]FK-506 was a potent inhibitor of IL-2 secretion in JURKAT cells stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (See FIG. 1 and the following description under "Figure Legends"), although 5 times less potent than the parent compound.
In order to examine the subcellular distribution of [3 H]FK-506 binding, JURKAT cells were incubated with [3 H]FK-506 for 30 min., washed, lysed in hypotonic buffer and fractionated into cytosol, nuclei and membrane fractions. Greater than 95% of the [3 H]FK-506 is specifically associated with the cytoplasmic compartment and the remainder distributed about equally between nuclei and plasma membranes (See following Table 1). This is similar to the reported subcellular distribution of CsA which is largely concentrated in the cytosol where it is found associated with a specific binding protein, cyclophilin (M. M. Merker et al., J. Immunol. 132, 3064 (1984)). In view of this, we prepared cytosol from JURKAT cells incubated with [3 H]FK-506 and subjected it to size fractionation by HPLC gel filtration (See FIG. 3 and the following description under "Figure Legends").
A single peak of radioactivity was observed eluting at a position corresponding to a molecular weight of 10-12 Kd (FIG. 3). The formation of this complex does not require the intact cell since incubation of JURKAT cytosol with [3 H]FK-506 and subsequent HPLC gel filtration yields the same complex (See FIG. 4 and the following description under "Figure Legends"). Inclusion of a 200 fold molar excess of unlabeled FK-506 in the reaction mixture inhibits the formation of this complex by 90% while CsA is without effect (FIG. 4 inset), suggesting that FK-506 binds to a protein other than cyclophilin.
To rule out the possibility that distinct binding sites for both CsA and FK-506 are present on cyclophilin, we assayed highly purifie cyclophilin for [3 H]CsA and [3 H]FK-506 binding using the LH-20 assay developed for cyclophilin (R. E. Handschumacher et al., Science 226, 544 (1984). We could not detect specific binding of [3 H]FK-506 to Purified calf thymus cyclophilin (See following Table 2). Further characterization of the cytoplasmic component (s) responsible for binding reveals that [3 H]FK-506 binding is heat stable (Table 2), while binding of [3 H]CsA to crude JURKAT cyclophilin is completely aborgated under these conditions. In order to further delineate CsA and FK-506 binding, we fractionated JURKAT cytosol utilizing HPLC gel filtration and assayed individual fractions for both [3 H]FK-506 binding using the LH-20 assay. Under our conditions, the peak of [3 H]CsA binding elutes at a position corresponding to a molecular weight of 15-17,000, well ahead of the peak of 3H]FK-506 binding (See FIG. 5 and the following of description under "Figure Legends") again indicating that both activities are distinct.
Incubation of fractions containing 3H]FK-506 binding activity with proteinase K (Boehringer Mannheim) completely abolished the binding reaction, suggesting that FK-506 forms a complex with a protein.
Binding of [3 H]FK-506 to crude cytosol is saturatable with half maximal binding observed at 4.0 nM (See FIG. 6 and the following description under "Figure Legends"). This value is similar to the concentration of [3 H]FK-506 inhibiting IL-2 secretion in JURKAT (1.1 nM) suggesting that the binding observed is physiologically relevant.
Inhibition of IL-2 secretion in JURKAT cells stimulated with PMA and ionomycin by FK-506 and [3 H]dihydro FK-506. JURKAT cells (equivalent to those available from the ATCC, supra) were cultured in RPMI 1640 media (Cellgro, Mediatech, Wash. D.C.) containing 1% Pen/Strep (Gibco), 1 mM Glutamine and 10% heat-inactivated fetal calf serum (Hazleton). For assay of IL-2 secretion by JURKAT cells, cells were cultured for 16 hr. in 96-well microtiter plates (Costar) at 1×10 cells/ml in the above media containing 10 ng/ml of PMA (Sigma) and 5 μM ionomycin (Calbiochem) along with the concentrations of FK-506 or [3 H]FK-506 indicated. Supernatants (50-100 μl ) was assayed for IL-2 using a commercially available ELISA assay (Collaborative Research Inc.). --□--□--□--, FK-506; -- -- -- --, [3 H]-dihydro FK- 506.
Fractionation of cytoplasmically associated [3 H]FK-506 by HPLC gel filtration. Jurkat cells were incubated with 6.2 nM [3 H]FK-506 for 30 min. at 37° C. Cells were pelleted as before and resuspended in 250 μl of 10 mM Tris-HCl, pH 7.5 containing 1 mM MgCl2. After 10 min at 4° C., the cells were lysed by rapidly forcing through a 25 g needle (See M. M. Merker et al., J. Immunol. 132, 3064 (1984). The lysate was centrifuged at 100,000×g in a Beckman TL-100 ultracentrifuge and 100 μl of supernatant was subjected to HPLC gel filtration on a Bio-Sil TSK-250 column (7.5×300 mm, Bio-Rad Laboratories) at a flow rate of 1 ml/min. Fractions (0.4 ml) were collected and 100 μl of each counted in 10 ml of Aquasol-2. The arrows indicate the elution position of various molecular weight standards (numbers are molecular weight×102 ) fractionated under identical conditions.
Direct binding of [3 H]FK-506 to a low molecular weight cytosolic component. A cell-free extract was prepared from JURKAT cells by lysing 40×10 cells in 0.5 ml of 10 mM Tris-HCl, PH 7.5 containing 1 mM MgCl as described in the legend for FIG. 3. 200 μg of protein (Bradford assay, BSA standard, (see M. M. Bradford, Anal. Biochem. 72, 248 (1976) was incubated with 16 nM [3 H]FK-506 for 10 min at 37° C. 50 μl of the reaction mixture were fractionated by HPLC gel filtration as described in the legend for FIG. 2. The figure inset shows the results of a similar fractionation of the following: --□--□--58 --, 200 μg of cytosol and 16 nM [3 H]FK-506 alone; -- -- -- --, cytosol, [3 H]FK-506 and a 200 fold molar excess of FK-506 and -- -- -- --, cytosol, [3 H]FK-506 and a 200-fold molar excess of CsA.
Separation of JURKAT cyclophilin from [3 H]FK-506 binding activity by HPLC gel filtration. 1.0 mg of JURKAT cytosol, prepared as previously described, was subjected to HPLC gel filtration. In order to maximize the separation of low molecular weight components, a Bio-Sil TSK-125 column (7.5×600 mm) was used. 0.4 ml fractions were collected at a flow rate of 1.0 ml/min. 100 μl of each fraction was incubated with 0.5 nM [3 H]CsA or [3 H]FK-506 and assayed for binding to cytosol components as previously described (6). -- -- -- --, [3 H]CsA binding; -- -- -- --, [3 H]FK-506 binding.
Binding of [3 H]FK-506 to JURKAT cytosol as a function of [3 H]FK-506 concentration. Binding of [3 H]FK-506 was assayed by the LH-20 assay described in Table 2 with 4.9 μg of JURKAT cytosol and the concentrations of [3 H]FK-506 indicated. Nonspecific binding, assessed by the amount of [3 H]FK-506 bound in the presence of 200-fold molar excess of unlabeled FK-506, has been subtracted but never exceeded 5% of the total cpm bound.
Depicted is a summary of the PPIase assay used to measure the cis-trans isomerization of the proline-alanine peptide bond in the peptide, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide.
Illustrated is HCB protein PPIase activity as a function of HCB protein concentration (A). Symbols ( ) represent the results of experiments with 2 different preparations of HCB protein. (B) Comparison of cyclophilin PPIase activity (□◯) and HCB protein PPIase activity ().
Illustrated is the inhibition of HCB protein PPIase activity of dihydro FK-506; -- -- , CsA; , (A). (B) Illustrates the inhibition of cyclophilin PPIase activity by CSC; -- -- -- --, FK-506; -- --.
TABLE 1______________________________________Subcellular distribution of [3 H]FK-506in JURKAT cells [3 H]FK-506Fraction (1) Bound (cpm) % of Total (2)______________________________________nuclear pellet 3,581 1.7plasma membrane 1,824 0.9cytosol 207,879 97.4______________________________________ (1) 10 × 106 JURKAT cells were incubated with 6 nM [3 H]FK506 and lysed as described in the legend to FIG. 2. The crude lysate was centrifuged at 2,000 × g for 10 min. to pellet nuclei. The supernatant was centrifuged at 150,000 × g for 20 min. in a Beckman TL100 ultracentrifuge to pellet crude membranes. Each cell fraction was solubilized in 1% Triton X100 and counted in 10 ml of Aquasol2. (2) The total cpm recovered from all subcellular fractions represents 83% of the total cpm associated with intact cells.
TABLE 2______________________________________Lack of [3 H]FK-506 binding to purifiedcyclophilin and effects of heating on [3 H]FK-506and [3 H]CsA binding to JURKAT cytosol [3 H]FK-506 [3 H]CsA Bound (cpm)* Bound (cpm)*______________________________________cyclophilin, 0 20,988100 ng (1)JURKAT cytosol, 13,585 5,98840 μg (2)JURKAT cytosol 15,315 040 μg 56 C.,15 min. (2)______________________________________ *Binding of [3 H]FK506 and [3 H]CsA to either purified cyclophilin or JURKAT cytosol was assayed by the LH20 assay previously described (see R. E. Handschumaker et al., supra). Assays (100 μ l) contained 10 mM sodium phosphate, pH 7.4, 5 mM mercaptoethanol and 6.0 nM of either [ H]FK506 (specific activity = 49 mCi/mg) or [3 H]CsA (specific activity = 44 mCi/mg) along with purified cyclophilin or 0.5 nM of either compound with crude JURKAT cytosol. Binding is reported as cpm specifically bound (total cpm bound cpm bound in the presence of a 200 fold molar excess of unlabeled compound). (1) Cyclophilin was purified from calf thymus as described (See M. W. Harding et al., supra) and had a specific activity of 68 μg CsA bound/mg protein. (2) JURKAT cytosol was prepared as described in the legend to FIG. 2. For experiments with heat treated JURKAT cytosol, protein was adjusted to 1.0 mg/ml with 10 mM sodium phosphate buffer, pH 7.4 and heated for the time indicated. Precipitated protein was removed by centrifugation and equivalent volumes assayed for [3 H]FK506 and [3 H]CsA binding.
(1) Cyclophilin was purified from calf thymus as described (See M. W. Harding et al., supra) and had a specific activity of 68 μg CsA bound/mg protein.
(2) JURKAT cytosol was prepared as described in the legend to FIG. 2. For experiments with heat treated JURKAT cytosol, protein was adjusted to 1.0 mg/ml with 10 mM sodium phosphate buffer, pH 7.4 and heated for the time indicated. Precipitated protein was removed by centrifugation and equivalent volumes assayed for [3 H]FK-506 and [3 H]CsA binding.
The partially determined amino terminal amino acid sequence of the HCB protein is: H2 N-Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gly-Asp-Gly-Arg-Thr-Phe-Pro-Lys-Arg-Gly-Gln-Thr-X-Val-Val-His-Tyr-Thr-Gly-Met-Leu-Glu-Asp-Gly-Lys-Lys-Phe-Asp, as determined by gas phase sequencing on an Applied Biosystems 477A Protein Sequencer and a Milligen/Biosearch Prosequencer 6000 Series. The "X" designation refers to an amino acid which has not been definitively identified as yet, which may be cysteine or histidine.
The HCB protein, per se or immobilized, can be used as a specific binding partner to a variety of binding ligands for diagnostic, purification or investigatory procedures. A preferred immobilizing matrix is cyanogen bromide activated Sepharose (Pharmacia) to which the HCB protein can be covalently linked, forming an affinity chromatography column.
The HCB protein is of physiological importance because of the high specificity for binding active forms of the immunosuppressant, FK-506. The above described immobilized affinity matrix can be prepared which reversibly binds FK-506 in a complex, which can be eluted with aqueous buffer reagents of increasing ionic strength. The formed affinity matrix can also be used to detect FK-506-like macrolide substances by displacement of tritiated (3 H)-FK-506. This would include FK-520, FK-523, FK-525 and other FK-506 analogs as disclosed in EPO Publication No. 0184162.
The matrix also provides a method to identify and/or quantify FK-506 in serum and other body fluids as well as detect FK-506 like cellular constituents which may be natural ligands.
Still further, the matrix is useful in screening candidate chemical structures that, like FK-506, can have immunosuppressive activity and, therefore, is useful in the development of other classes of drugs that function through the action of this protein.
Since the HCB protein has been purified to homogeneity, oligonucleotide probes can be used to identify the gene thereby allowing the protein to be produced by known recombinant DNA techniques.
A typical procedure for the purification of the HCB protein is described in following Example 1.
Since, the HCB protein has an affinity for the immunosuppressant FK-506, and its active analogs, it or certain derived chemicals and/or natural derivatives thereof, including subfragments of the whole protein, can be used as a specific binding partner for these ligands in numerous receptor binding procedures known in the art.
Similarly, it can be used to purify a desired ligand from a composition containing ligand. For example, the HCB protein can be used for purifying FK-506 or related structures from a yeast fermentation broth in which the FK-506 is produced. Further, it can be used to select compounds which bind to the HCB protein as a screening test for identifying new immunosuppressant drugs. In these various procedures, it is preferred, although not required, to immobilize the HCB protein. This can be accomplished by any procedure known in the art. A particularly useful support for immobilizing proteins is cyanogen bromide treated Sepharose (CNBR-activated Sepharose 4B, Pharmacia, Piscataway, N.J.). The immobilized HCB protein is prepared by mixing the protein under basic conditions with the cyanogen bromide-activated Sepharose. The binding of the HCB protein to this matrix can result in a marked stabilization of the bound activity through a three-dimensional stabilization achieved by multiple bonds through the amino groups of the HCB protein.
The preferred immobilized HCB protein can also be used diagnostically for the determination of the concentration of FK-506 and its metabolites from physiological fluids, e.g. body fluids, and tissue extracts as for example in patients who are undergoing FK-506 immunosuppressive therapy.
The protein can also be used in an assay to bind FK-506 type macrolide compounds and biologically useful ligands, by allowing the HCB protein and FK-506 type macrolide/ligand to form a complex in an excess of the macrolide, then eluding the mixture through a column and analyzing the concentration of the pure complex Spectrophotometrically or by scintillation counting. By this methodology, pure samples of the complex can be formed, from which the macrolide or biologically useful ligand can be isolated by interrupting the binding with an e.g. strong ionic salt solution and followed by conventional chromatographic separation.
Various changes and modifications can be made in the products and processes of the present invention without departing from the spirit and scope thereof. The various embodiments which have been set forth herein and the following example are for the purpose of further illustrating the invention but are not intended to limit it.
Human Jurkat cells were cultured in RPMI 1640 media containing 1% Pen/Strep, 1 mM glutamine and 10% heat-inactivated fetal calf serum. Approximately 70×109 cells were washed in PBS and pelleted by low speed centrifugation. The cell pellet was resuspended in 4 volumes of lysis buffer containing; 10 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM 2-mercaptoethanol, 1 mM PMSF and 1 mM EDTA. The cells were disrupted by dounce homogenization (20 strokes) and centrifuged at 500×g for 20 min to remove nuclei and cell debris. The supernatant (S-100) was centrifuged at 100,000×g for 1 hour to remove crude membranes. The S-100 (127 ml, 8.06 mg/ml protein determined by the Bradford assay with BSA as a standard) was transferred to a glass flask and heated for 20 min, with gentle shaking, in a 56° C. water bath. Precipitated protein was removed by centrifugation and the supernatant (114 ml, 2.59 mg/ml) concentrated by filtration through an Amicon YM5 membrane and dialyzed for 8 hrs against several liters of buffer containing; 5 mM 2-mercaptoethanol, 1 mM PMSF and 1 mM EDTA. The peak of activity, which eluted at approximately 50 mM KH2 PO4, was dialyzed against buffer containing; 20 mM NaH2 SO4, 50 mM NaH2 SO4 pH 6.8, 5 mM 2 -mercaptoethanol, 1 mM PMSF and 1 mM EDTA and concentrated as before. The protein (2 ml, 0.64 mg/ml) was size fractionated by HPLC on a TSK-125 gel filtration column (21×600 mm, Bio-Rad). The peak of activity was pooled and dialyzed against buffer containing; 5 mM NaH2 PO4, pH 6.8, 5 mM 2-mercaptoethanol, 1 mM PMSF and 1 mM EDTA. The protein was concentrated to 2.2 ml (0.64 mg/ml) and fractionated by weak ion exchange on a SynChropak CM 300 column (4.6×250 mm, SynChrom, Inc.). Protein was eluted isocratically with buffer of the same composition as the dialysis buffer. The peak of activity was concentrated to 0.77 ml (0.17 ng/ml) and stored at 0° C. SDS-PAGE of the CM 300 material revealed a single band (by combined silver and Coomassie blue staining) at a molecular weight of 10-11,000. Amino terminal sequence analysis of the same material revealed a single terminus.
Purified HCB protein was mixed with tritrated dihydro FK-506, in the wells of a microtiter plate and incubated at R.T. for 5 min. The mixtures were loaded onto 2 ml minicolumns of Sephadex LH-20 (Pharmacia). The elutions were made with 0.5 ml of 30 mM phosphate buffer, pH 7.2 with 0.02% sodium azide and collected. The amount of bound tritrated dihydro FK-506 was quantitated by the use of a scintillation counter.
Two papers [Takahashi, et al., Nature, 337, 473 (1989) & Fischer, et al., Nature, 337, 476 (1989)] demonstrate that cyclophilin is a peptidyl-proline isomerase (PPIase). The function of this enzyme is the catalysis of cis-trans isomerization of proline residues in proteins; a step important for the folding of proteins into their native conformations. It has further been demonstrated that cyclosporin A (CsA) inhibits the PPIase activity of cyclophilin. It has been suggested that cyclophilin PPIase activity may be part of an important signal transduction pathway in lymphocytes and other cells.
In view of the similarities between cyclophilin and the HCB protein (e.g. cytosolic localization, low molecular weight, immunosuppressant binding, etc.), we assayed the pure, homogeneous HCB protein of the instant invention for PPIase activity under the same conditions used for cyclophilin. In addition, since the results were not a prior predictable, we examined the effects of CsA and FK-506 in these assays.
The assay used was essentially the same as described by K. Lang and F. Schmidt [Nature, 333%, 453 (1988)]. Briefly, the PPIase assay measures the "cis" to "trans" isomerization of the proline-alanine peptide bond in the peptide, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. When in the "trans" form, the peptide is readily cleaved by chymotrypsin and the appearance of p-nitroanilide can be quantitated spectrophotometrically at 405 nm. The assay exhibits a high blank since 88% of the substrate is present in the "trans" form at equilibrium. In addition, the assay exhibits an appreciable nonenzymatic rate of "cis" to "trans" conversion (k=0.05 sec-1 at 25° C. in 100 mM Tris-HCl, pH 7.8.). A summary of the assay is given in FIG. 6.
Reaction mixtures (1.0 ml) contained, 0.1 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Bachem) (30 μl addition of 2.1 mM stock in DMSO), 100 mM Tris-HCl, pH 7.8, cyclophilin as purified by the method described by Harding et al., J. Biol. Chem. 261, p. 8547 (1986) or FK-506-binding protein and where indicated either CsA, FK-506 or dihydro FK-506. After 1.0 minutes, 30 ul of 2.0 mg/ml chymotrypsin (Sigma) in 100 mM Tris-HCl, pH 7.8 was added. After brief mixing, the absorbance was monitored at 405 nm in either a Perkin-Elmer 4B or Beckman DU 68 spectrophotometer. Absorbance measurements were taken at 3 or 5 second intervals for 4 minutes. A plot of [steady state]-Abs405 [time=t] versus time was made on semi-log paper. From the straight line, the time constant for Abs405 [steady state]-Abs405 [time=t] to fall by 1/e was calculated. The reciprocal of this constant yields the rate constant k.
Two preparations of HCB protein isolated from different batches of JURKAT cells as described in Example 1 were assayed for PPIase activity as described above. As illustrated in FIG. 7A, the HCB protein exhibits a low, but significant PPIase activity. The increase in isomerization rate is proportional to the concentration of HCB protein. Cyclophilin PPIase activity is approximately 25 fold higher than that of the HCB protein (FIG. 7B). The reason for the differences in activity may reflect substrate specificity or reaction conditions.
Dihydro-FK-506 specifically inhibits HCB protein PPIase activity (FIG. 8A). CsA, at a concentration of 1 μM was without effect. CsA, however, specifically inhibits cyclophilin PPIase activity (FIG. 8B). FK-506 at a concentration of 1 μM had no effect on cyclophilin PPIase activity.
The data presented demonstrates that the HCB protein, like cyclophilin, unexpectedly has been discovered to possess Peptidyl-proline isomerase activity. Although the activity associated with the HCB protein is significantly less than that of cyclophilin, it is specifically inhibited by FK-506 and not CsA, suggesting that it does not represent cyclophilin contamination. The association of PPIase activity with both cyclophilin and the HCB protein implies that this activity is important during T-cell activation. Furthermore, screening assays can be devised using the assay described above, by one skilled in the art, to identify specific inhibitors which are novel immunosuppressive agents.
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|U.S. Classification||530/350, 435/233, 530/810, 530/827|
|International Classification||C12N9/90, C12N15/90|
|Cooperative Classification||Y10S530/81, Y10S530/827, C12N9/90, C12N15/90|
|European Classification||C12N15/90, C12N9/90|
|Jul 25, 1991||AS||Assignment|
Owner name: MERCK & CO., INC. A CORPORATION OF NJ, NEW JERS
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:SIEKIERKA, JOHN J.;HUNG, HSUEN-YUN;STARUCH, MARIE J.;AND OTHERS;REEL/FRAME:005775/0777
Effective date: 19890620
|Aug 15, 1995||FPAY||Fee payment|
Year of fee payment: 4
|Aug 18, 1999||FPAY||Fee payment|
Year of fee payment: 8
|Sep 26, 2003||FPAY||Fee payment|
Year of fee payment: 12

domain: chemistry


Source: US2012008736A An X-ray diffraction contrast tomography system (DCT) comprising a laboratory X-ray source (2), a staging device (5) rotating a polycrystalline material sample in the direct path of the X-ray beam, a first X-ray detector (6) detecting the direct X-ray beam being transmitted through the crystalline material sample, a second X-ray detector (7) positioned between the staging device and the first X-ray detector for detecting diffracted X-ray beams, and a processing device (15) for analysing detected values. The crystallographic grain orientation of the individual grain in the polycrystalline sample is determined based on the two-dimensional position of extinction spots and the associated angular position of the sample for a set of extinction spots pertaining to the individual grain.
|Publication status||Published - 2012|
Bibliographical noteDTU reference nummer: 92676-11
Poulsen, H. F., & Lauridsen, E. M. (2012). IPC No. G01N23/083. X-ray diffraction contrast tomography (DCT) system, and an X-ray diffraction contrast tomography (DCT) method. (Patent No. WO2012003839).


text:
Your tongue isn’t the only thing you can burn at Starbucks. The java chain is reportedly rolling out a digital music service that’ll allow you to sample and burn MP3s onto a CD while sipping a latte.
Posted January 17 2015 — 4:48 AM EST
- Jason Reitman hosting 'Mad Men' live read, finale screening
- Hear Barenaked Ladies' '90s alt-ballad 'Say What You Want'
- 'The Originals' star: Season 2 finale sets up 'a whole new show'
- Jenny Slate, Ari Graynor to star in FX road-trip comedy fro 'Obvious Child' team'
- D'Angelo announces U.S. dates for Second Coming tour
- 'Penny Dreadful' showrunner John Logan teases season 2's 'ferocious enemy'
- Nick Broomfield talks 'Tales of the Grim Sleeper'

domain: entertainment


text:
Star Magic Circle 2013 member Alex Diaz, told PEP.ph (Philippine Entertainment Portal) about his love for the arts in a recent interview.
Unlike other young celebrities, he was quite open about discussing his way of expressing art by using his body as his canvass.
At the moment, he has six different ink designs on his body.
He narrated how he told his dad about it, “I was, like, 'Papa... love you. May tattoos ako!'
“Nagalit siya, sabi niya, ‘Ano? Saan? Ilan iyong tattoo mo?’
“Sabi ko, ‘Six po. Six tattoos po.’
“Nagalit siya sa akin, tapos maya-maya sabi niya parang, ‘Okay lang.’”
The 17-year-old shared that ever since he was young, he was already entertaining the thought of getting a tattoo in the future.
“Ine-express ko naman, e. Kahit bata pa po, lagi kong sinasabing, ‘Dad, gusto ko ng tattoos… gusto ko ng tattoos.’”
ALEX AND HIS TATTOOS. Alex showed some of his tattoos to PEP and explained what each one meant.
The power button tattoo on his right wrist means, “I always believed kasi na you should— especially if you know the tattoos, especially if you’re gonna put words— it should always be positive words that you believe will happen.
“So, I put power in my body para ma-represent na parang mga events in my life.”
On his right arm is the chemical symbol for adrenaline. Alex explains why he chose this: “Kasi, iyon nga po, like, it represents my personality kasi super hyper ako. I’ve always stayed as ganoon.”
Pointing to another tattoo on his right forearm, Alex exclaimed, “That’s for my mom!
“Kasi, ano po, she went to Canada na to study. Mag-isa siya doon, so I really worry about her.”
The ink reads "Find your way home” and beside it is a drawing of an anchor.
“It just means a nautical symbol, like when you find your way home, and then just keep grounded.
“'Cause... you know how the anchor puts down the ship? Just keep grounded no matter what?
“Kasi in showbiz, di ba? When all the lights surround you, minsan nakakalimutan niyo iyong pinanggalingan niyo po, so definitely, it’s just a reminder [to] always keep… always keep down to earth.”
Alex also has tattoo on the lower right part of his chest that says, “As I lay me down to sleep/ I pray the Lord my soul to keep/ If I die before I wake/ I pray the Lord my soul to take.”
He says there is nothing religious at all about this tattoo. In fact, Alex cited, “I don’t have a specific religion."
However, he still believes that there is a higher power that governs us all, and this tattoo reminds him of that belief.
"I do believe that the big guy upstairs is taking care of all of us here.”


text:
Pittsburgh spanish Restaurant Reviews
Welcome to our Pittsburgh spanish restaurant reviews guide where you'll find top restaurants off the beaten path, both locally owned and chain. In fact, some of the best restaurants in the Pittsburgh just might be the little mom and pop operations flying under the radar. For some of the best picks in Pittsburgh, you've come to the right place.
Our Pittsburgh restaurant reviews guide can help you make sense of the thousands of options out there. Happy eating!
Also check out our Best Restaurants in Pittsburgh 2016 Guide
“I stopped at cuzamil downtown last week for a quick lunch during my stay in pittsburgh. I was surprised to find a full service restaurant between Wendy's and McDonals and behind the construction of the new subway station. I was thinking to order plain enchiladas to be safe, since you can never go w... ” (02/01/2005)
“I was turned on to this spot by my son. Upon entering, I thought that I was back in Barcelona. Sampled several dishes from seafood to meat to poultry, all superb. With the exception os Los Caracoles in Barcelona, the best Spanish on this side of the pond.”

domain: travel


text:
Jared Keith Wiechert
Address: 66187 Prairie View Dr.
Goshen, Indiana 46526
About this Song
Haunting atmospherics and hypnotic dissonant church organ slowly builds, a spacious acoustic drum kit comes in. Dark, nightmarish, dreamy and euphoric. A somber sense of longing and melancholy. This eerie futuristic track is also great for building suspense & tension in horror, sci-fi & thrillers.


text:
Corrupt cops. Bantering bank robbers. Starry-eyed lovers. A Saturday Night Live cast member flailing for laughs.
A cursory glance at the weekend's box-office winners confirms the thinking movie lover's worst fear: The world may have changed, but Hollywood's idea of what we want to see remains the same.
Not to worry. It's mid-October, and that means the Fort Lauderdale International Film Festival mini-festivals are upon us.
FLIFF, the world's longest film festival, owes that dubious distinction to its three far-flung mini-fests. The first of these satellite events, the Asian Pacific Film Festival, launches tonight with India's Vanaprastham. While the film hasn't yet screened for us, Film Threat raves about the picture, calling it exotic, lushly photographed and sublimely acted. It's the tale of an impoverished theater performer and the rich woman who falls in love not with him, but with a character he plays. Immersed in the rich traditions of South Indian theater and culture, Vanaprastham merges dance, music, sung dialogue and pantomime.
If the titles of some of the Asian Pacific fest's other offerings sound a little familiar, perhaps it's because this isn't their first trip to South Florida. Both Tony Bui's Three Seasons and Takeshi Kitano's Kikujiro played the Miami Film Festival (the former in February 1999, the latter the following year). They bowed in Fort Lauderdale-area art houses a few months later. Hirokazu Koreda's lyrical Maborosi premiered locally in 1999. All are fine films that merited more attention than they received on their first visits to Fort Lauderdale.
While Vanaprastham kicks off the Asian Pacific Film Festival tonight at 7 at the AMC Ridge Plaza, 9200 State Road 84, Davie, the balance of the A-Pac films screen this weekend and next at FLIFF's Cinema Paradiso, 503 SE Sixth St., downtown Fort Lauderdale.
As luck would have it, two of FLIFF's most promising Asian films aren't part of the Asian Pacific Film Festival. They do, however, turn up at the Pembroke Pines and Boca Raton Mini Fests which run from Oct. 24-28, the former at Regal's Westfork cinema, 15977 Pines Blvd., Pembroke Pines, and the latter at Regal's Shadowood multiplex, 9889 Glades Road, Boca Raton.
Japanese iconoclast Masato Harada first dazzled Fort Lauderdale Film Festival audiences in 1998 with a pair of exquisite but radically different films -- Bounce and Kamikaze Taxi. His latest, Inugami, spins humor, tragedy and myth out of the unlikely coupling of a 40-ish spinster and a handsome young schoolteacher. Nominated for a Golden Bear at Berlin and awarded the top prize at Newport, Inugami has already stirred up quite a buzz on the film festival circuit.
Yoji Yamada's Jugosai takes a more lighthearted tack. It chronicles the journey of a ninth-grader who leaves his comfortable home in the suburbs of Yokohama and hitchhikes to a tiny island at the southern tip of Japan in search of a 7,000-year-old cedar tree. Along the way he learns valuable life lessons during encounters with a series of colorful characters.
Harada isn't the only FLIFF director whose work has charmed audiences at previous events. Italian filmmakers Andrea and Antonio Frazzi opened last year's fete with The Sky Is Falling, which went on to claim the FLIFF award for best foreign film. They return this year with Almost America, about an Italian woman in the 1950s who, following a devastating flood that wipes out her village, uproots her family and moves to Canada in search of the husband who went there years earlier to establish a new home.
Individual tickets to all films cost $7. For more information call 954-525-3456, or check the festival Web site, www.fliff .com.


Market leading manufacturer of custom wheels and accessories for enthusiasts asked The Highland Group to optimize its operation and help position the organization for growth.
- Grow annual revenue by $40 million.
- Develop an effective order entry process that reflects true customer demand.
- Increase throughput by 8%.
- Grew revenues by $42 million after the first year.
- Delivered project ROI of 5:1, exceeding promised savings.
Optimize Operation & Position for Growth
The world’s largest and market leading manufacturer of custom wheels and accessories for enthusiasts and the racing industry asked The Highland Group to optimize its operation and put in place processes that would position the organization for growth.
After working with leadership to define high-level Driver Goals, The Highland Group analyzed the operation and key vendors to identify constraints, inefficiencies and other opportunities for improvement. The team reengineered several processes and implemented tools to track demand, sales, orders, inventory and vendor performance.
- Identified and prioritized molds for repair, determined individual vendor capacity, and developed and implemented a purchase order management process to track molds out for repair.
- Conducted an order review collection process to identify orders that had not been fulfilled, orders that had not been invoiced, and invoices that had not been paid.
- Reengineered the order management process.
- Developed and implemented a vendor management process, determined individual vendor capacity, worked with the vendors to improve their processes, increasing repair capacity by 33%.
- Partnered with vendors to develop and implement a source inspection program.
- Developed and implemented a warranty management process that included the establishment and population of a warranty database.
- Developed a database of product sales history, defined excess and slow moving inventory, conducted a special sales promotion to move the inventory.
- Developed and implemented a rolling demand forecast model.
Grew revenues after the first year by


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Mar 24, 2014, 3:05 PM EST
If you happen to run into Oilers coach Dallas Eakins today, don’t bother asking how his weekend went.
Following Saturday’s 8-1 home loss to Calgary — a game in which someone threw an Oilers jersey onto the ice for the second time this season — Eakins admitted the last 48 hours were tough to swallow.
“I didn’t even leave the house yesterday,” he said, per Sportsnet. “I wanted to spend the day with my girls. You’re living in a depressed state.”
To say Eakins’ first year in Edmonton has been disappointing is a major understatement. The Oilers have a worse winning percentage (.410) than they had in either of the previous two years (.469 and .451, respectively), and are on pace to draft in the top-10 for the sixth year in a row.
The 8-1 loss came just two days after another brutal performance, a 3-1 loss at home to Buffalo — the only team in below Edmonton in the standings. Needless to say, losing by a combined 11-2 score to the Flames and Sabres — at Rexall, no less — has taken its toll on Eakins.
“I wanted to leave that [Flames] game behind,” he said. “However we have to deal with it today and then we’ll move on.”
Like Charlie Brown being followed around by that dang raincloud, Eakins just can’t escape the bad vibes. During Monday’s practice, he watched as goalie Viktor Fasth was run over by Anton Lander and was forced to leave the ice. Fasth, 2-0-1 in his brief time with Oilers, reportedly suffered a sprained neck in the collision.
Good times in Edmonton!
- In praise of the Hamburglar 11
- Leafs’ Panik latest to be fined for diving 6
- Connor McDavid is just on fire right now 17
- Video: Zidlicky’s first goal with Detroit gives Red Wings OT win over Rangers 35
- Video: P.K. Subban illustrates hockey instincts through GoPro camera perspective 5
- Video: Lundqvist could be back in 10 to 14 days 7
- Isles’ Martin suspended one game for ‘reckless’ knee on Daley 14
- More bad luck for the B’s — Connolly out 6 weeks with fractured finger 23
- Fehr willing to give Panthers ‘a reasonable opportunity’ to redevelop market 53
- Video: Isles’ Martin to face hearing for ‘stupid’ knee-on-knee hit 39
- Wild beat Avs, dying seconds fight set stage for rematch (173)
- Petrovic: Chicago will ‘probably seek revenge’ for Kane injury tonight (82)
- Winners and losers of the 2015 trade deadline (78)
- Trade: Blackhawks acquire Vermette from Arizona for Dahlbeck, first rounder (74)
- Wings get Zidlicky from Devils (74)

domain: news


Taraba State Polytechnic is one of the fastest growing institutions in Nigeria. In this piece, ANDREW OJIH takes a look at Tetfund intervention and the leadership style of its rector, Dr Ayuba Abershi leading to the current height.
The Taraba State Polytechnic in Suntai, like most other academic institutions in the state has, no doubt, suffered serious infrastructural deficit over the years with staff, including the rector inhabiting offices that were devoid of conveniences even as the institution once looked like an abandoned community secondary school.
In the last few years however, the interventions from Tertiary Education Trust Funds (TETFund) has changed the narratives.
In recent times, the polytechnic under Dr Ayuba Abarshi has witnessed drastic infrastructural transformation, with more befiting buildings and conducive atmosphere for teaching and learning.
Until recently, the main office block of the polytechnic was an eyesore as it was bereft of any inconveniences.
However with assistance from TETfund, decent and befiting office spaces for lecturers and other staff of the polytechnic have been provided.
The institurion’s director of works, Mr Samuel told Blueprint that before now, the situation was actually very bad. “In most cases, we had to rush home in the middle of the day to use conveniences at home or other public conveniences. And because the offices were also very small, we had to stockpile documents and other things such that the offices were simply chocking. It was hell working under these conditions back then.
“With the provision of these new offices now, there are times I simply work till very late in the night without even realising how late it is. The offices now are habitable, spacious, decent and very comfortable. It has also greatly increased our productivity to a very large extent.”
Mr Terfa kunde, a student of the polytechnic who graduated a few years ago confided in this correspondent that the lecture rooms at the state poly were more of cages than what are lecture rooms.
“One of the major problems we had in the school was lecture rooms. They were just so small, disorganised and so poorly ventilated such that each lecture was nearly a nightmare. The last time I went to the school, I saw that they have a lot of new lecture halls and have even renovated most of the old ones. I think that would have great impact on teaching and learning now.”
Investgation revealks that the newly-completed science and environmental buildings, the technical workshop, the farm house, and other structures have not only ensured sufficient space for learning and administrative works, but have also largely increase the general outlook of the school.
Just recently, more than 13 members of staff completed their Doctorate degrees while several are undergoing training in various disciplines. A lot more have been sponsored for Masters degrees and several seminars and workshops have been organised by the school to train and retrain the lecturers on new trends in the teaching profession and other areas. This is aimed at ensuring that the staff remain in tune with emerging trends and contemporary developments in their respective fields.
Over the years, the school perimeter has remained completely porous thereby allowing encroachment from the surrounding villages with unhindered trespass on the institution’s property. To that extent, students have been reportedly attacked right on campus while some female students were usually raped.
To address the challenge, the polytechnic just completed the construction of about 10 kilometre perimeter fence around the school with about four gates for ease of access by the school community. Since the completion of the perimeter fence with the gates manned by security personnel, there has been any reported reported attack on students on the campus.
Currently, the school is undergoing the construction of a 300 bed space facility for female students with sponsorship from Tetfund.
According to Miss Juliet Ibrahim who is a student of the polytechnic, the hostel is a major plus as accommodation has remained a major challenge especially from students coming outside the state.
She said, “It is always very difficult to get accommodation around here and even when you do, it is still very expensive. So, if the school is able to provide accommodation on campus for most of the students, the pressure on accommodation outside would be eased to some extent. In any case, it is more convenient to stay in school and enjoy other benefits such as light and power and the relative security the campus offers.”
According to the rector Dr Abarshi, “The management and board of the polytechnic actually sat and decided that there was need to upgrade the school in all its ramifications. Consequently we set out to do all we could given the scarce resources. “Fortunately, Tetfund has been of tremendous help. They have funded most of the projects on both campuses and that has drastically changed the outlook of the school.
“However, this is work in progress and there is still so much to be done. We have some ongoing projects and others that we are still hoping to secure funding to execute. In the end, our ultimate aim is to make sure that we position the school to provide the best learning environment and experience for our students and make teaching and working a pleasant experience for our staff. We just want to be among the leading polytechnics in the country and beyond.”
No doubt, when the name Dr Abershi was mentioned as the rector of the polytechnic, little did the institution and its host communities know that a serial achiever was appointed to pilot the affairs of the institution.
The little they heard about him gave them a glimmer of hope that he would build on what his predecessor did.
As soon as he took over the mantle of leadership as the rector, he gave a marching orders to graduate assistants to proceed further in their studies. This includes other ranks that were yet to bag their doctorates.
Investigation shows that the extent of staff development programmes put in place in the polytechnic is unprecedented.
For instance, the only thing staff needs in order to enjoy training is employment letter. As a result of this staff development approach, the polytechnic boasts of many Masters degree holders as well as doctoral degree holders. This is also extended to non-teaching staff in the institution.
It is as result of this that many non-teaching staff with excellent results and are interested in the field of academics have been converted to academic staff.
According to a staff member who preferred anonymity, “At the time Dr Ayuba Abershi came, we had barely up to six PhD holders, but at the moment, there are many doctoral degree holders and other ranks as a result of harvests of promotions engendered by Dr Abershi when he came.
“He believes that there is no reason to deny any hardworking qualified staff his or her promotion on some flimsy grounds.
“Many staff members of the institution can attest to these facts. Very few members, if any, can say have not enjoyed any of Dr Abershi’s goodies,” he said.
Paulina Iliyamu, a diplome student in the Department of Communications told Blueprint that the smooth running of academic programmes in the polytechnic has attracted students’ population into the school.
Shee said, “In terms of infrastructure, the rector has turned the polytechnic into a construction site as massive infrastructural projects are currently ongoing in the school.
“We are lucky to have someone like him at the helms of affairs. If we had him from the beginning to lay the foundation, this poly would have been far from where it is currently,” he said.
Steady growth/ economic activities
Residents of Jalingo, the state capital have expressed joy that the steady and uninterrupted growth in the polytechnic under the leadership of Dr Abershi is a boost to the economic activities of the host communities.
The residents commended the rector through Tetfund for providing infrastructural development to the institution thereby giving room for expansion of economic activities.
They expressed satisfaction that Dr Abeshi has make a good turn around in the history of the polytechnic that makes it a first class institution.
They argued that Tetfund intervention and his achievements have earned the institution national and international recognitions.


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The Power of Nutrition
Nutricia believes in the power of nutrition to make a positive and proven difference to the health of patients. We develop and deliver nutritrional products and services that support each specific stage of a patient's journey.
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Examples of research and development being conducted right now include the development of innovative products to meet the needs of frail elderly patients, research into the optimal management of infants with cow's milk allergies and research into the role of nutrition to help patients in the earliest stages of Alzheimer's disease.
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In pediatric care, we have developed a range of highly specialized nutritional products for children with inborn errors of metabolism, faltering growth or cow’s milk allergies. Furthermore, Nutricia organizes events where families can connect with each other, and offers support services to help families living with these medical conditions on a daily basis.
As our population ages, many more people are becoming vulnerable to the effects of old age, including frailty and disease. At Nutricia we offer healthy and balanced nutrition to elderly patients, a key factor for their overall care. Our portfolio also includes products and services to support health care providers and patients in the dietary management of chronic disease like cancer and stroke.
We see the world through the eyes of the patients we serve.
We see the world through the eyes of the patients who use our products and the health care professionals who prescribe them. It’s only through appreciating their needs that we can provide the necessary support and solutions.
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Nutricia develops nutritional solutions in specific disease areas. In pediatric care, we pioneer products and services for cow’s milk allergy, faltering growth and metabolic disorders. Our nutritional solutions for adults aim to support malnourished patients with chronic diseases such as Alzheimer’s disease, cancer and stroke, and geriatric syndromes, such as frailty, in specific stages of their disease therapy.
Neocate Splash is an innovation launched in 2017 in the USA specifically for children and teens with cow’s milk allergy. It is the first and only hypoallergenic and ready-to-use amino acid-based formula for children and teenagers with cow’s milk allergy or multiple food protein allergies. Neocate Splash is nutritionally complete and can be consumed on the go, without the hassle of measuring and mixing a hypoallergenic formula.
Small can be big
Closely working with the patient communities we serve has allowed Nutricia to be at the forefront of product innovation. It has also been a source of inspiration for products and services that help patients comply with their medically prescribed nutritional regimen.
For young patients whose growth is faltering due to disease or disability, we developed Fortini Compact Multi Fibre. These children often struggle to finish a whole bottle of standard pediatric oral nutritional supplements to meet their nutritional needs. We found a way to compact the same amount of energy, protein and fiber into a smaller bottle, helping them drink up.
Focus on taste
Nutricia’s efforts to pioneer nutritional discoveries also focus on products that taste good, a key factor that influences patient compliance with treatment, for both children and adults.
Our nutritional supplements for children with severe food allergies are produced in a variety of flavors, helping children accept the constraints of their diet. The products are also presented in small and colorful pouches, helping young patients focus less on the “medical” aspect of their diets.
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Health care costs are rising due to the increasing burden of aging and chronic disease on health care systems. Patients often require specialized care that is provided in hospitals or other care institutions, yet they would rather be cared for in the comfort of their home, living independently as long as they can. In countries such as the Netherlands, Poland and the UK, Nutricia brings medical nutrition products and specialized services to patients’ doorsteps, supporting health care professionals in delivering better quality and more cost-efficient care for their patients.
Patients prefer to be cared for in the comfort of their home, living indepently as long as they can.
WORK WITH PARTNERS
Partnering for a better food experience
We know we cannot achieve our ambition of establishing medical nutrition as an integral part of health care alone. That is why we work with partners to improve the quality of nutritional care that patients receive. Nutricia’s partnership with the Alicia Foundation demonstrates this approach. The Alicia Foundation is a center for scientific and culinary research dedicated to promoting healthier eating that also focuses on cuisine adapted to the specific needs of patients using medical nutrition products.
Together with the Alicia Foundation we have trained chefs from all around the world to prepare dishes especially adapted for patients with swallowing difficulties (dysphagia) using Nutilis, a thickening medical nutrition product from Nutricia. These dishes are safe to swallow, nutritious and tasty. Dysphagia can occur after suffering a stroke, but it can also occur due to old age or as a consequence of certain types of cancer. With our education programs for patients and caregivers, we strive to make safe, nutritious food accessible to the greatest number of patients, helping them meet their nutritional needs while enjoying the foods they love together with their families.
Digital platforms for carers and profesionals
Our products are only one aspect of the care process: Patients and their families also need help, support and reassurance to cope with these medical conditions. Nutricia recently launched Neocate Footsteps in the United States. A digital platform designed for doctors and the parents of children with difficult medical conditions, Neocate Footsteps helps clinicians and families manage a child’s condition day to day, including insurance; dietary guidelines and recipes; contact with nutritionists and other families; and practical advice as the child enters daycare, begins school or simply goes on an outing.
For parents and caregivers of children with cow’s milk allergies, we partnered with Allergy U.K. to provide support services such as the website cowsmilkallergy.co.uk. On the platform, caregivers can find important information about food allergies and the diagnosis of food-allergic conditions. In 2017, we paved the way for the launch of a truly innovative, personalized chatbot service to support parents in the process of diagnosing cow’s milk allergies in their children. This enhanced our ability to support patients and, ultimately, deliver better care.

domain: medical


Allahabad High Court Reserves Order On CAA 'Name And Shame' Hoardings
Allahabad High Court on Sunday reserved its order on CAA ‘Name And Shame' case. The court will pronounce its verdict on Monday. The hoardings put up by Chief Minister Yogi Adityanath-led Uttar Pradesh government that holds photographs and other details of all the allegedly accused in violence during the protests against the Citizenship Amendment Act (CAA). Watch the full story.
Updated : 08 March 2020, 06:43 PM


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Amy WinehouseBorn 14 September 1983. Died 23 July 2011
Amy Winehouse Biography (Wikipedia)
Amy Jade Winehouse (14 September 1983 – 23 July 2011) was an English singer and songwriter. She was known for her deep, expressive contralto vocals and her eclectic mix of musical genres, including soul, rhythm and blues and jazz.
A member of the National Youth Jazz Orchestra during her youth, Winehouse's influences were Sarah Vaughan and Dinah Washington. She signed to Simon Fuller's 19 Management in 2002 and soon recorded a number of songs before signing a publishing deal with EMI. She also formed a working relationship with producer Salaam Remi through these record publishers. Winehouse's debut album, Frank, was released in 2003. Produced mainly by Remi, many of the album's songs were influenced by jazz and, apart from two covers, were co-written by Winehouse.
Frank was a critical success in the UK and was nominated for the Mercury Prize. The song "Stronger Than Me" won her the Ivor Novello Award for Best Contemporary Song from the British Academy of Songwriters, Composers, and Authors. Winehouse's follow-up album, Back to Black (2006), was an international success. At the 2007 Brit Awards it was nominated for British Album of the Year, and she received the award for British Female Solo Artist. The song "Rehab" won her a second Ivor Novello Award. At the 50th Grammy Awards in 2008, she won five awards, tying the then record for the most wins by a female artist in a single night and becoming the first British woman to win five Grammys, including three of the General Field "Big Four" Grammy Awards: Best New Artist, Record of the Year and Song of the Year (for "Rehab"), as well as Best Pop Vocal Album.
- Mark Ronson: From The Hearthttps://ichef.bbci.co.uk/images/ic/240x135/p07qphfd.jpghttps://ichef.bbci.co.uk/images/ic/240x135/p07qphfd.jpg2019-10-11T11:06:00.000ZMark Ronson looks back on recording with Amy Winehouse.https://www.bbc.co.uk/music/audiovideo/popular/p07qsm64
Mark Ronson: From The Heart
- Amy Winehouse - Tears Dry On Their Own (Later Archive 2006)https://ichef.bbci.co.uk/images/ic/240x135/p04dvbw2.jpghttps://ichef.bbci.co.uk/images/ic/240x135/p04dvbw2.jpg2016-11-01T13:52:00.000ZAmy Winehouse performs Tears Dry On Their Own on Later... with Jools Holland in 2006https://www.bbc.co.uk/music/audiovideo/popular/p04dvcj9
Amy Winehouse - Tears Dry On Their Own (Later Archive 2006)
- Amy Winehouse - Rehab (Later Archive 2006)https://ichef.bbci.co.uk/images/ic/240x135/p04dfrhd.jpghttps://ichef.bbci.co.uk/images/ic/240x135/p04dfrhd.jpg2016-10-28T12:11:00.000ZAmy Winehouse performs Rehab on Later... with Jools Holland in 2006https://www.bbc.co.uk/music/audiovideo/popular/p04dfrwm
Amy Winehouse - Rehab (Later Archive 2006)
- Craig speaks to Amy Winehouse's best friend, Juliette Ashbyhttps://ichef.bbci.co.uk/images/ic/240x135/p042cln4.jpghttps://ichef.bbci.co.uk/images/ic/240x135/p042cln4.jpg2016-07-23T18:35:00.000ZCraig speaks to Juliette Ashby about her friendship with Amy Winehouse, on the fifth anniversary of the singer's passing.https://www.bbc.co.uk/music/audiovideo/popular/p042clp0
Craig speaks to Amy Winehouse's best friend, Juliette Ashby
- Archive Interview: Amy Winehouse, 2006https://ichef.bbci.co.uk/images/ic/240x135/p042cd7s.jpghttps://ichef.bbci.co.uk/images/ic/240x135/p042cd7s.jpg2016-07-23T16:50:00.000ZCraig chats to Amy about 'It's a Wonderful Life' and her 2006 album 'You Know I'm No Good'.https://www.bbc.co.uk/music/audiovideo/popular/p042cd8d
Archive Interview: Amy Winehouse, 2006
- Amy Winehouse in conversation with Janice Longhttps://ichef.bbci.co.uk/images/ic/240x135/p01bqljc.jpghttps://ichef.bbci.co.uk/images/ic/240x135/p01bqljc.jpg2016-04-04T11:31:00.000ZJanice looks back at Amy Winehouse's 2003 visit to the studio shortly before the release of her first album, Frank.https://www.bbc.co.uk/music/audiovideo/popular/p03pzz77
Amy Winehouse in conversation with Janice Long
- Juliette Ashby Interview & Sessionhttps://ichef.bbci.co.uk/images/ic/240x135/p032vpdz.jpghttps://ichef.bbci.co.uk/images/ic/240x135/p032vpdz.jpg2015-09-18T15:01:00.000ZJuliette Ashby and her band play live in session for The Craig Charles Funk & Soul Show.https://www.bbc.co.uk/music/audiovideo/popular/p032vpps
Juliette Ashby Interview & Session
- Amy Winehouse enter Michael Ball's Singers Hall of Famehttps://ichef.bbci.co.uk/images/ic/240x135/p02hhdyd.jpghttps://ichef.bbci.co.uk/images/ic/240x135/p02hhdyd.jpg2015-01-25T20:50:00.000ZAmy Winehouse is inducted into Michael Balls Singers Hall of Fame!https://www.bbc.co.uk/music/audiovideo/popular/p02hhdyp
Amy Winehouse enter Michael Ball's Singers Hall of Fame
- What role did the press play in Amy's downfall?https://ichef.bbci.co.uk/images/ic/240x135/p0151qr5.jpghttps://ichef.bbci.co.uk/images/ic/240x135/p0151qr5.jpg2013-02-13T15:56:00.000ZContributors Adele Mandy and Rosie Stokoe talk about the conflicting roles of the press in the rise and fall of singer Amy Winehouse.https://www.bbc.co.uk/music/audiovideo/popular/p0151qrf
What role did the press play in Amy's downfall?
- Amy was ''her own worst enemy''https://ichef.bbci.co.uk/images/ic/240x135/p0151qky.jpghttps://ichef.bbci.co.uk/images/ic/240x135/p0151qky.jpg2013-02-13T15:49:00.000ZAlison Duncan, who went to see Amy Winehouse when she was first starting out, talks about the tragedy of her talent being destroyed by addiction.https://www.bbc.co.uk/music/audiovideo/popular/p0151ql0
Amy was ''her own worst enemy''
Amy Winehouse Tracks


David Carr of the NYT provided an interesting suggestion, that perhaps news can be sold the way music is sold--to an iPod-like device.
Rich Gordon of Poynter begs to differ and highlights one very important point: Unlike music, news stories don't get replayed.
Says Gordon: "Even if you imagine a future in which the Kindle (or some superior electronic reading device) is as ubiquitous and easy to use as the iPod/iTunes package, there's one critical reason to think people won't pay for news content like they do for music: When people discover a song they like, they want to hear it over and over again. This was the principle that drove AM radio decades ago, and it drives iTunes today. As much as I love great journalism, it's awfully rare that I want to read any article more than once."
I totally agree with Gordon. The future of the news industry lies with the free model. You can't fight consumer expectation, which is that news should be free. That has already been burned into the consumer psyche.
What you can do is to try to monetize the traffic. Online advertising is but one way. Creative sponsorship is something that can really pay for a lot of news content. Then, there is e-commerce. If you have a lot of traffic, you can tap on that traffic and offer them stuff to buy online.
The business model possibilities are great but first of all, you need massive traffic to make them viable. And the only way to get such huge traffic is to give your news away for free.


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Nozmo King was formed in 1967 in Adelaide, South Australia. The band was formed by Mike McCabe after the English invasion. The group was moderately successful but only lasted for a short three years.
The name of the band was inspired by frequent advertisements and signs for 'No Smoking'. The band played music by The Beatles, The Hollies and mainly the popular contemporary songs of the day. Unfortunately the group disbanded in 1970.
- Mike McCabe - vocals, guitar
- Jim Hermel - guitar, vocals
- Bill Coleman - bass
- Leigh Langdon - drums
*Coleman was called up for National Service and was replaced by John Chamings.

domain: history